PREVENTION OF BONE LOSS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/290,129, filed on December 16, 2021, the entire contents of which are incorporated herein in their entirety by this reference.

BACKGROUND

Cancer is one of the leading casues of death worldwide. For example, pancreatic cancer has an incidence rate nearly comparable to its mortality rate. The 5-year survival rate has remained less than 6% for the past three decades, demonstrating the aggressiveness and lethal nature of this disease.

Cancer is a risk factor for bone loss and fracture. While the mechanism of bone loss is not well understood, the bone loss includes a decrease in both mineral content and protein matrix components of the bone, and leads to an increased fracture rate of, predominantly, femoral bones and bones in the forearm and vertebrae. These fractures, in turn, lead to an increase in general morbidity, a marked loss of stature and mobility, and, in many cases, an increase in mortality resulting from complications. Unchecked, bone loss can lead to osteoporosis and/or osteopenia. Osteopenia is reduced bone mass due to a decrease in the rate of osteoid synthesis to a level insufficient to compensate normal bone lysis. Osteoporosis is a major debilitating disease whose prominent feature is the loss of bone mass (decreased density and enlargement of bone spaces) without a reduction in bone volume, producing porosity and fragility.

Thus, there is a great need in the art for compositions and methods for preventing and/or treating bone loss conditions, including cancer-associated bone loss conditions.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery that administration of NK cells (e.g., super-charged NK cells), certain probiotic bacteria, sonicated osteoclasts, or any combination thereof prevents and/or treats bone loss conditions, e.g., cancer- associated bone loss conditions. Since NK cells are also effective in killing cancer cells, the immunotherapy comprising NK cells (e.g., super-charged NK cells) provides an unexpected and surprising therapeutic benefit in preventing and/or treating either (1) bone loss conditions, or (2) both cancer proliferation and cancer-associated bone loss conditions. The stage of differentiation of, e.g., pancreatic tumors has a profound effect on the function of NK cells. Specifically, the stem-like/poorly differentiated tumors are preferentially targeted by the NK cells, and an intact immune system is required for the elimination of tumors. However, tumors have been shown to cause immune suppression, in particular, NK suppression. For example, NK cells from cancer patients and humanized mice implanted with tumors lose their ability to kill and differentiate tumors. Inability of the NK cells from cancer patients to curtail tumor growth through cancer cell lysis and differentiation of tumors is a profound deficiency that can be remedied by administering the super-charged NK cells. In the exemplary case provided herein, the role of the supercharged NK cells and interferon gamma (IFN-y) in mediating tumor dissemination and bone quality alteration related to pancreatic cancer is elucidated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram illustrating the regulatory interactions that maintain homeostasis of the skeletal systems.

FIG. 2 shows a schematic diagram illustrating the roles of natural killer cells in cancer.

FIG. 3 shows a schematic diagram illustrating the BLT-NSG human immune cell reconstitution.

FIG. 4 shows that greater than 90% of tissue infiltrated immune cells in BLT-NSG mice are of human immune cells. In the pancreas of hu-BLT mice: IgG shows 99.9 % of human immune cells, m-CD45 99 % (top panels). In the liver of hu-BLT mice: Ig G presents 99.9% of human immune cells and m-CD45 of 98 % (bottome panels).

FIGs. 5A-5B show the ex vivo assessment of bone architecture by micro-CT analysis. FIG. 5 A shows the representative images of MP2. FIG. 5B shows the representative images of MP2 + NK + AJ2.

FIGs. 6A-6B show the phenotypic characteristics of bone marrow, spleen, peripheral blood, pancreas in hu-BLT mice. Lack of tumor growth, metastasis and longterm survival of NSG mice after orthotopic implantation of NK-supematant differentiated MP2 tumors in pancreas. MP2 tumors were differentiated by the NK-supernatants. Patient- derived differentiated PL12 (2 / | 0 ⁶ ) (n = 3), NK-differentiated MP2 tumors (diff-MP2) (5 x 10 ⁵ ) (n = 3), and MP2 tumors (3 x 10 ⁵ ) (n = 3) were implanted into the pancreas of NSG mice. OCs were generated from hu-BLT bone marrow monocytes and human peripheral blood monocytes. NK cells purified from hu-BLT splenocytes were pre-treated with IL-2 (1000 U/mL) and anti-CD16mAb (3 pg/mL) for 18 hours and then either cultured alone or with hu-BLT-OCs or human OCs in the presence of sAJ2 (NK: OCs: sAJ2; 2: 1 :4) and the numbers of expanding NK cells were counted on days 6, 10, 14, 18 and 22. At each day of culture equal numbers of NK cells from each group were cultured and cell growth determined (FIG. 6A). The supernatants from the NK cells and OCs cultures in the presence of sAJ2, were harvested on days 6, 10, 14, 18 and 22, and the levels of IFN-y were determined using single ELISA (FIG. 6B).

FIG. 7 shows that a single injection of super-charged NK-cells with/without feeding with AJ2 inhibited tumor growth due to differentiation of tumors in hu-BLT mice. Implantation of tumor cells in the pancreas and tail vein injection of super-charged NK that were carried out. At the time of sacrifice mice were bled and the levels of IFN-y in the serum were determined using multiplex array (n = 3).

FIG. 8 shows that a single injection of super-charged NK-cells with/without feeding with AJ2 inhibited tumor growth due to differentiation of tumors in hu-BLT mice. Hu-BLT mice were implanted with 1 * 10 ⁶ tumor cells in the pancreas, and after 1-2 weeks mice received 1.5 x 10 ⁶ super-charged NK cells via tail vein injection, and disease progression was monitored for another 3-5 weeks. Mice were also fed AJ2 (5 billion/dose) starting 1-2 weeks before tumor implantation, and thereafter every 48 h throughout the experiment. Implantation of tumor cells in the pancreas and tail vein injection of super-charged NK cells were carried out, and at the time of sacrifice mice were bled and the levels of IFN-y in the serum were determined using multiplex array (n = 3).

FIG. 9 shows the Hu-BLT mice that were implanted with MP2 tumors and injected with NK cells and fed with AJ2. Upon sacrifice, tumors were resected, and single cell cultures were prepared, and equal numbers of tumors were cultured on day 7 or 11 and the levels of IFN-y were determined in culture supernatants (n = 2/each experimental condition).

FIG. 10 shows the Hu-BLT mice that were implanted with MP2 tumors and injected with NK cells or implanted with NK-differentiated tumors. At the end of the experiment pancreatic tumors were harvested and tumor growth was assessed on days 7, 11 and 14, and on day 7 attached tumors from each well were counted and equal numbers of tumors from each group were re-cultured and tumor growth in each well was determined every 3 days (n = 12/each experimental condition, one representative experiment is shown in the figure).

FIGs. 11A-11C show that the NK cell cytotoxicity and ability to secrete IFN-y are severely decreased in pancreatic cancer patients.

FIGs. 12A-12L show that the injection of super-charged NK-cells with/without feeding with AJ2 restored and increased IFN-y secretion and/or cytotoxic function of NK cells from different tissues of tumor-bearing hu-BLT mice. Upon sacrifice, PBMCs were isolated from blood and treated with IL-2 (1000 U/mL) before they were used in cytotoxicity assay against OSCSCs using 4 h ⁵¹ Cr release assay. Lytic units 30/10 ⁶ cells were determined using inverse number of NK cells required to lyse 30% of the target cells x 100. Procedures were carried out before the PBMCs were isolated and treated with (1000 U/mL) and the supernatants were harvested and IFN-y secretion was determined using ELISA (n = 4/each experimental condition) (FIG. 12B, FIG. 12C). Procedures were carried out before spleens were harvested, and single-cell suspensions were prepared. Splenocytes were treated with IL-2 (1000 U/mL) before they were used for cytotoxicity against OSCSCs using 4 h ⁵¹ Cr release assay. Lytic units 30/10 ⁶ cells were determined using inverse number of NK cells required to lyse 30% of the target cells x 100 (n = 4/each experimental condition). Procedures were carried out as previously described before the supernatants were harvested from day 3 or 7 cultures of splenocytes, and IFN-y secretion was determined using ELISA (n = 5/each experimental condition). NK-enriched cells were isolated from splenocytes and were cultured with IL-2 (1000 U/mL) before they were used for cytotoxicity against OSCSCs using 4 h ⁵¹ Cr release assay. Supernatants were harvested from day 3 or 7 NK-enriched cultures and IFN-y secretion was determined using ELISA (n = 6/each experimental condition). The CD3+ T-cells were isolated from splenocytes and were cultured with IL-2 (100 U/mL), and on day 3 or day 7 the supernatants were harvested and IFN-y secretion was determined using ELISA (n = 4/each experimental condition). BM cells were harvested and treated with IL-2 (1000 U/mL) for 7 days before they were used for cytotoxicity against OSCSCs using 4 h ⁵¹ Cr release assay. Supernatants were harvested on day 3 or day 7 of BM cultures and IFN-y secretion was determined using ELISA (n = 6/each experimental condition) (K,L). The following symbols represent the levels of statistical significance within each analysis, *** (p-value < 0.001), **.

FIGs. 13A-13B show that the mice fed with AJ2 presented increased trabecular bone formation when compared to the CTRL (control) group. FIGs. 14A-14B show that the MP2 tumor-bearing mice injected with NK cells and fed with AJ2, presented statistically significant higher trabecular bone volume when compared to MP2 tumor and MP2+AJ2 group, respectively.

FIGs. 15A-15B show that the stem-like/undifferentiated tumors implanted in hu- BLT mice injected with cultures of NK cells and fed with or without AJ2 presented similar bone formation when compared to the control group.

FIG. 16 shows that there was a remarkable correlation between induction and secretion of IFN-y and the bone morphology. In the MP2 tumor-bearing mice injected with NK cells and fed with AJ2 group there was increased IFN-y in serum, cell cultures from pancreatic tumors, NK cells purified from splenocytes, PBMCs, splenocytes cell cultures, and bone marrow cells. On the contrary, MP2 tumor-bearing hu-BLT mice showed decreased IFN-y in the same compartments.

FIG. 17 shows that the histological analysis of the AJ2 sample exhibited increased bone formation when compared to the Control and MP2 samples. The histomorphometric values for the MP2+NK +AJ2 sample could not be confirmed due to staining failure.

FIG. 18 shows that the MP2 tumor sample exhibits positive TRAP staining.

FIG. 19 shows the decreased bone in OSCSC-implanted BLT mice and its restoration by either feeding AJ2 or injection with super-charged NK cells.

FIG. 20 shows the decreased bone in MP2 poorly differentiated pancreatic tumor implanted BLT mice and its restoration by either feeding AJ2 and/or injection with supercharged NK cells.

FIG. 21A-FIG. 21G show increased IFN-y and decreased IL-10 secretions by sAJ4 treated PBMCs in comparison to sAJ3 and sAJ2 treated PBMCs. PBMCs were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with sAJ3 (PBMC:sAJ3, 1 :20) or with sAJ4 (PBMC:sAJ4, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (PBMC:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (PBMC:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y (FIG. 21A), TNF-a (FIG. 21B), IL-6 (FIG. 21C), and IL-10 (FIG. 21D) secretion using multiplex assay. PBMCs were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with a combination of IL-2 (1000 U/ml) and anti- CD3/28 antibody (25 pl/ml) or with a combination of IL-2 (1000 U/ml) and sAJ2 (PBMC:sAJ2, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (PBMC:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (PBMC:sAJ4, 1 :20) or with sAJ2 (PBMC:sAJ2, 1 :20) or with sAJ3 (PBMC:sAJ3, 1 :20) or with sAJ4 (PBMC:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y secretion using single ELISA. One of the representative experiments is shown (FIG. 2 IE), and cumulative samples are shown (n=5) (FIG. 2 IF). PBMCs were treated as described in FIG. 2 IE for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL-10 secretion using specific single ELISAs, and ratio of IFN-y to IL-10 was determined (FIG. 21G). **(p value 0.001-0.01), *(p value 0.01-0.05).

FIG. 22A-FIG. 221 show increased IFN-y and decreased IL- 10 secretions by sAJ4 treated NK cells in comparison to sAJ3 and sAJ2 treated NK cells. NK cells were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with sAJ3 (NK:sAJ3, 1 :20) or with sAJ4 (NK:sAJ4, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (NK:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (NK:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from NK cells to determine IFN-y (FIG. 22A) and IL- 10 (FIG. 22B) secretion using multiplex assay. NK cells were treated as described in FIG. 22A for 18 hours, and the number of cells secreting IFN-y in the NK cells were determined as spot counts using ELISpot assay (FIG. 22C), and the supernatants were harvested to determine IFN-y using single ELISA (FIG. 22D). NK cells were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with a combination of IL-2 (1000 U/ml) and sAJ2 (NK:sAJ2, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (NK:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (NK:sAJ4, 1 :20) or with sAJ2 (NK:sAJ2, 1 :20) or with sAJ3 (NK:sAJ3, 1 :20) or with sAJ4 (NK:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from NK cells to determine IFN-y secretion using single ELISA. One of the representative experiments (FIG. 22E), and the combination of samples are shown (n=5) (FIG. 22F). NK cells were treated as described in FIG. 22E for 18 hours before the supernatant was harvested to determine IFN- y secretion using multiplex assay (n=5) (FIG. 22G). NK were treated as described in FIG. 22E for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL- 10 secretion using specific single ELISAs, and ratio of IFN-y to IL- 10 was determined (FIG. 22H). NK were treated as described in FIG. 22E for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL-10 secretion using multiplex assay, and ratio of IFN-y to IL- 10 was determined (n=4) (FIG. 221). ***(p value <0.001), *(p value 0.01-0.05).

FIG. 23A-FIG. 23B show increased IFN-y secretion levels in sAJ4 treated NK cells and monocytes in comparison to sAJ3 treated cells. NK cells and monocytes of healthy individuals were isolated from PBMCs as described in Example 4. NK cells or monocytes or co-culture of NK and monocytes (NK: monocytes; 1 : 1) treated with sAJ3 (NK:sAJ3, 1 :20) or sAJ4 (NK:sAJ4, 1 :20) or a combination of IL-2 (1000 U/ml) and sAJ3 (NK:sAJ3, 1 :20) or a combination of IL-2 (1000 U/ml) and sAJ4 (NK:sAJ4, 1 :20) or a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) and sAJ3 (NK:sAJ3, 1 :20) or or a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) and sAJ4 (NK:sAJ4, 1 :20) for 18 hours before the supernatants were harvested to determine IFN-y using specific ELISA (FIG. 23 A). NK cells and monocytes were treated as described in FIG. 23 A for 18 hours before the supernatant was harvested to determine IFN-y and IL- 10 secretion using specific single ELIS As, and ratio of IFN-y to IL- 10 was determined (FIG. 23B).

FIG. 24 shows increased IFN-y by sAJ4 treated CD8+ T cells in comparison to sAJ3 treated CD8+ T cells. CD8+ T cells were left untreated or treated with IL-2 (100 U/ml) or IL-2 (100 U/ml) and anti-CD3/28 mAbs (25 pl/ml), or IL-2 (100 U/ml) and sAJ2 (CD8+ T:sAJ2, 1 :20) or IL-2 (100 U/ml) and sAJ3 (CD8+ T:sAJ3, 1 :20) or IL-2 (100 U/ml) and sAJ4 (CD8+ T:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from NK cells to determine IFN-y using specific ELISAs.

FIG. 25 shows schematic representation of AJ3 function in ALS. AJ3 will be effective in alleviating auto-immunity, in particular in ALS since it will greatly regulate the levels and function of IFN-y, decreasing over activation and death of motor neurons. Gene deletion or mutation may decrease MHC-class I expression on some motor neurons activating NK cells. Both cytokine and receptor mediated cross-linking will greatly sway the activation of the NK cells towards greater IFN-y secretion in the presence of no or decreased IL-10. Induction of IFN-y secretion by the NK cells will not only expand CD8+ T cells but also will differentiate and increase MHC-class I expression on motor neurons, allowing the mutated motor neurons to become susceptible to CD8+ T cell activation and effector function, in which case further IFN-y secretion could exacerbate the death of not only mutated motor neurons, but also the non-mutated bystander cells, through overactivation and induction of cell death. Treatment with AJ3 will regulate the increase in the secretion of IFN-y by increased induction of IL-10, decreasing the activation of NK cells and CD8+ T cells, and minimizing or even halting the death of motor neurons and slowing the progression of the disease. In the absence of disease, the default function of AJ3 formulation is towards increase in anti-inflammatory IL- 10 induction.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are compositions and methods that are useful in preventing and/or treating bone loss conditions (e.g., cancer-associated bone loss conditions), in inducing bone formation, and/or inhibiting or preventing bone loss.

This disclosure provides the correlation and potential impact of the immune system, specifically NK cells and interferon gamma, in bone quality alteration related to cancer, e.g., pancreatic cancer in hu-BLT mice. Specifically, the studies show how MP2 tumors injected in hu-BLT mice affected the bone structure. The analysis show how the MP2 tumor-bearing mice that are injected with NK cells and fed with and without AJ2 influenced the bone structure.

As demonstrated herein, the suppression of NK cell cytotoxicity and decreased secretion of IFN-y in tumor-bearing mice within all tissue compartments are restored by the super-charged NK cells. There was a remarkable correlation between the micro-CT analysis results, the induction and secretion of IFN-y, and bone morphology. The MP2 tumorbearing mice injected with NK cells’ cultures and fed with AJ2 presented increased bone formation with statistically significant higher trabecular bone volume compared to MP2 tumor-bearing mice group and MP2 tumor-bearing mice fed with AJ2 group, respectively. On the contrary, MP2 tumor-bearing mice showed decreased IFN-y and decreased bone formation.

Consistent with the micro-CT findings, histological analysis of the AJ2 treatment group exhibited increased bone formation when compared to the Control and MP2 tumor groups. TRAP staining demonstrated more osteoclast activity and bone resorption in the MP2 tumor sample compared to the rest of the samples.

Therefore, it is demonstrated herein that IFN-y (1) induces secretion by NK cells, (2) inhibits tumor growth, and (3) decreases skeletal complications of malignancy by directly acting on host cells to inhibits osteoclast formation and function.

In this study, the enhanced osteolytic lesion formation in BLT tumor-bearing mice and NK cells’ ability to secrete IFN-y to significantly reduce bone loss in tumor-bearing mice indicate a direct anti-osteoclastogenic role for IFN-y in the setting of cancer-induced bone disease. Furthermore, the present study further indicates that IFN-y directly promotes bone formation.

Overall, the present data demonstrate that the injection of NK cells into tumorbearing mice increased IFN-y secretion in hu-BLT mice. The data also indicate that IFN-y has direct anti-tumor effects and can suppress tumor-induced bone loss by directly targeting host osteoclasts to inhibit osteolysis.

Accordingly, the present work provides a novel report of bone quality alteration related to pancreatic cancer in hu-BTL mice and the role of IFN-y secreted by NK cells in the suppression of tumor-induced bone loss and induction of bone formation.

NK and T cells make up significant percentages of lymphocytes in peripheral blood mononuclear cells (PBMCs). NK cells are mainly known as the effectors of innate immunity due to their lack of antigen-specificity. CD4+ and CD8+ T cells mediate the adaptive cellular immunity, which closely collaborate with the innate immune system. NK cells and CD8+ T cells play significant role in cancer control, and NK and CD8+ T cellbased immunotherapies are among the leading standards in cancer therapeutics. Decreased function of these two lymphocytes result in poor prognosis in cancer patients. It has been shown that NK cells could activate and induce the proliferation of T cells, and also could kill chronically activated leukocytes. Increased levels of tumor-infiltrating CD8+ T cells were found to be associated with complete responses to standard chemotherapeutic regimens, and the presence of CD8+ memory T cells is associated with patient survival. It was found that NK cells can accelerate CD8+ T cells responses against viral infections, such as those caused by cytomegaloviruses.

AJ2 is a combination of different strains of gram-positive probiotic bacteria selected based on their superior ability to induce optimal secretion of both pro-inflammatory and anti-inflammatory cytokines in NK cells. Here, provided herein are evaluations of Al-Pro (AJ3), CA/I-Pro (AJ4), in addition to and incomparison to NK-CLK (AJ2). Sonicated bacteria (e.g., AJ2) was used for in-vitro studies and live bacteria (e.g., AJ2) for in-vivo studies, and similar effect on immune cells was seen. A combination of osteoclasts (OCs) and sonicated AJ2 (sAJ2) gave rise to generate super-charged NK (sNK) cells with great potential to kill and differentiate tumors. The in-vivo studies have demonstrated that supercharged NK cells regulate the balance of T cell subsets, cytokine secretions, and cytotoxic activity of immune cells in various tissue compartments of mice. In addition, it was demonstrated that super-charged NK cells lyse activated CD4+ T and not CD8+ T cells, thus selecting and preferentially expanding CD8+ T cells.

Toll-like receptors (TLRs) are known to recognize common microbial patterns and, plays crucial role in innate immune response and initiation of adaptive immune response. NK cells were found to express TLR mRNA for TLR1-10. NK cells were found to produce higher levels of IFN-y and also increased cytotoxic activity after TLR2, TLR3, TLR4 and TLR5 stimulation. TLRs 2, 4, 5 and 11 on the cell surface recognize bacterial lipoproteins, lipopolysaccharide (LPS), flagellin and profilin, respectively. TLRs 3, 7, 8 and 9 are expressed in endosomal compartments and recognize viral and bacterial nucleic acids. In addition, heat shock proteins (HSPs) and extracellular matrix components, such as fibronectin and hyaluronan can also activate the innate immune system through TLR2 and TLR4. Moreover, DNA complexes may activate TLR3, TLR7 and TLR9. TLR expression has been clearly shown on the surface of innate immune cells such as monocytemacrophages and dendritic cells and therefore, TLRs have traditionally been considered to play an important role in indirectly controlling T cell responses through the activation of innate immune cells through TLRs. However, recent studies indicated that T cells can also express TLRs. At the moment it is not clear how TLRs contribute to the activation of T cells, however, the present disclosure indicates that functional modulation of T cells by probiotic bacteria, AJ3 and AJ4 is much less than that seen by the NK cells, even though there is still an increase in activation and regulation by the AJ4 and AJ3, respectively. Therefore, there are direct and indirect activation of T cells by AJ3 and AJ4 probiotic bacteria of T cells. Accordingly, in certain aspects, further provided herein are compositions comprising probiotic bacteria and methods of using them. In particular, the activities and functions of different formulations of probiotic bacteria (e.g., AJ2, AJ3, AJ4) have beem explored. To compare the activation by the probiotic bacteria to either cytokine or receptor cross linking; PBMC, NK and CD8+ T cells were left untreated or treated with IL-2 or IL- 2+anti-CD16mAbs or IL-2+anti-CD3/CD28mAbs in the presence and absence of sAJ2, sAJ3 and sAJ4. IL-2+anti-CD16mAbs activation of PBMCs and NK cells had the highest IFN-y/IL-10 ratio whereas IL-2 combination with sAJ4 had the next highest followed by IL-2+sAJ2 and the lowest was seen with IL-2+sAJ3. Interestingly, IL-2+anti- CD3/CD28mAbs had lower IFN-y/IL-10 in PBMCs when compared to either IL-2 alone or IL2+anti-CD16mAbs. Accordingly, the highest secretion of IFN-y was seen when the PBMCs and NK cells were treated with IL-2+sAJ4, intermediate for IL-2+sAJ2 and the lowest IL-2+sAJ3. Indeed, IFN-y secretion by IL-2+sAJ4 exceeded much higher than that of the levels of IFN-y secretion by IL-2 or IL-2+anti-CD16mAbs or IL-2+anti- CD3/CD28mAbs in PBMCs and NK cells. The levels of IFN-y secretion without IL-2 remained much lower and the highest was seen when sAJ4 was used to activate immune cells, and the ratio of IFN-y to IL- 10 remained much lower in all treatments when compared to those treated in the presence of IL-2. Of note is the difference between NK cells and CD8+ T cells in which synergistic induction of IFN-y by IL-2+sAJ4 was significantly higher than those seen by CD8+ T cells indicating either lack of TLR receptors for response or augmented secretion of IL- 10 which regulate the secretion of IFN-y in CD8+ T cells. sAJ3 probiotic bacteria had the lowest IFN-y/ILlO ratios and triggered much lower induction of IFN-y, Thus, sAJ3 probiotic bacteria was formulated to augment antiinflammatory cytokine IL- 10 to counter the aggressive nature of pro-inflammatory cytokine IFN-y induced by NK and CD8+ T cells in ALS patients.

Therefore, sAJ3 will alleviate auto-immunity seen in ALS by regulating the levels and function of IFN-y, thereby decreasing overactivation and death of motor neurons.

By contrast, AJ4, which is particularly effective in activating NK cells and inducing IFN-g production, will treat cancer and bone loss (including cancer-associated bone loss).

Definitions

The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The term “administering” is intended to include routes of administration which allow an agent to perform its intended function. Examples of routes of administration for treatment of a body which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal, etc.), oral, inhalation, and transdermal routes. The injection can be bolus injections or can be continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. The agent also may be administered as a prodrug, which is converted to its active form in vivo.

The amount or level of IFN-y or other cytokine/chemokine is “significantly” higher or lower than the normal amount, if the amount is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least, about, or no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more than that amount. Alternately, the amount of the cytokines and/or chemokines can be considered “significantly” higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of the cytokines and/or chemokines. Such “significance” can also be applied to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell growth, and the like.

As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the patient, which may include synergistic effects of the two agents). For example, the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic agents.

The term “control” refers to any reference standard suitable to provide a comparison to the expression products in the test sample. Such a control may comprise any suitable sample, including but not limited to a sample from a control diseased patient (e.g., those afflicted with a bone loss condition) (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the diseased patient (e.g., those afflicted with a bone loss condition), cultured primary cells/tissues isolated from a subject such as a normal subject or the diseased patient (e.g., those afflicted with a bone loss condition), adjacent normal cells/tissues obtained from the same organ or body location of the diseased patient, a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository. In other embodiments, the control may comprise a reference standard IFN-y level from any suitable source, including but not limited to a previously determined IFN-y level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, certain bone density) or receiving a certain treatment (for example, cancer therapy or bone loss therapy). It will be understood by those of skill in the art that such control samples and reference standard IFN-y levels can be used in combination as controls in the methods of the present invention. In some embodiments, the control may comprise normal or non-diseased cell/tissue sample. In other embodiments, the control may comprise a level for a set of patients, such as a set of diseased patients (e.g., those afflicted with a bone loss condition), or for a set of patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome. In other preferred embodiments, the control may comprise normal cells, cells from patients treated with bone loss therapy, and cells from patients having benign cancer and the associated bone loss condition. In still other embodiments, the control may also comprise a measured value for example, healthy or diseased individuals who were not treated with the agents of the present disclosure, or healthy or diseased individuals who were administered with other bone loss therapy. In other embodiments, the control comprises a ratio of IFN-y and other cytokine/chemokine levels, including but not limited to the level of one cytokine against the level of another cytokine, e.g., the ratio of the level of IFN-y and the level of IL-10. In particularly preferred embodiments, the control comprises a control sample which is of the same lineage and/or type as the test sample.

The term “immune cell” refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

The term “immune response” refers to a response mediated by any or all immune cells. The “immune response” includes T cell mediated and/or B cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.

The term “inhibit” includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. In some embodiments, cancer is “inhibited” if at least one symptom of the bone loss condition is alleviated, terminated, slowed, or prevented. As used herein, a bone loss condition is also “inhibited” if recurrence of the bone loss condition is reduced, slowed, delayed, or prevented. Similarly, a biological function, such as the function of a protein, is inhibited if it is decreased as compared to a reference state, such as a control like a wild-type state. The term “preventing” is art-recognized, and when used in relation to a disease such as cancer or a bone loss condition (e.g., cancer-associated bone loss condition), is well understood in the art, and includes administration of a composition which reduces the severity of or the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the agent. Thus, prevention of a bone loss condition or bone loss includes, for example, delaying the appearance of reduction in bone density in a population of patients receiving a prophylactic treatment relative to an untreated control population, e.g., by a statistically and/or clinically significant amount.

A “therapeutically effective amount” of a substance or cells is an amount capable of producing a medically desirable result in a treated patient, e.g., increase in bone density, decrease in bone loss, with an acceptable benefit: risk ratio, preferably in a human or nonhuman mammal.

The term “treating” includes prophylactic and/or therapeutic treatments. The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the subject one or more agents of the present disclosure. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal), then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition); whereas, if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

All numerical ranges provided herein are understood to be shorthand for all of the decimal and fractional values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9 and all intervening fractional values between the aforementioned integers such as, for example, 1/2, 1/3, 1/4, 1/5, 1/6, 1/8, and 1/9, and all multiples of the aforementioned values. With respect to subranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

Bone Loss Condition

There are three major types of bone cells: osteocytes, osteoblasts and osteoclasts. Osteocytes are the most abundant cell type in bone (Nijweide et al. (1996) Principles of Bone Biology (Bilezikian, Riasz and Rodan, eds.), Academic Press, New York, N.Y., pp. 115-126), with approximately ten times more osteocytes than osteoblasts (Parfitt et al. (1977) Clin. Orthop. Rel. Res. 127:236-247), and with osteoblasts far more abundant than osteoclasts. Each of these different types of bone cell has a different phenotype, morphology and function. Osteocytes are localized within the mineral matrix at regular intervals, and arise from osteoblasts. During their transition from osteoblasts, osteocytes maintain certain osteoblastic features, but acquire several osteocyte-specific characteristics. Mature osteocytes are stellate shaped or dendritic cells enclosed within the lacuno-canalicular network of bone. Long, slender cytoplasmic processes radiate from the central cell body, with most of the processes perpendicular to the bone surface. The processes connect the osteocyte to neighboring osteocytes and to the cells lining the bone surface.

The functions of osteocytes include: to respond to mechanical strain and to send signals of bone formation or bone resorption to the bone surface, to modify their microenvironment, and to regulate both local and systemic mineral homeostasis. Increasing evidence indicates that osteocytes may regulate physiological local bone remodeling, in part through their cell death and apoptosis that trigger osteoclasts formation and bone resorption, and in part by secreting sclerostin, a molecule specifically produced by osteocytes that acts as an inhibitor of bone formation (Giuliani et al. (2015) in Bone Cancer (Second Edition), Chapter 42, pp 491-500).

Osteoblasts are the skeletal cells responsible for bone formation, and thus synthesize and regulate the deposition and mineralization of the extracellular matrix of bone (Aubin and Liu, (1996) Principles of Bone Biology (Bilezikian, Riasz and Rodan, eds.), Academic Press, New York, N.Y., pp. 51-67).

Osteoclasts are multinucleated giant cells with resorbing activity of mineralized bone (Suda et al., (1996) Principles of Bone Biology (Bilezikian, Riasz and Rodan, eds.), Academic Press, New York, N.Y., pp. 87-102). The term “bone loss condition” refers to a condition that occurs when the body doesn’t make new bone as quickly as it reabsorbs old bone. In some embodiments, “bone loss conditions” include bone diseases, such as osteopenia, osteolysis, osteoporosis, osteoplasia (osteomalacia), and Paget's disease of bone. In other embodiments, “bone loss conditions” include bone loss that is associated with other diseases, such as diabetes, chronic renal failure, hyperparathyroidism, and cancer (e.g., multiple myeloma and breast cancer), which result in abnormal or excessive bone loss. The present invention is directed to methods of treating and/or preventing bone loss conditions, such as osteoporosis and osteopenia and other diseases where inhibiting bone loss may be beneficial, including Paget's disease, malignant hypercalcemia, periodontal diseasejoint loosening and metastatic bone disease, as well as reducing the risk of fractures, both vertebral and nonvertebral.

Osteopenia refers to bone density that is lower than normal density but not low enough to be classified as osteoporosis. Osteopenia is reduced bone mass due to a decrease in the rate of osteoid synthesis to a level insufficient to compensate normal bone lysis. Osteopenia is commonly seen in people over age 50 that have lower than average bone density but do not have osteoporosis.

Osteoporosis is a structural deterioration of the skeleton caused by loss of bone mass resulting from an imbalance in bone formation, bone resorption, or both, such that the resorption dominates the bone formation phase, thereby reducing the weight-bearing capacity of the affected bone. In a healthy adult, the rate at which bone is formed and resorbed is tightly coordinated so as to maintain the renewal of skeletal bone. However, in osteoporotic individuals an imbalance in these bone remodeling cycles develops which results in both loss of bone mass and in formation of microarchitectural defects in the continuity of the skeleton. These skeletal defects, created by perturbation in the remodeling sequence, accumulate and finally reach a point at which the structural integrity of the skeleton is severely compromised and bone fracture is likely. Although this imbalance occurs gradually in most individuals as they age (“senile osteoporosis”), it is much more severe and occurs at a rapid rate in postmenopausal women. In addition, osteoporosis also may result from nutritional and endocrine imbalances, hereditary disorders and a number of malignant transformations.

Bone loss is also an important consideration for treatment among cancers, particularly among multiple myeloma, breast cancer, and pancreatic cancer. Osteoplasia, also known as osteomalacia (“soft bones”), is a defect in bone mineralization (e.g., incomplete mineralization), and classically is related to vitamin D deficiency (1,25-dihydroxy vitamin D3). The defect can cause compression fractures in bone, and a decrease in bone mass, as well as extended zones of hypertrophy and proliferative cartilage in place of bone tissue. The deficiency may result from a nutritional deficiency (e.g., rickets in children), malabsorption of vitamin D or calcium, and/or impaired metabolism of the vitamin.

Bone Loss Therapy

Current treatments for osteoporosis or osteopenia are based on inhibiting further bone resorption, e.g., by 1) inhibiting the differentiation of hemopoietic mononuclear cells into mature osteoclasts, 2) by directly preventing osteoclast-mediated bone resorption, or 3) by affecting the hormonal control of bone resorption. Drug regimens used for the treatment of osteoporosis include calcium supplements, estrogen, calcitonin, estradiol, and diphosphonates. Vitamin D3 and its metabolites, known to enhance calcium and phosphate absorption, can also be used. Similarly, parathyroid hormone (PTH, such as the 84-amino acid PTH peptide or fragments thereof, such as the teriparatide first 1-34 amino acids of human PTH, can also be used (see, for example, U.S. Pat. Publ. 2018/0028622 and U.S. Pat. 8,110,547, each of which is incorporated in their entirety herein by this reference).

Probiotic bacteria

In some embodiments, the instant invention is drawn to a method comprising administering to the subject a composition comprising at least one probiotic bacterial strain, capable of regulating NK cell function. In other embodiments, a method may comprise contacting the NK cells in vivo, in vitro, or ex vivo with at least one probiotic bacterial strain in order to activate NK cells prior to administration to the subject.

Such probiotic bacteria induce significant production or secretion of various cytokines/chemokines, e.g., IFN-y, Gro-alpha, IL-10, and TNF-a. In addition, such probiotic bacteria induce significant activation and/or expansion of NK cells.

Preferred probiotic bacteria species of the present disclosure include Streptococcus (e.g., S. thermophiles'), Bifidobacterium (e.g., B. longum, B. breve, B. infantis), and/or Lactobacillus genera (e.g., L. acidophilus, L. helveticus, L. bulgaricus, L. rhamnosus, L. plantarum, and L. easel). The compositions and methods of the present disclosure comprise at least one probiotic bacterial strain, preferably a combination of two or more different bacterial strains, to a subject, preferably a mammal (e.g., a human). Such administration may be systemically or locally (e.g., directly to intestines, e.g., orally or rectally) performed. The preferable administration route for probiotic bacteria is oral administration. Other routes (e.g., rectal) may be also used. For administration, either the bacteria (e.g., in a wet, sonicated, ground, or dried form or formula), the bacterial culture medium comprising the bacteria, or the bacterial culture medium supernatant (not containing the bacteria), may be administered. The bacteria may be alive, partially alive, or dead. The bacteria may be sonicated, ground, wet, or dry (e.g., freeze-dried).

In some embodiments, the composition (bacterial, pharmaceutical, and/or nutraceutical) of the present disclosure comprises at least about 1 x 10 ⁴ , 1 x 10 ⁵ , 1 x 10 ⁶ , 2 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 5 x 10 ⁷ , 1 x 10 ⁸ , 5 x 10 ⁸ , 10 x 10 ⁸ , 100 x 10 ⁸ , IxlO ⁹ , 5xl0 ⁹ , 10xl0 ⁹ , 100 x 10 ⁹ , HO x 10 ⁹ , 120 x 10 ⁹ , 130 x 10 ⁹ , 140 x 10 ⁹ , 150 x 10 ⁹ , 160 x 10 ⁹ , 170 x 10 ⁹ , 180 x 10 ⁹ , 190 x 10 ⁹ , 200 x 10 ⁹ , 210 x 10 ⁹ , 220 x 10 ⁹ , 230 x 10 ⁹ , 240 x 10 ⁹ , 250 x 10 ⁹ , 260 x

10 ⁹ , 270 x 10 ⁹ , 280 x 10 ⁹ , 290 x 10 ⁹ , 300 x 10 ⁹ , 310 x 10 ⁹ , 320 x 10 ⁹ , 330 x 10 ⁹ , 340 x 10 ⁹ ,

350 x 10 ⁹ , 360 x 10 ⁹ , 370 x 10 ⁹ , 380 x 10 ⁹ , 390 x 10 ⁹ , 400 x 10 ⁹ , 410 x 10 ⁹ , 420 x 10 ⁹ , 430 x 10 ⁹ , 440 x 10 ⁹ , 450 x 10 ⁹ , 460 x 10 ⁹ , 470 x 10 ⁹ , 480 x 10 ⁹ , 490 x 10 ⁹ , or 500 x 10 ⁹ , but no more than 510 x 10 ⁹ , 520 x 10 ⁹ , 530 x 10 ⁹ , 540 x 10 ⁹ , 550 x 10 ⁹ , 600 x 10 ⁹ , 650 x 10 ⁹ , 700 x 10 ⁹ , 750 x 10 ⁹ , 800 x 10 ⁹ , 850 x 10 ⁹ , 900 x 10 ⁹ , 950 x 10 ⁹ , or 1000 x 10 ⁹ total CFU of bacteria per gram of the composition.

In some embodiments, the composition comprises at least about 180 x 10 ⁹ but no more than about 270 x 10 ⁹ total CFU of bacteria per gram of the composition. In preferred embodiments, the composition comprises about 250 x 10 ⁹ total CFU of bacteria per gram of the composition.

AJ2 probiotic bacteria

AJ2 is a combination of 7 strains of gram-positive probiotic bacteria with the ability to induce synergistic production of IFN-y when added to IL-2 -treated or IL-2 + anti-CD16 monoclonal antibody -treated NK cells (anti-CD16mAb). The combination of strains was used to provide bacterial diversity in addition to synergistic induction of a balanced pro and anti-inflammatory cytokine and growth factor release in NK cells. Moreover, the quantity of each bacteria within the combination of strains was adjusted to yield a closer ratio of IFN-y to IL- 10 to that obtained when NK cells are activated with IL-2 + anti-CD16mAb in the absence of bacteria. The rationale behind such selection was to obtain a ratio similar to that obtained with NK cells activated with IL-2 + anti-CD16mAb in the absence of bacteria since such treatment provided significant differentiation of the cells.

AJ3 probiotic bacteria

In some embodiments, the composition (bacterial, pharmaceutical, and/or nutraceutical) of the present disclosure comprises at least two bacterial strains selected from: Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium inf antis. In some embodiments, one or more bacterial strains are intact. In some embodiments, one or more bacterial strains are sonicated. In preferred embodiments, the composition is an AJ3 composition comprising Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium infantis.

In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, but no more than about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bacteria in the composition are Bifidobacterium Longum.

In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%, but no more than about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bacteria in the composition are Bifidobacterium breve.

In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, but no more than about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bacteria in the composition are Bifidobacterium infantis.

In some embodiments, the percent bacteria refers to the percentage of the colony forming unit (CFU) of said bacteria relative to the total CFU of bacteria in the composition.

In some embodiments, the bacteria in the composition comprise about 50% (or about 40% to about 60%) Bifidobacterium Longum, about 10% (or about 1% to about 20%) Bifidobacterium breve, and about 40% (or about 30% to about 50%) Bifidobacterium infantis, wherein the percent bacteria refers to the percentage of the CFU of said bacteria relative to the total CFU of bacteria in the composition.

AJ4 composition

In some embodiments, the composition (bacterial, pharmaceutical, and/or nutraceutical) of the present disclosure comprises at least two bacterial strains selected from: Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei. In some embodiments, one or more bacterial strains are intact. In some embodiments, one or more bacterial strains are sonicated. In preferred embodiments, the composition is an AJ4 composition comprising Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei.

In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, but no more than about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bacteria in the composition are Streptococcus thermophiles.

In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%, but no more than about 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bacteria in the composition are Lactobacillus acidophilus. In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,

26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,

41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,

56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, but no more than about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bacteria in the composition are Lactobacillus plantarum.

In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,

26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%, but no more than about 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,

52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,

67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,

97%, 98%, or 99% of the bacteria in the composition are Lactobacillus paracasei.

In some embodiments, the percent bacteria refers to the percentage of the colony forming unit (CFU) of said bacteria relative to the total CFU of bacteria in the composition.

In some embodiments, the bacteria in the composition comprise about 30% (or about 20% to about 40%) Streptococcus thermophiles, about 20% (or about 10% to about 30%) Lactobacillus acidophilus, about 40% (or about 30% to about 50%) Lactobacillus plantarum, and about 10% (or about 1% to about 20%) Lactobacillus paracasei, wherein the percent bacteria refers to the percentage of the CFU of said bacteria relative to the total CFU of bacteria in the composition.

Osteoclasts

Osteoclasts are multinuclear cells, have the unique ability to degrade bone to initiate normal bone remodeling and mediate bone loss in pathologic conditions by increasing their resorptive activity. Osteoclasts are derived from hematopoietic stem cells, precursors in the myeloid/ monocyte lineage that circulate in the blood after their formation in the bone marrow, and are tartrate-resistant acid phosphatase (TRAP)-positive cells.

Osteoclasts’ differentiation is controlled by interactions between osteoblasts and/or stromal cells and pre-osteoclasts. M-CSF and RANKL (the essential factors expressed by osteoblasts, stromal cells, and lymphocytes) are required for osteoclasts formation. M-CSF is a cytokine released from osteoblasts as a result of endocrine stimulation from parathyroid hormone. It binds to receptors on osteoclast precursor cells (OPC) and induces differentiation into osteoclasts. M-CSF is required for both the proliferative and differentiation phase of osteoclast development. RANKL is critical for osteoclastogenesis and bone resorption. RANKL interacts with its receptor RANK (receptor activator of NF- kB), a transmembrane receptor that is a member of the tumor necrosis factor (TNF) receptor superfamily and is expressed on the surface of pre-osteoclasts and mature osteoclasts. Osteoprotegerin (OPG) is a soluble decoy receptor which is produced by osteoblasts and can block osteoclast formation in vitro and bone resorption in vivo by binding to RANKL and reducing its ability to bind to RANK.

When osteoclasts are activated for resorption, a tight attachment to the bone surface is made via a membrane domain called the sealing zone (SZ). The formation of this region involves the rearrangement of the cytoskeleton and the formation of an F-actin ring. When resorption begins, the area of membrane within the actin ring forms the ruffled border. This is a highly-convoluted membrane domain which provides a large surface area for the release of protons and proteolytic enzymes that dissolve the bone matrix.

Bone resorption is necessary for many skeletal processes. It is an obligatory event during bone growth, tooth eruption and fracture healing, and is also necessary for the maintenance of an appropriate level of blood calcium. Bone resorption is tightly coupled to bone formation in the healthy skeleton, however several diseases manifest as a result of an imbalance between resorption and formation. Osteopetrosis is a disease caused by a lack of osteoclast activity, leading to an increase in bone mass, whereas osteoporosis is a disease caused by osteoclast over activity, therefore leading to reduced bone mass and an increased risk of fracture.

Bone homeostasis is achieved when there is a balance between osteoblast bone formation and osteoclast bone resorption. Osteoclasts also express many ligands for receptors present on activated NK cells. They reported that osteoclasts express ULBP-1, ULBP-2/5/6 and ULBP-3, but little or no MIC-A, MIC-B, or MHC class Llike ligands for NKG2D, the activating receptor of NK cells.

Osteoclasts (OCs), in comparison to dendritic cells (DCs) and monocytes, are significant activators of NK cell expansion and function (Tseng et al. (2015) Oncotarget 6(24):20002-25). Additionally, osteoclasts secrete significant amounts of IL-12, IL-15, IFN- y and IL- 18, which are known to activate NK cells; osteoclasts also express important NK- activating ligands. Accordingly, osteoclasts expand and activate NK cells to levels that are higher than those established by other methodologies.

IFN-y interaction with Osteoclasts and other immune cells

IFN-y is a multifunctional cytokine produced mainly by NK cells and activated T cells that plays a critical role in host immune responses against pathogens and cancer. Previously, it has been reported that IFN-y can inhibit the critical osteoclast regulator, receptor activator of NFkB ligand (RANKL), by activating ubiquitin-mediated degradation of its signaling pathway adaptor protein TRAF-6.

Further studies showed that IFN-y could also be used to inhibit experimental tooth movement and bone erosion by decreasing osteoclastic activity. It has been shown that IFN- y participates in the regulation of RANKL signaling and bone destruction. Bone and immune system are functionally interconnected. Immune and bone cells derive from same progenitors in the bone marrow, they share a common microenvironment and are being influenced by similar mediators, different immune cells such as macrophages, T and B lymphocytes, mast cells, natural killer cells (NK), etc. have been shown to influence bone cells as well (FIG. 1).

Immune cells and their products (cytokines) play an important role in the regulation of skeletal development and function, particularly of the osteoclast, which implies that immune cell dysfunction may be involved in the pathogenesis of certain skeletal disorders. IFN-y, produced by both NK cells and Thl lymphocytes, has been shown to inhibit osteoclastogenesis in vitro.

However, the in vivo effects of IFN-y on bone tissue are less clear since often provide a contrasting effect when compared to in vitro studies. Reduced functioning of osteoclast and NK cell function coexist in osteopetrotic mutant rat. OC progenitor activity is positively regulated by TNF-a and negatively regulated by IFN-y. IFN-y binds to its receptor on osteoclasts, degrades RANKL signaling and thus inhibits the activation of osteoclasts and protects our bones from being resorbed. This cytokine is produced predominantly by NK and natural killer T (NKT) cells involved in the innate immune response, and by CD4+ Thl and CD8+ cytotoxic T lymphocyte (CTL) effector T cells, once antigen-specific immunity develops. ITIM-bearing NK receptor, positively regulates osteoclasts differentiation, immunoreceptor tyrosine-based activation motif (ITAM)- mediated signaling is critical for osteoclast differentiation. Crosstalk between the skeletal system and T cells, is termed as osteoimmunology. RANKL expressed by CD4+ and CD8+ T-cells can induce osteoclast genesis, providing a link between immune and skeletal system. Osteoclasts produce chemokines that recruit CD8 positive T cells. Osteoclasts induced the secretion of IL-2, IL-6, IFN-y and induced the proliferation of CD8 positive T cells. CD8 positive T cells activated by osteoclasts expressed FoxP3, CTLA4, and receptor activator of NF-kB ligand. Anti- CD3/CD28- stimulated y6 T cells or CD4+ T cells inhibit human osteoclast formation and resorptive activity in vitro. Cytokine production by CD3/CD28-stimulated y6 T cells and observed a lack of IL- 17 production, with activated y6 T cells producing abundant interferon (IFN)-y. Neutralization of IFN-y markedly restored the formation of osteoclasts from precursor cells and the resorptive activity of mature osteoclasts, suggesting that IFN-y is the major factor responsible for the inhibitory role of activated y6 T cells on osteoclastogenesis and resorptive activity of mature osteoclasts.

Based on the observation that bone destruction in rheumatoid arthritis is always caused by an excessive activation of the immune system, researchers identified a close relationship between immune system and osteoclasts, which is termed osteoimmunology. In fact, RANKL is secreted by activated T-cells, and dysregulation of RANKL leads to defective formation of lymph nodes and lymphocyte differentiation as well as impaired osteoclastic bone resorption. A number of molecules known to be involved in the regulation of immune system, including TNF alpha, IL-1, IL-7, IL-17, IL-6, IFN-y, IFN- alpha also play critical roles in osteoclastogenesis.

IFN-y, also known as immune interferon, is the only type II IFN and was discovered in 1965. It is secreted predominantly by T-cells, natural killer cells, and some other cells such as macrophages, dendritic cells and B cells. IFN-y signal transduction is mediated by binding with IFNGR1 and IFNGR2 resulting in activation of intracellular molecular signaling networks such as JAK-STAT pathway and STAT-independent pathways such as the MAP kinease, NF-kB, and PI3K pathways.

Briefly, following binding with IFN-GRs, JAK1 and JAK2 facilitate transphosphorylation of the JAKs and the receptor subunits are activated. Subsequently, STAT1 is recruited to the receptor and becomes phosphorylated, resulting in active form of the STAT known as IFN-y activated factor (GAF). GAF then binds to the IFN-y activation site (GAS) on the promoter of IFNinducible genes leading to target gene expression. Although they share considerably overlapping functions with type I IFNs, IFN-y uniquely regulates a variety of autoinflammatory and autoimmune diseases, such as systemic lupus erythematosus, hemophagocytic lymphohistiocytosis (HLH), and macrophage activation syndrome (MAS).

IFN-y has long been used as an immunosuppressive. Further studies showed that IFN-y could also be used to inhibit experimental tooth movement and bone erosion by decreasing osteoclastic activity. It has been shown that IFN-y participates in the regulation of RANKL signaling and bone destruction.

IFN-y has also recently been found to participate in a number of signaling pathways in osteoclastogenesis. Li et al. revealed that IFN-y mediates a previously unknown feedback loop that exits between osteoclasts and activated T-cells by inducing indoleamine 2,3- dioxygenase (IDO) expression in osteoclasts. Ji et al. identified another mechanism through which IFN-y regulates osteoclastogenesis. In contrast to previous studies, they found that IFN-y alone did not affect TRAF6 expression in human osteoclast precursors, whereas, IFN-y cooperated with TLRs to suppress RANK expression by inhibiting colony stimulating factor 1 receptor (c-Fms), a potent stimulator of RANK expression. In addition to directly inhibiting osteoclast formation. IFN-y was recently reported to promote osteoblastogenesis and bone formation both in vitro and in vivo. The anabolic effect of IFN-y is mediated by increasing both osteoblastogenesis and osteoclastogenesis with a predominant stimulatory effect on the osteoblast lineage, thus increasing bone mass and rescuing oophorectomized (OVX) mice from osteoporosis. In addition, IFN-y significantly increases the expression of osteogenic markers in differentiating mesenchymal stem cell (MSC) into osteoblasts in vitro, including runt-related transcription factor 2 and osteopontin.

Overall, the effects of IFN-y on bone remain complex with some investigators reporting contrasting findings about its effect on osteoclastogenesis and bone resorption. High doses of IFN-y have been used as treatment in patients with osteopetrosis to induce bone resorption and reduce bone mass. Other studies report that IFN-y directly inhibits osteoclast differentiation and induce osteoclast apoptosis. In addition, the effect of IFN-y on osteoclast differentiation and function could be affected, among others, by estrogen deficiency, inflammation, and bacterial toxins.

Interfereon-gamma (IFN-y) and its Antitumor Effects

IFN-y INDUCES APOPTOSIS OF CANCER CELLS It has been demonstrated that high doses of IFN-y could induce apoptosis in NSCLC cell-lines, namely A549 and H460, by activating JAK-STAT1 -caspase signaling. Western blot analyses showed that STAT1 forced transcription and synthesis of caspase 3 and caspase 7, which further initiated apoptotic processes in cancer cells (Song et al. (2019) Cancer Res, 81771781 : 1-29. Additionally, it was shown that IFN-y can increase the motility of antigen-specific CD8+ T-cells to the antigen-expressing (target) cells and enhance the killing capacity of target cells. When IFN-y+/+ and IFN-y-/- CD8 T-cells were incubated with the target cells, significantly higher effectiveness of IFN-y competent cells was observed. Addition of anti-IFN-y-antibody to the co-culture system markedly reduced target cell killing. Interestingly, IFN-y can selectively induce apoptosis in stem-like colon cancer cells through JAK-STAT1-IRF1 signaling in a dose-dependent manner. Kundu et al. reported that precise neutralization of cytokine from IL-12 family, namely p40 monomer, induces IL-12-IFN-y signaling cascade in prostate cancer both in vitro and in vivo, which subsequently leads to cancer cells death and tumor regression. They found that anti-p40 antibody treatment significantly elevated the expression of apoptosis-related genes such as caspase 3, caspase 7, caspase 8, caspase 9, BAD, BID, cytochrome C, BAK, and p53 (Kundu et al. (2017) PNAS 114(43): 11482-7). Consistently, in NSCLC cells lines, namely H1975, HCC827, and H1437, IFN-y induced programmed cell death through the activation of caspases downstream of JAK-STAT1 signaling. Similar results have been reported in melanoma cells wherein the activation of caspase 3 was IFN-y/IRF3/ISG54 dependent.

OTHER IFN-y-DEPENDENT TUMOR-SUPPRESSIVE MECHANISMS

Although, IFN-y can directly affect the viability of tumor cells, increasing evidence points to interactions with surrounding stromal cells for effective rejection of solid tumors. For instance, immunohistology analyses of large tumor sections revealed that IFN-y could reduce the number of endothelial cells and induce blood vessel destruction and later promote tumor tissue necrosis. In fact, Kammertoens et al. showed responsiveness of cancer endothelial cells by highlighting the necessary role that IFN-y plays in the regression of solid tumors. By using electron microscopy they observed that IFN-y-exposed endothelial cells became round, condensed, and more occluded, which reduced blood flow in tumor tissues and subsequently, prompted tumor ischemia (Kammertoen et al. (2017) Nature 2017;545:98-102). Similarly, by interacting with stromal fibroblasts IFN-y downregulated the expression of vascular endothelial growth factor A, a growth factor critical for tumor neovascularization. Therefore, it is equally important to investigate IFN- y-mediated effects on tumor stromal cells, especially in solid, well-established tumors.

Interplay between IFN-y and macrophages in an inflamed setting has previously been described and has raised questions regarding their interaction in the tumor microenvironment (TME). Unsurprisingly, crosstalk between IFN-y and Ml -like immunostimulatory tumor-associated macrophages (TAMs) was sufficient to inhibit tumor growth in Lewis lung carcinoma and colon adenocarcinoma. Generated Ml-like TAMs secreted CXCL9, CXCL10, and CD86, which stimulated the recruitment of cytotoxic T lymphocytes (CTLs) to the TME as well as their activation. Recruited CTLs produced IFN- y that was proven to be critical for sustaining Ml TAM activity and tumor inhibition. Reprograming of IL33-/- Tregs was also linked to higher IFN-y production and thus, improved the immune response in tumor tissue.

IFN-y interacts with distinct cytokines from the TME to induce cancer growth arrest. Synergistically with TNF, IFN-y stimulates the senescence of tumor cell growth through stabilization of pl6INK4a - Rb pathway. This effect is mediated by activation of STAT1 and TNF receptor 1 and is maintained permanently in vitro and in vivo. Together with inducing apoptosis or senescence, IFN-y can shift tumors to a dormant state. As recently shown IFN-y - mediated upregulation of IDO 1 increased the intracellular concentration of kynurenine (kyn, IDO1 - catalyzed tryptophan metabolite), which activated aryl hydrocarbon receptor (AhR). AhR moved to the nucleus and directly upregulated transcription of cell cycle-regulatory molecule, p27. Thus, IDOl-Kyn-AhR- p27 pathway was proposed as a mechanism which explains how high concentration of IFN- y induces tumor dormancy. The existence of IL-12-IFN-y relationship has also been described. As the IL-12 producers, dendritic cells (DCs) stimulate NK cells to secrete IFN- y, therefore, survival of tumor-bearing mice was improved and number of metastasis was reduced. Moreover, IFN-y produced by NK cells altered tumor structure and limited the number of metastasis by increasing the expression of the extracellular matrix protein, fibronectin 1 (Glasner et al. (2018) Immunity 48: 107-19).

IFN-y CONTRIBUTES TO THE EFFICIENCY OF CANCER IMMUNOTHERAPY

The discovery of antibodies targeting immune checkpoint molecules, such as programmed cell death protein 1 (PD-1), its ligand PD-L1, and cytotoxic T-lymphocyte- associated protein 4 (CTLA-4), provided hope for patients with chemo-resistant and latestage tumors. However, their efficiency has only been proven in a small portion of treated patients. IFN-y is believed to be one of the critical factors determining the success of immunotherapy. By analyzing gene expression profiles from tumor tissue samples, Ayers et al. reported that metastatic melanoma, head and neck squamous cell carcinoma, and gastric cancer patients who responded to anti-PD-1 therapy had higher expression scores for IFN- y-related genes when compared to non-responders. They proposed that the detected IFN-y signature (IDO1, CXCL10, CXCL9, HLA-DRA, STAT1, and IFNG) can be a prediction marker for the clinical response to immune checkpoint inhibitors (Ayers et al. (2017) J Clin Invest. 127(8):2930-40). Similarly, a four-gene IFN-y signature (IFNG, CD274, LAG3, and CXCL9) has been suggested as identifying pattern for urothelial and NSCLC patients who can benefit from the anti-PD-Ll antibody durvalumab. Moreover, successful anti-PD-1 treatment depends on intratumoral crosstalk between IL-12 and IFN-y. After binding to PD- 1, this antibody stimulates CD8+ T-cells to secrete IFN-y, which activates its receptor on DCs, thus increasing the production of IL- 12 in the TME. The newly generated interleukin acts back on CD8+ T cells to further stimulate IFN-y production and enhance cytotoxic tumor cell function. Therefore, a combination of anti-PD-1 antibody and induction of INF -y via the compositions of the present disclosure would be particularly useful in preventing and/or treating cancer. Alternative mechanism by which IFN-y contributes to efficiency of cancer immunotherapy was described by Wang et al. In that model, tumor-infiltrating CD8+ T-cells secreted IFN-y in response to nivolumab, an anti-PD-Ll antibody. The released IFN-y mediated lipid peroxidation and ferroptosis in tumor cells by reducing the uptake of cystine and excretion of glutamate, resulting in tumor cells death both in vitro and in vivo. Mechanistically, type II interferon activated the JAK1-STAT1 signaling pathway, which further downregulated the transcription of SLC7A11 and SLC3 A2 proteins of the glutamate-cystine antiporter system. Likewise, the clinical benefits of cancer immunotherapy were reduced in nivolumab -treated mice bearing INFGR-/- tumors.

Thibaut et al. recently suggested a model in which tumor-reactive T-cells secrete IFN-y, which diffuses extensively to alter the TME in distant areas. The prolonged activity of IFN-y has been shown to be crucial for antitumor immune response as shown by induction of PD-L1 expression and inhibition of tumor growth (Hoekstra et al. (2020) Nat Cancer 1 (3):291 -301 ). Furthermore, Zhang et al. proposed that IFN-y may be a good therapeutic option for improving the efficacy of PD-1 blockade therapy for pancreatic cancer by preventing the trafficking of CXCR2+ CD68+ immunosuppressive macrophages to the TME by blocking the CXCL8-CXCR2 axis (Zhang et al. (2020) J Immunother Cancer 8(1): 1-15).

The efficiency of anti-CTLA-4 therapy was also IFN-y dependent. Whole exome sequencing data showed that melanoma tumors resistant to immunotherapy had defects in IFN-y signaling, namely loss of IFNGR1, IRF-1, JAK2 and IFNGR2 genes, as well as amplification of SOCS1 and PIAS4 inhibitory genes. Therefore, the combination of immune checkpoint inhibitors and IFN-y can be a good strategy to increase the overall efficiency of cancer immunotherapy. Indeed, two such clinical trials have already been initiated testing the combination of nivolumab or pembrolizumab with IFN-y (NCT02614456 and NCT03063632, respectively). Other studies suggest that disruption of IFN-y signaling in tumor cells could boost tumor growth and impact the efficiency of given immune checkpoint inhibitor therapy. Amplification of IFN-y-pathway inhibitory molecules or downregulation and loss of its receptor and downstream signaling mediators are common mechanisms for tumor cells to avoid generated immune response. It was recently shown that aging can also consistently attenuate IFN-y signaling in triple-negative breast cancer patients and limit the efficiency of immune checkpoint blockade (ICB) therapy. Another hypothesis is that enhanced intratumoral production of IFN-y can improve the potency of ICB therapy in patients with cancer. For example, pharmacological blockade or partial genetic deletion of CBM complex (CARMAl-BCLlO-MALTl) in Tregs reprogram them to secrete IFN-y which results in tumor regression. In addition, combination of CBM inhibition and anti-PD-1 antibodies enabled tumor control in MC38 colon carcinoma-bearing mice who were resistant to anti-PD-1 monotherapy. Similarly, tumor regression has been observed only in melanoma-bearing mice treated with PD-1 targeted therapy together with antibodies against neuropilin- 1. Neuropilin- 1 is a protein found on most of the tumor-infiltrating Tregs, important for their suppressive function. Notably, Neuropilin-1 deletion in Tregs led to increased expression of Thl cell markers such as T- bet and IFN-y. Treg-secreted IFN-y drove intratumoral fragility of the remaining immune- suppressive Tregs via hypoxia-inducible factor 1 -alpha (HIFla) which stimulated host immunity to eliminate cancer cells. Similarly, it was suggested that metastatic potential of tumor cells after receiving immunotherapy was due to reduction of IFN-y in the TME and augmented activity of integrin avP3 signaling axis. Collectively, the presence of IFN-y in the TME is required for optimal antitumor responses in cancer patients receiving mono- or combined immune checkpoint inhibitors (see e.g., Jorgovanovic et al. (2020) Biomarker Research 9:49 and references therein).

NK cells

Natural Killer (NK) cells are granular lymphocytes that function at the interference of innate and adaptive immunity. Discovered in the early 1970’s by accident when investigators were studying specific cytotoxic effects of lymphocytes, it was not until the 1980’s that they became generally accepted despite the accumulated evidence. NK cells are a subset of 8 cytotoxic lymphocytes able to recognize and lyse tumor cells and virus infected cells without prior sensitization. Traditionally they have been classified as effectors of innate immunity due to the lack of antigen specific cell surface receptors. NK cells are known to mediate direct and antibody dependent cellular cytotoxicity (ADCC) against tumors as well as to regulate the function of other cells through the secretion of cytokines and chemokines. NK cells derive from CD34+ hematopoietic stem cells (HSC’s) found in the bone marrow. They can be found throughout the body in the spleen, liver, placenta, and peripheral blood. Human NK cells are defined phenotypically by the surface expression of CD56 and CD 16, and by their lack of CD3 surface expression. CD56 is a human neural-cell adhesion molecule, but its function on human NK cells is yet to be understood. Although the function of CD56 is unknown, its expression correlates with the expression of other surface markers that confer important functional properties to NK cells. Two subsets of NK cells have been identified based on surface expression of CD56 and CD 16. The major subset of NK cells, about 90% of human NK cells, is defined by low expression of CD56 (CD56dim) and high expression of CD16 (CD16 bright). The minor subset makes up approximately 10% of human NK cells and is defined by high expression of CD56 (CD56 bright) and low or lack of CD 16 (CD 16 dim) expression. The CD56dim CD 16 bright cells were found to be the more cytotoxic subset of human NK cells. On the other hand, CD56bright CD16dim/- NK cells were found to secrete more cytokines such as interferon-y (IFN-y), tumor necrosis factor-a (TNF-a), TNF-P, granulocyte macrophage colony stimulating factor (GM-CSF), interleukin- 10 (IL- 10), and IL 13 after being stimulated with pro-inflammatory cytokines.

NK cells develop in the bone marrow and constitute about 5-10% of total lymphocytes in the peripheral circulation and secondary lymphoid organs. Effector function of NK cells include direct natural cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), as well as secretion of inflammatory cytokines and chemokines that indirectly regulate the functions of other immune cells. NK cells mediate cytotoxicity against transformed tumor cells, as well as healthy cells, by releasing pre-formed granules of proteins, known as perforin and granzyme B, which can induce apoptosis, or programmed cell death in target cell. NK cells have also been identified within inflamed synovial fluid and express RANKL and M-CSF which during their interaction with monocytes can trigger the formation of osteoclasts in a process that is RANKL and M-CSF dependent.

NK cell cytotoxic activity in cancer patients is severely reduced, correlating with the decreased expression of NK cell activating receptors even at the early stages of disease, and are further diminished in advanced cancers. Patients’ NK cells are significantly defective in their function, and the defect is seen at both preneoplastic and neoplastic stages of pancreatic cancer. Patients’ NK cells do not recover their full functional potential even when the best activating conditions are provided for their expansion and function. Moreover, patients’ NK cells under the expansion conditions give rise to T cell expansion at a much faster rate, with a subsequent decrease in the percentages of NK cells when compared to those from healthy individuals.

Therefore, the use of allogeneic NK cells from healthy individuals is preferable than autologous NK cells for therapeutic purposes. The use of allogeneic NK cells in hematologic malignancies following HLA-haploidentical HSCT in clinical trials has previously been reported. In addition, allogeneic NK cells exert less collateral damage due to graft versus host disease (GVHD) when used therapeutically in solid tumors.

In search of a more potent therapeutic dose of NK cells, a novel strategy to expand highly functional NK cells has been established, coined as super-charged NK cells, by employing osteoclasts as feeder cells in the presence of a combination of sonicated probiotic bacteria (sAJ2). The potency and effectiveness of super-charged NK cells are significantly superior to those established by other methodologies or when compared to primary activated NK cells. The term super-charged was used to describe the magnitude and superiority of their functional potential in lysing and differentiating CSCs/poorly differentiated tumors.

NK cells have the following roles: 1) Selection and differentiation of healthy stem cells. 2) Regulate inflammation, 3) activate CAR NK, CAR T, and T cells, 4) control the tumors through tumor differentiation, 5) kill cancer stem cells, 6) kill through oncologic viruses. 7) act synergistically with radiation and chemotherapy, 8) kill through antibodies and checkpoint inhibition, 9) kill suppressor cells, 10) exert preferential expansion of CD8+ T cells, and 11) gene knockout recognition (FIG. 2).

NK cell immunotherapy

NK cells have very diverse biological functions including significant roles in defense against tumor cells. Based on knowledge of NK cell function and evidence that they become nonfunctional in cancer patients, several approaches have been proposed for the use of NK cells in immunotherapy:

1) Cytokines

Many cytokines such as IL-2, IL-21, IL-12, IL-15, and IFN- y have been known to activate and boost NK cells function. Cytokines can be used to boost NK cells for immunotherapeutic means, both in vitro and in vivo.

2) Antibodies

Antibodies can be applied to NK cell immunotherapy based on different approaches. NK cells can target tumor cells coated with IgG antibodies through the ADCC. There are several monoclonal antibody treatments available specific to different tumor antigens such as the use of anti-CD20 for the treatment of B cell lymphoma or anti-Her2 for the treatment of Her2-overexpressing invasive breast cancer. Antibodies can be also being used to block NK cells’ checkpoint inhibitors. Studies have shown that NK cells express checkpoint molecules such as PD-1, CTLA-4, TIM-3 and TIGIT.

3) Adoptive transfer of NK cells

Transferring functionally competent NK cells as an immunotherapy approach has been established for many years. NK cells can be harvested from different sources, and their functional competencies may vary depending on the strategies used to separate, activate or expand them. Sources of natural killer cells for adoptive transfer: a) NK cells isolated from Peripheral Blood Mononuclear Cells (PBMCs)

NK cells can be isolated from autologous and allogeneic PBMCs. The strategies to expand and activate NK cells from PBMCs are different. Investigators have tried to Expand NK cells directly from PBMCs without isolating the NK cells population, or by depleting CD3+ cells, or selecting CD16+, or CD 16+ and CD56+ cells. Different cells have been used as feeder cells to improve NK cells expansion. Irradiated K562 and OK432 are two of the most popular feeder cells for NK expansions. Different Cytokines and other activators also have been applied to expand NK cells in vitro. IL-2, IL-21, IL- 15 and IL- 18 are some of the cytokines used for this means.

However, a novel strategy has been established to expand NK cells up to 21,000- to 132,000-fold in 20 days. In this technique, NK cells are treated with IL-2 and CD 16 antibody and probiotic bacteria and Osteoclasts are used as feeder cells. The Expanded NK cells, called “Super-Charged NK cell” have high cytotoxicity and IFN- y secretion.

Autologous NK cells as a source of therapy has not been very effective. It has been shown that NK cells from cancer patients are less functional both in terms of cytotoxicity and of IFN- y secretion in vitro. In an in vivo study adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression.

In another study autologous NK cells have also demonstrated a limited effect on tumor suppression in malignant glioma. Allogenic NK cells can be a better resource for NK cell therapy since NK cells from healthy donors have better functionality than NK cells in cancer patient, besides the KIR receptor from the donor mismatch the MHC class I of the recipient letting NK cells to skip some of the inhibitory processes. b) Stem cell- derived NK cells

Due to the pluripotency of stem cells, using them as a precursor of NK cells has become one of the interesting sources of NK cells. Generation of NK cells from ESCs and iPSCs enerally requires two steps. First, CD34+ hematopoietic precursors must be generated. The CD34+ cells are then sorted and differentiated into NK cells with cytokines and feeder cells (usually murine stromal cells). When NK cells were generated from hESCs, they were mostly CD56+CD45+ NK cells, which also expressed inhibitory and activating receptors typically found on adult NK cells.

These NK cells were also able to mediate cytotoxicity against of leukemic cells, K562 (erythroleukemia), and several solid tumors, including breast cancer (MCF7), testicular embryonal carcinoma (NTERA2), prostate cancer (PC3), and glioma (U87) cell lines. A studied showed that when efficacy of Induced pluripotent stem (IPS) cell-derived natural killer cells with NK cells isolated from peripheral blood that had been activated and expanded in long-term culture, and overnight activated Peripheral blood isolated NK cells, were compared, NK cells derived from IPS mediate anti-ovarian cancer killing in NSG mouse at least as well as NK cells isolated from blood. The Kaufman research group established a feeder-free, sorting-free approach to generate NK cells from human ESCs. These authors used a spin-embryoid body system with BMP4 and VEGF to derive hematopoietic progenitors. After 11 days of culture, the spin-EBs were transferred to the NK cell culture containing the cytokines IL-3, IL-15, IL-7, SCF and Flt3L for 28 days. This feeder-free system can generate NK cells that have no difference from those derived from the murine embryonic liver cell line EL08-1D2 as feeder cell. Meanwhile, this group established a clinical-scale derivation of NK cells from ESCs and iPSCs without cell sorting and in the absence of feeder cells. c) NK cells isolated from Umbilical Cord blood.

Umbilical cord blood (UCB) has become a known source for NK cells. The NK cells from UCB and peripheral blood (PB) have some differences. UCB NK cells express similar levels of CD56, NKp46, NK30 and NKG2D as PB NK cells but lower levels of CD 16 CD2, CD 11 a, CD 18, CD62L), KIRs, DNAM-1, NKG2C, IL-2R, and CD57 and CD8. UCB has a higher percentage of NK cells but these cells have lower cytotoxicity in comparison to NK cells from PB which could be due to lower levels of Granzyme B and perforin in CB NK cells. Studies have shown that with proper signaling NK cells from UCB can be expanded to create many cells with proper function. d) Genetically modified NK cells

The genetic modification can be used to promote the efficacy of NK cells by different means. NK cells can be genetically modified to secrete cytokines in favor of their survival and activation. Engineering Chimeric antigen receptor (CAR) NK cells has currently become the topic of interest. Currently, several tumor antigen-binding domains have been designed as CAR extracellular domains and tested.

Methods for Detecting the Amount/Activity of NK cells and Cytokines/Chemokines (e.g., IFN-y)

The activity or level of NK cells can be detected by any means known in the art. For example, the NK cell level in PBMC can be determined by detecting the presence/absence of surface markers that phenotypically define the NK cells (e.g., the surface expression of CD56 and CD 16, and by their lack of CD3 surface expression - see the section “NK Cells” above). Such detection of NK cells can utilize any methods known in the art, including those described below for detecting a cytokine/chemokine. The NK cell level and/or activity can also be detected by the amount of IFN-y or other cytokines/chemokines secreted by NK cells. Alternatively, NK cells can be isolated from PBMC using e.g., using isolation kits from Stem Cell Technologies (Vancouver, BC, Canada), and their cytotoxicity activity can be measured using e.g., ⁵¹ Cr release assay (see below). NK cell’s ADCC activity can also be used as a measure of the NK cell amount and/or activity.

The activity or level of a cytokine or chemokine (e.g., IFN-y) can be detected and/or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means well-known to those of skill in the art. Accordingly, any method known in the art for detecting polypeptides can be used. Such methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference). Preferred are binder-ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.

For example, ELISA and RIA procedures may be conducted such that a desired cytokine/chemokine standard is labeled (with a radioisotope such as ¹²⁵ I or ³⁵ S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabeled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, the cytokine/chemokine in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme- labeled anti-biomarker protein antibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable.

The above techniques may be conducted essentially as a “one-step” or “two-step” assay. A “one-step” assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. A “two-step” assay involves washing before contacting, the mixture with labeled antibody. Other conventional methods may also be employed as suitable.

In certain embodiments, a method for measuring the cytokine/chemokine levels comprises the steps of: contacting a biological specimen with an antibody or variant (e.g., fragment) thereof which selectively binds the cytokine/chemokine, and detecting whether said antibody or variant thereof is bound to said sample and thereby measuring the levels of the fFN-y or cytokine/chemokine.

Enzymatic and radiolabeling of a cytokine/chemokine and/or the antibodies may be effected by conventional means. Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.

It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient.

It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support.

Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.

Other techniques may be used to detect a cytokine/chemokine according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti-cytokine/chemokine antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including ¹²⁵ I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.

Immunohistochemistry may be used to detect expression of IFN-y or cytokine/chemokine, e.g., in a biopsy sample. A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabeling. The assay is scored visually, using microscopy.

Antibodies that may be used to detect a cytokine/chemokine include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the biomarker protein to be detected. An antibody may have a Kd of at most about 10' ⁶ M, 10' ⁷ M, 10' ⁸ M, 10' ⁹ M, 10' ¹⁰ M, 1O' ^U M, 10" ¹² M. The phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant. An antibody may bind preferentially to the biomarker protein relative to other proteins, such as related proteins.

Antibodies may be commercially available or may be prepared according to methods known in the art.

Antibodies and derivatives thereof that may be used encompass polyclonal or monoclonal antibodies, chimeric, human, humanized, primatized (CDR-grafted), veneered or single-chain antibodies as well as functional fragments, /.<?., cytokine/chemokine-binding fragments, of antibodies. For example, antibody fragments capable of binding to a biomarker protein or portions thereof, including, but not limited to, Fv, Fab, Fab' and F(ab')2 fragments can be used. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab')2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and hinge region of the heavy chain.

In some embodiments, agents that specifically bind to IFN-y or a cytokine/chemokine other than antibodies are used, such as peptides. Peptides that specifically bind to a cytokine/chemokine is well known in the art (e.g., receptor fragment for the cytokine/chemokine), and can also be identified by any means known in the art. For example, specific peptide binders of a biomarker protein can be screened for using peptide phage display libraries.

Sampling Methods

In some embodiments, NK cell or cytokine/chemokine (e.g., IFN-y) amount and/or activity measurement(s) in a sample from a subject is compared to a pre-determined control (standard) sample. The sample from the subject is typically from blood or tissue. The control sample can be from the same subject or from a different subject. The control sample is typically a normal, non-diseased sample. However, in some embodiments, such as for staging of disease or for evaluating the efficacy of treatment, the control sample can be from a diseased subject.

The control sample can be a combination of samples from several different subjects. In some embodiments, the NK cell or cytokine/chemokine amount and/or activity measurement(s) from a subject is compared to a pre-determined level. This pre-determined level is typically obtained from normal samples. A pre-determined NK cell or cytokine/chemokine amount and/or activity measurement(s) may be determined in populations of patients with or without a disease (e.g., bone loss condition and/or cancer). The pre-determined NK cell or cytokine/chemokine amount and/or activity measurement(s) can be a single number, equally applicable to every patient, or the pre-determined NK cell or cytokine/chemokine amount and/or activity measurement(s) can vary according to specific subpopulations of patients. Age, weight, height, and other factors of a subject may affect the pre-determined NK cell or cytokine/chemokine amount and/or activity measurement s) of the individual. Furthermore, the pre-determined cytokine/chemokine amount and/or activity can be determined for each subject individually. In some embodiments, the amounts determined and/or compared in a method described herein are based on absolute measurements.

In other embodiments, the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratios e.g., NK cell or cytokine/chemokine level, and/or activity before a treatment vs. after a treatment, and the like). For example, the relative analysis can be based on the ratio of pre-treatment cytokine/chemokine measurement as compared to post-treatment cytokine/chemokine measurement. Pre-treatment NK cell or cytokine/chemokine measurement can be made at any time prior to initiation of anti-cancer therapy or an anti-inflammation therpay. Posttreatment cytokine/chemokine measurement can be made at any time after initiation of anticancer therapy or an bone loss therapy. In some embodiments, post-treatment NK cell or cytokine/chemokine measurements are made 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more after initiation of the administration of the compositions of the present disclosure.

In some embodiments of the present invention the change of NK cell or cytokine/chemokine amount and/or activity measurement(s) from the pre-determined level is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 fold or greater, or any range in between, inclusive. Such cutoff values apply equally when the measurement is based on relative changes, such as based on the ratio of pre-treatment NK cell or cytokine/chemokine measurement as compared to post-treatment NK cell or cytokine/chemokine measurement.

Biological samples can be collected from a variety of sources from a patient including a body fluid sample, cell sample, or a tissue sample. “Body fluids” refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In some embodiments, the subject and/or control sample is selected from the group consisting of cells, cell lines, histological slides, paraffin embedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow. In some embodiments, the sample is serum, plasma, or urine. In other embodiments, the sample is serum.

The samples can be collected from individuals repeatedly over a longitudinal period of time (e.g., once or more on the order of days, weeks, months, annually, biannually, etc.). Obtaining numerous samples from an individual over a period of time can be used to verify results from earlier detections and/or to identify an alteration in biological pattern as a result of, for example, disease progression, drug treatment, etc. For example, subject samples can be taken and monitored every month, every two months, or combinations of one, two, or three month intervals according to the present invention. In addition, the cytokine/chemokine amount and/or activity measurements of the subject obtained over time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject’s own values, as an internal, or personal, control for long-term monitoring.

Sample preparation and separation can involve any of the procedures, depending on the type of sample collected and/or analysis of cytokine/chemokine measurement(s). Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of samples, concentration of sample proteins, extraction and purification of lipids.

Pharmaceutical Compositions

The present invention provides pharmaceutically acceptable compositions of the compositions disclosed herein. As described in detail below, the pharmaceutical compositions of the present invention may be formulated for administration in solid or liquid form, including those adapted for the following exemplary routes: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; or (2) parenteral administration, for example, by subcutaneous, intratumoral, intramuscular or intravenous injection as, for example, a sterile solution or suspension.

Cells (e.g., NK cells, super-charged NK cells, osteoclasts) can be administered at 1, 10, 1000, 10,000, 0.1 x 10 ⁶ , 0.2 x 10 ⁶ , 0.3 x 10 ⁶ , 0.4 x 10 ⁶ , 0.5 x 10 ⁶ , 0.6 x 10 ⁶ , 0.7 x 10 ⁶ , 0.8 x 10 ⁶ , 0.9 x 10 ⁶ , 1.0 x 10 ⁶ , 5.0 x 10 ⁶ , 1.0 x 10 ⁷ , 5.0 x 10 ⁷ , 1.0 x 10 ⁸ , 5.0 x 10 ⁸ , 1.0 x 10 ⁹ or more, or any range in between or any value in between, cells per kilogram of subject body weight. The number of cells injected may be adjusted based on the desired level of administration in a given amount of time. Generally, 1 * 10 ⁵ to about 1 x 10 ⁹ cells/kg of body weight, from about 1 * 10 ⁶ to about 1 x 10 ⁸ cells/kg of body weight, or about 1 x 10 ⁷ cells/kg of body weight, or more cells, as necessary, may be injected. In some embodiments, injection of at least about 100, 1000, 10,000, O.lxlO ⁶ , 0.5xl0 ⁶ , l.Ox lO ⁶ , 2.0x l0 ⁶ , 3.0x l0 ⁶ , 4.0x l0 ⁶ , or 5.0x l0 ⁶ total cells relative to an average size mouse is effective. Cells (e.g., NK cells, super-charged NK cells, osteoclasts) can be administered in any suitable route as described herein, such as by infusion. Cells (e.g., NK cells, super-charged NK cells, osteoclasts) can also be administered before, concurrently with, or after, other bone loss therapy and/or anticancer agents.

Administration can be accomplished using methods generally known in the art. Agents, including cells, may be introduced to the desired site by direct injection, or by any other means used in the art including, but are not limited to, intravascular, intracerebral, parenteral, intraperitoneal, intravenous, epidural, intraspinal, intrastemal, intra-articular, intra-synovial, intrathecal, intra-arterial, intracardiac, intratumoral, or intramuscular administration. For example, subjects of interest may be administered with cells by various routes. Such routes include, but are not limited to, intravenous administration, subcutaneous administration, administration to a specific tissue (e.g, focal transplantation), injection into the femur bone marrow cavity, injection into the spleen, injection into the tumor, administration under the renal capsule of fetal liver, and the like. In certain embodiments, the NK cells (e.g., super-charged NK cells) of the present invention are injected to the subject intratumorally or subcutaneously. Cells may be administered in one infusion, or through successive infusions over a defined time period sufficient to generate a desired effect. Exemplary methods for transplantation, engraftment assessment, and marker phenotyping analysis of transplanted cells are well-known in the art (see, for example, Pearson et al. (2008) Curr. Protoc. Immunol. 81 : 15.21.1-15.21.21; Ito et al. (2002) Blood 100:3175-3182; Traggiai et al. (2004) Science 304: 104-107; Ishikawa et al. Blood (2005) 106: 1565-1573; Shultz et al. (2005) J. Immunol. 174:6477-6489; and Holyoake et al. (1999) Exp. Hematol. 27: 1418-1427).

Two or more cell types can be combined and administered, such as NK cells (e.g., super-charged NK cells) and osteoclasts, and the like. The ratio of NK cells (e.g., supercharged NK cells) to other cell types (e.g., osteoclasts) can be 1 : 1, but can modulated in any amount desired (e.g, 1 : 1, 1.1 : 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1, 2: 1, 2.5: 1, 3: 1, 3.5: 1, 4:1, 4.5: 1, 5: 1, 5.5: 1, 6: 1, 6.5: 1, 7:1, 7.5: 1, 8: 1, 8.5: 1, 9: 1, 9.5:1, 10: 1, or greater).

Engraftment of transplanted cells may be assessed by any of various methods, such as, but not limited to, bone density, tumor volume, cytokine levels, time of administration, flow cytometric analysis of cells of interest obtained from the subject at one or more time points following transplantation, and the like. For example, a time-based analysis of waiting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 days or more can signal the time for sample harvesting. Any such metrics are variables that can be adjusted according to well-known parameters in order to determine the effect of the variable on a response to the agents of the present disclosure. In addition, the transplanted cells can be co-transplanted with other agents, such as cytokines, extracellular matrices, cell culture supports, and the like.

An agent (including cells, e.g., NK cells, super-charged NK cells, osteoclasts) can be administered to an individual in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).

Compositions described herein (e.g., probiotic bacteria, e.g., AJ2, AJ3, AJ4) may be used for oral administration to the gastrointestinal tract, directed at the objective of introducing the probiotic bacteria to tissues of the gastrointestinal tract. The formulation for a therapeutic composition of the present invention may also include other probiotic agents or nutrients which promote spore germination and/or bacterial growth. An exemplary material is a bifidogenic oligosaccharide, which promotes the growth of beneficial probiotic bacteria. In certain embodiments, the probiotic bacterial strain is combined with a therapeutically-effective dose of an (preferably, broad spectrum) antibiotic, or an antifungal agent. In some embodimetns, the compositions described herein are encapsulated into an enterically-coated, time-released capsule or tablet. The enteric coating allows the capsule/tablet to remain intact (i.e., undisolved) as it passes through the gastrointestinal tract, until after a certain time and/or until it reaches a certain part of the GI tract (e.g., the small intestine). The time-released component prevents the “release” of the probiotic bacterial strain in the compositions described herein for a pre-determined time period.

The therapeutic compositions of the present invention may also include known antioxidants, buffering agents, and other agents such as coloring agents, flavorings, vitamins or minerals.

In some embodiments, the therapeutic compositions of the present invention are combined with a carrier which is physiologically compatible with the gastrointestinal tissue of the species to which it is administered. Carriers can be comprised of solid-based, dry materials for formulation into tablet, capsule or powdered form; or the carrier can be comprised of liquid or gel-based materials for formulations into liquid or gel forms. The specific type of carrier, as well as the final formulation depends, in part, upon the selected route(s) of administration. The therapeutic composition of the present invention may also include a variety of carriers and/or binders. A preferred carrier is micro-crystalline cellulose (MCC) added in an amount sufficient to complete the one gram dosage total weight. Carriers can be solid-based dry materials for formulations in tablet, capsule or powdered form, and can be liquid or gel-based materials for formulations in liquid or gel forms, which forms depend, in part, upon the routes of administration. Typical carriers for dry formulations include, but are not limited to: trehalose, malto-dextrin, rice flour, microcrystalline cellulose (MCC) magnesium sterate, inositol, FOS, GOS, dextrose, sucrose, and like carriers. Suitable liquid or gel-based carriers include but are not limited to: water and physiological salt solutions; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol, propylene glycol, and the like). Preferably, water-based carriers possess a neutral pH value (i.e., pH 7.0). Other carriers or agents for administering the compositions described herein are known in the art, e.g., in U.S.Patent No. 6,461,607.

The phrase “pharmaceutically acceptable” is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of one or more bacterial strains as disclosed herein.

In some embodiments, the composition (e.g., bacterial composition, pharmaceutical composition, nutraceutical composition) comprises at least one carbohydrate. A “carbohydrate” refers to a sugar or polymer of sugars. The terms “saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula CnHznOn. A carbohydrate may be a monosaccharide, a disaccharide, tri saccharide, oligosaccharide, or polysaccharide. The most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates may contain modified saccharide units such as 2’-deoxyribose wherein a hydroxyl group is removed, 2’ -fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2’- fluororibose, deoxyribose, and hexose). Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.

In some embodiments, the composition comprises at least one lipid. As used herein a “lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans). In some embodiments the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16: 1), margaric acid (17:0), heptadecenoic acid (17: 1), stearic acid (18:0), oleic acid (18: 1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20: 1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EP A), docosanoic acid (22:0), docosenoic acid (22: 1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0). In some embodiments the composition comprises at least one modified lipid, for example a lipid that has been modified by cooking.

In some embodiments, the composition comprises at least one supplemental mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.

In some embodiments, the composition comprises at least one supplemental vitamin. The at least one vitamin can be fat-soluble or water soluble vitamins. Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin Bl 2, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.

In some embodiments, the composition comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.

In some embodiments, the excipient comprises a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.

In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol. In some embodiments, the composition comprises a binder as an excipient. Nonlimiting examples of suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.

In some embodiments, the composition comprises a lubricant as an excipient. Nonlimiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.

In some embodiments, the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.

In some embodiments, the composition comprises a disintegrant as an excipient. In some embodiments the disintegrant is a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth. In some embodiments the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments, the composition is a food product (e.g., a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed. Specific examples of the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies.

Nutraceutical Composition

A nutraceutical composition is a pharmaceutical alternative which may have physiological benefits. In some embodiments, a nutraceutical composition is a food (or part of a food) that provides medical or health benefits, including the prevention and/or treatment of a disease. See, e.g., Brower (1998) Nat. Biotechnol. 16:728-731; Kalra (2003) AAPS Pharm Sci. 5(3):25. In other embodiments, a nutraceutical composition is a dietary or nutritional supplement.

Accordingly, a nutraceutical composition of the invention can be a food product, foodstuff, functional food, or a supplement composition for a food product or a foodstuff. As used herein, the term food product refers to any food or feed which provides a nutritional source and is suitable for oral consumption by humans or animals. The food product may be a prepared and packaged food (e.g., mayonnaise, salad dressing, bread, or cheese food) or an animal feed (e.g., extruded and pelleted animal feed, coarse mixed feed or pet food composition). As used herein, the term foodstuff refers to a nutritional source for human or animal oral consumption. Functional foods refer to foods being consumed as part of a usual diet but are demonstrated to have physiological benefits and/or reduce the risk of chronic disease beyond basic nutritional functions.

Food products, foodstuffs, functional foods, or dietary supplements may be beverages such as non-alcoholic and alcoholic drinks as well as liquid preparations to be added to drinking water and liquid food. Non-alcoholic drinks are for instance soft drinks; sport drinks; fruit juices, such as orange juice, apple juice and grapefruit juice; lemonades; teas; near-water drinks; and milk and other dairy drinks such as yogurt drinks, and diet drinks. In other embodiments, food products, foodstuffs, functional foods, or dietary supplements refer to solid or semi-solid foods. These forms can include, but are not limited to, baked goods such as cakes and cookies; puddings; dairy products; confections; snack foods (e.g., chips); or frozen confections or novelties (e.g., ice cream, milk shakes); prepared frozen meals; candy; liquid food such as soups; spreads; sauces; salad dressings; prepared meat products; cheese; yogurt and any other fat or oil containing foods; and food ingredients (e.g., wheat flour). In some embodiments, the food products, foodstuffs, functional foods, or dietary supplements may be in the form of tablets, boluses, powders, granules, pastes, pills or capsules for the ease of ingestion.

It is understood by those of skill in the art that in additional to isolated, and optionally purified and/or sonicated compositions of the present disclosure and other ingredients can be added to food products, foodstuffs, or functional foods described herein, for example, fillers, emulsifiers, preservatives, etc. for the processing or manufacture of the same. Additionally, flavors, coloring agents, spices, nuts and the like may be incorporated into the nutraceutical composition. Flavorings can be in the form of flavored extracts, volatile oils, chocolate flavorings, peanut butter flavoring, cookie crumbs, crisp rice, vanilla or any commercially available flavoring.

Emulsifiers can also be added for stability of the nutraceutical compositions. Examples of suitable emulsifiers include, but are not limited to, lecithin (e.g., from egg or soy), and/or mono- and di-glycerides. Other emulsifiers are readily apparent to the skilled artisan and selection of suitable emulsifier(s) will depend, in part, upon the formulation and final product. Preservatives can also be added to the nutritional supplement to extend product shelf life. Preferably, preservatives such as potassium sorbate, sodium sorbate, potassium benzoate, sodium benzoate or calcium disodium EDTA are used.

In addition, the nutraceutical composition can contain natural or artificial (preferably low calorie) sweeteners, e.g., saccharides, cyclamates, aspartamine, aspartame, acesulfame K, and/or sorbitol. Such artificial sweeteners can be desirable if the nutraceutical composition is intended to be consumed by an overweight or obese individual, or an individual with type II diabetes who is prone to hyperglycemia.

Moreover, a multi-vitamin and mineral supplement can be added to the nutraceutical compositions of the present invention to obtain an adequate amount of an essential nutrient, which is missing in some diets. The multi-vitamin and mineral supplement can also be useful for disease prevention and protection against nutritional losses and deficiencies due to lifestyle patterns. As described herein, modulation of commensal bacterial populations can provide additional benefit against the development and progression of inflammatory diseases, autoimmune diseases, and cancer. Accordingly, particular embodiments of the invention provide for the nutritional source of the nutraceutical to modulate endogenous commensal bacterial populations. Such modulation can be achieved by modification of gut pH, consumption of beneficial bacteria (e.g., as in yogurt), by providing nutritional sources (e.g., prebiotics) that select for particular populations of bacteria, or by providing antibacterial compounds. Such modulation can mean an increase or decrease in the gut microbiota populations or ratios. In particular embodiments, the absolute or relative numbers of desirable gut microorganisms is increased and/or the absolute or relative numbers of undesirable gut microorganisms is decreased. For example, it is contemplated that there are a variety of nutritional sources exhibiting antibacterial activity that can be used to modulate gut microbiota populations. For example, garlic has been shown to produce the compound allicin (allyl 2-propenethiosulfinate), which exhibits antibacterial activity toward E. coli (Fujisawa, et al. (2009) Biosci. Biotechnol. Biochem. 73 (9): 1948- 55; Fujisawa, et al. (2008) J. Agric. Food Chem. 56(11):4229-35). Similarly, rosemary extracts and other essential oils have been shown to contain antibacterial activity (Klancnik, et al. (2009) J. Food Prot. 72(8): 1744-52; Si, et al. (2006) J. Appl. Microbiol. 100(2):296- 305). Extracts of the edible basidiomycete, Lentinus edodes (Shiitake), have also been shown to possess antibiotic activity (Soboleva, et al. (2006) Antibiot. Khimioter. 51 (7):3-8; Hirasawa, et al. (1999) Int. J. Antimicrob. Agents 11(2): 151-7). Moreover, purple and red vegetable and fruit juices exhibit antibacterial activities (Lee, et al. (2003) Nutrition 19:994- 996).

Furthermore, it is contemplated herein that the food products, foodstuffs, functional foods, or dietary supplements may be combined with antibiotics to control the gut microbiota populations.

The nutraceutical composition of the present invention can be provided in a commercial package, alone, or with additional components, e.g., other food products, food stuffs, functional foods, dietary supplement. Desirably, the commercial package has instructions for consumption of the instant nutraceutical, including preparation and frequency of consumption, and use in the prevention or treatment of inflammatory diseases, autoimmune diseases and cancer. Moreover, in particular embodiments, the commercial package further includes a natural product (e.g., the food, extracts, antibiotics, and oils) that modulates endogenous commensal bacterial populations. A package containing both a nutraceutical of the present disclosure in combination with said natural product can contain instructions for consuming the natural product, e.g., in advance (e.g., 2, 4, 6 or 8 or more hours) of consuming the nutraceutical in order to enhance the activity of the nutraceutical composition.

Subjects

In certain embodiments, the subject suitable for the compositions and methods disclosed herein is a mammal e.g., mouse, rat, primate, non-human mammal, domestic animal, such as a dog, cat, cow, horse, and the like), and is preferably a human. In some embodiments, the subject is healthy (e.g., as a prevention of a bone loss condition). In some embodiments, the subject is afflicted with a disease (e.g., a cancer and/or bone loss condition). It is important to point out that the agents and methods of the present disclosure are effective and beneficial to any subject (healthy or diseased) who is in need of prevention and/or treatment of a bone loss condition, irrespective of whether the subject is afflicted with a cancer.

In other embodiments, the subject is an animal model of a cancer and/or bone loss condition. For example, the animal model can be an orthotopic xenograft animal model of human oral squamous carcinoma, or comprising cancer stem cells (CSCs)/undifferentiated tumors. In some embodiments, the subject is an animal model of an inflammatory disease or an autoimmune disease.

In some embodiments, the subject has not undergone treatment, such as bone loss therapy or a cancer therapy (chemotherapy, radiation therapy, targeted therapy, and/or anti- immune therapy - such as NK cell-related immunotherapies). In still other embodiments, the subject has undergone treatment, such as bone loss therapy or a cancer therapy (chemotherapy, radiation therapy, targeted therapy, and/or anti-immune therapy - such as NK cell-related immunotherapies).

In certain embodiments, the subject has had surgery to remove cancerous or pre- cancerous tissue. In other embodiments, the cancerous tissue has not been removed, e.g., the cancerous tissue may be located in an inoperable region of the body, such as in a tissue that is essential for life, or in a region where a surgical procedure would cause considerable risk of harm to the patient.

In certain embodiments, the subject is in need of an NK cell activation. In certain embodiments, the subject is in need of an increased level of certain cytokines (e.g., cytokines or chemokines described herein). In certain embodiments, the subject would benefit from the compositions or methods of the present disclosure, irrespective of whether they would need e.g., an NK cell activation.

The methods of the present invention can be used to treat and/or determine the responsiveness to a composition comprising at least one of probiotic bacteria, alone or in combination with other NK immunotherapies, of many different cancers in subjects such as those described herein.

Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (Lou Gehrig's Disease; ALS), also known as motor neurone disease, is considered the most common form of a motoneuron disease with an onset in adult age of, in average, about 50-60 years and an incidence of 1 :50,000 per year.

ALS results in the death of neurons controlling voluntary muscles. ALS is characterized by stiff muscles, muscle twitching, and gradually worsening weakness due to muscles decreasing in size. It may begin with weakness in the arms or legs, or with difficulty speaking or swallowing. About half of the people affected develop at least mild difficulties with thinking and behavior and most people experience pain. Most eventually lose the ability to walk, use their hands, speak, swallow, and breathe. ALS is a progressive disease with a fatal outcome due to gradual paralysis of all voluntary muscles throughout the body, whereby the breathing and swallowing muscles become affected early on already.

The cause is not known in 90% to 95% of cases, but is believed to involve both genetic and environmental factors. The remaining 5-10% of cases are inherited from a person's parents. The most common familial forms of ALS in adults are caused by mutations of the superoxide dismutase gene, or SOD1, located on chromosome 21. The underlying mechanism involves damage to both upper and lower motor neurons.

No cure for ALS is known. The goal of current treatment is to improve symptoms. A medication called riluzole may extend life by about two to three months. Non-invasive ventilation may result in both improved quality and length of life. Mechanical ventilation can prolong survival but does not stop disease progression. A feeding tube may help. The disease can affect people of any age, but usually starts around the age of 60 and in inherited cases around the age of 50. The average survival from onset to death is two to four years, though this can vary. About 10% survive longer than 10 years. Most die from respiratory failure. Amyotrophic Lateral Sclerosis (ALS) Therapies

Two drugs, riluzole and edaravone, are currently available to delay the progression of the disease. Riluzole prolongs ALS survival; it increases survival rates at 12 months by 10% and prolongs survival by 6 months. Similarly Edaravone is effective in treating ALS.

Masitinib is a tyrosine kinase inhibitor used to treat cancer in dogs. It was proven that mastinib inhibited glial cell activation in the appropriate rat model and increased survival.

Retigabine is an approved drug for epilepsy, and acts by binding to the voltagegated potassium channels and increasing the M-current, thus leading to membrane hyperpolarization. Retigabine is able to prolong motor neuron survival and decrease excitability, which is advantageous in the treatment of ALS, since it is believed that, in this disease, neurons are hyper-excitable, firing more than normal and ultimately leading to cell death. This drug is still under clinical trial for the treatment of ALS.

Tamoxifen is an antioestrogen drug, approved for the chemotherapy and chemoprevention of breast cancer. The repurposing of this drug for the treatment of ALS arose serendipitously, after the observation of a neurological improvement in patients and disease stabilization in ALS patients with breast cancer treated with tamoxifen. Its neuroprotective properties appear to be related to inhibition of protein kinase C, which is overexpressed in the spinal cord of ALS patients. Moreover, tamoxifen was found to be able to modulate a proteinopathy present in ALS, through its capacity to be an autophagy modulator.

Cancer

As described herein, the methods and compositions provided herein can be used for preventing or treating a cancer-associated bone loss. Notably, while eradication/treatment of cancer may halt further bone loss resulting from cancer, the eradication/treatment of cancer alone does not restore the bone density already lost due to cancer. Thus, the compositions and methods provided herein are necessary to restore the lost bone density. Accordingly, the compositions and methods provided herein carry out multiple functions in a subject afflicted with cancer: (1) prevent/treat cancer, which aids in preventing future bone loss due to cancer, (2) prevent future bone loss (i.e., cancer-independent loss) by regulating the bone homeostasis, and (3) actively restore the lost bone density. Importantly, functions (2) and (3) operate in an otherwise healthy subject without cancer, thereby preventing bone loss and increasing bone density. Cancer, tumor, or hyperproliferative disease refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. Cancers include, but are not limited to, B cell cancer, (e.g., multiple myeloma, Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphomas, Burkitt lymphoma, Waldenstrom's macroglobulinemia, Hairy cell leukemia, Primary central nervous system (CNS) lymphoma, Primary intraocular lymphoma, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis), T cell cancer (e.g., T-lymphoblastic lymphoma/leukemia, non-Hodgkin lymphomas, Peripheral T-cell lymphomas, Cutaneous T-cell lymphomas (e.g., mycosis fungoides, Sezary syndrome), Adult T-cell leukemia/lymphoma, Angioimmunoblastic T- cell lymphoma, Extranodal natural killer/T-cell lymphoma, Enteropathy-associated intestinal T-cell lymphoma (EATL), Anaplastic large cell lymphoma (ALCL), Hodgkin lymphoma), melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma (SCLC), bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

Cancer Therapies

The therapeutic agents of the present invention can be used alone or can be administered in combination therapy with, e.g., chemotherapeutic agents, hormones, antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy, and/or radiotherapy. The preceding treatment methods can be administered in conjunction with other forms of conventional therapy (e.g, standard-of-care treatments for cancer well-known to the skilled artisan), either consecutively with, pre- or post-conventional therapy. For example, agents of the present invention can be administered with a therapeutically effective dose of chemotherapeutic agent. In other embodiments, agents of the present invention are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent. The Physicians’ Desk Reference (PDR) discloses dosages of chemotherapeutic agents that have been used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art, and can be determined by the physician.

Immunotherapy is a targeted therapy that may comprise, for example, the use of cancer vaccines and/or sensitized antigen presenting cells. For example, an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site. The immunotherapy can involve passive immunity for shortterm protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). For example, anti-VEGF is known to be effective in treating renal cell carcinoma. Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a tumor or cancer.

Immunotherapy also encompasses immune checkpoint modulators. Immune checkpoints are a group of molecules on the cell surface of CD4+ and/or CD8+ T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response. Immune checkpoint proteins are well-known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, TMIDG2, KIR3DL3, and A2aR (see, for example, WO 2012/177624). Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer. In some embodiments, the composition of the present disclosure is administered in combination with one or more inhibitors of immune checkpoints, such as PD1, PD-L1, and/or CD47 inhibitors.

Adoptive cell-based immunotherapies can be combined with the therapies of the present invention. Well-known adoptive cell-based immunotherapeutic modalities, including, without limitation, irradiated autologous or allogeneic tumor cells, tumor lysates or apoptotic tumor cells, antigen-presenting cell-based immunotherapy, dendritic cell-based immunotherapy, adoptive T cell transfer, adoptive CAR T cell therapy, autologous immune enhancement therapy (AIET), cancer vaccines, and/or antigen presenting cells. Such cellbased immunotherapies can be further modified to express one or more gene products to further modulate immune responses, such as expressing cytokines like GM-CSF, and/or to express tumor-associated antigen (TAA) antigens, such as Mage-1, gp-100, and the like.

In other embodiments, immunotherapy comprises non-cell-based immunotherapies. In some embodiments, compositions comprising antigens with or without vaccineenhancing adjuvants are used. Such compositions exist in many well-known forms, such as peptide compositions, oncolytic viruses, recombinant antigen comprising fusion proteins, and the like. In some embodiments, immunomodulatory cytokines, such as interferons, G- CSF, imiquimod, TNF alpha, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In some embodiments, immunomodulatory interleukins, such as IL-2, IL-6, IL-7, IL- 12, IL- 17, IL-23, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In some embodiments, immunomodulatory chemokines, such as CCL3, CCL26, and CXCL7, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In some embodiments, immunomodulatory molecules targeting immunosuppression, such as STAT3 signaling modulators, NFkappaB signaling modulators, and immune checkpoint modulators, are used. The terms “immune checkpoint” and “anti-immune checkpoint therapy” are described above.

In still other embodiments, immunomodulatory drugs, such as immunocytostatic drugs, glucocorticoids, cytostatics, immunophilins and modulators thereof (e.g., rapamycin, a calcineurin inhibitor, tacrolimus, ciclosporin (cyclosporin), pimecrolimus, abetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (doca) aldosterone, a non-glucocorticoid steroid, a pyrimidine synthesis inhibitor, leflunomide, teriflunomide, a folic acid analog, methotrexate, anti-thymocyte globulin, antilymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, an opioid, an IMPDH inhibitor, mycophenolic acid, myriocin, fmgolimod, an NF- xB inhibitor, raloxifene, drotrecogin alfa, denosumab, an NF-xB signaling cascade inhibitor, disulfiram, olmesartan, dithiocarbamate, a proteasome inhibitor, bortezomib, MG132, Prol, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, non-steroidal anti-inflammatory drugs (NSAIDs), arsenic tri oxide, dehydroxymethylepoxy quinomycin (DHMEQ), 13 C(indole-3 -carbinol )/DIM(di- indolmethane) (13C/DIM), Bay 11-7082, luteolin, cell permeable peptide SN-50, IKBa - super repressor overexpression, NFKB decoy oligodeoxynucleotide (ODN), or a derivative or analog of any thereo, are used. In yet other embodiments, immunomodulatory antibodies or protein are used. For example, antibodies that bind to CD40, Toll-like receptor (TLR), 0X40, GITR, CD27, or to 4- IBB, T-cell bispecific antibodies, an anti-IL-2 receptor antibody, an anti-CD3 antibody, OKT3 (muromonab), otelixizumab, teplizumab, visilizumab, an anti-CD4 antibody, clenoliximab, keliximab, zanolimumab, an anti-CDl l a antibody, efalizumab, an anti-CD18 antibody, erlizumab, rovelizumab, an anti-CD20 antibody, afutuzumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, an anti-CD23 antibody, lumiliximab, an anti-CD40 antibody, teneliximab, toralizumab, an anti-CD40L antibody, ruplizumab, an anti-CD62L antibody, aselizumab, an anti-CD80 antibody, galiximab, an anti-CD147 antibody, gavilimomab, a B-Lymphocyte stimulator (BLyS) inhibiting antibody, belimumab, an CTLA4-Ig fusion protein, abatacept, belatacept, an anti- CTLA4 antibody, ipilimumab, tremelimumab, an anti-eotaxin 1 antibody, bertilimumab, an anti-a4-integrin antibody, natalizumab, an anti-IL-6R antibody, tocilizumab, an anti-LFA-1 antibody, odulimomab, an anti-CD25 antibody, basiliximab, daclizumab, inolimomab, an anti-CD5 antibody, zolimomab, an anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atorolimumab, cedelizumab, dorlimomab aritox, dorlixizumab, fontolizumab, gantenerumab, gomiliximab, lebrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab, aflibercept, alefacept, rilonacept, an IL-1 receptor antagonist, anakinra, an anti-IL-5 antibody, mepolizumab, an IgE inhibitor, omalizumab, talizumab, an IL 12 inhibitor, an IL23 inhibitor, ustekinumab, and the like.

Nutritional supplements that enhance immune responses, such as vitamin A, vitamin E, vitamin C, and the like, are well-known in the art (see, for example, U.S. Pat. Nos. 4,981,844 and 5,230,902 and PCT Publ. No. WO 2004/004483) can be used in the methods described herein.

Similarly, agents and therapies other than immunotherapy or in combination thereof can be used with in combination with an agent that increases the activity/amount of NK cells and/or IFN-y to treat a condition that would benefit therefrom. For example, chemotherapy, radiation, epigenetic modifiers (e.g., histone deacetylase (HD AC) modifiers, methylation modifiers, phosphorylation modifiers, and the like), targeted therapy, and the like are well-known in the art.

In some embodiments, chemotherapy is used. Chemotherapy includes the administration of a chemotherapeutic agent. Such a chemotherapeutic agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouridine, aphi dicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halichondrin, colchicine, and rhizoxin. Compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiments, PARP (e.g., PARP-1 and/or PARP-2) inhibitors are used and such inhibitors are well-known in the art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); INO-1001 (Inotek Pharmaceuticals Inc.); PJ34 (Soriano et al., 2001; Pacher et al., 2002b); 3 -aminobenzamide (Trevigen); 4-amino- 1,8-naphthalimide; (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re. 36,397); and NU1025 (Bowman et al.). The mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity. PARP catalyzes the conversion of .beta. -nicotinamide adenine dinucleotide (NAD+) into nicotinamide and poly-ADP -ribose (PAR). Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis (Bouchard V. J. et.al. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 2 Jun. 2001, pp. 97-110(14)). Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecule in the repair of DNA single- strand breaks (SSBs) (de Murcia J. et al. 1997. Proc Natl Acad Sci USA 94:7303-7307; Schreiber V, Dantzer F, Ame J C, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang Z Q, et al. (1997) Genes Dev 11 :2347-2358). Knockout of SSB repair by inhibition of PARP1 function induces DNA double-strand breaks (DSBs) that can trigger synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant H E, et al. (2005) Nature 434:913-917; Farmer H, et al. (2005) Nature 434:917-921). The foregoing examples of chemotherapeutic agents are illustrative, and are not intended to be limiting.

In other embodiments, radiation therapy is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., J. B. Lippencott Company, Philadelphia. The radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. The radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfm (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and 2B A-2-DMHA.

In other embodiments, hormone therapy is used. Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

In other embodiments, photodynamic therapy (also called PDT, photoradiation therapy, phototherapy, or photochemotherapy) is used for the treatment of some types of cancer. It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organisms when the organisms are exposed to a particular type of light.

In yet other embodiments, laser therapy is used to harness high-intensity light to destroy cancer cells. This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. It may also be used to treat cancer by shrinking or destroying tumors.

The immunotherapy and/or cancer therapy may be administered before, after, or concurrently with the agents/compositions described herein. The duration and/or dose of treatment with the compositions may vary according to the particular composition, or the particular combinatory therapy. An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan. The invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined by the methods of the invention is a factor in determining optimal treatment doses and schedules.

Exemplary Embodiments

1. A method of preventing and/or treating a bone loss condition in a subject, comprising administering to the subject an agent that increases (a) the amount and/or activity of the NK cells; and/or (b) the amount of IFN-y.

2. A method of preventing and/or treating a bone loss condition and a cancer in a subject, comprising administering to the subject an agent that increases (a) the amount and/or activity of the NK cells; and/or (b) the amount of IFN-y.

3. A method of inducing bone formation and/or preventing bone loss in a subject, comprising administering to the subject an agent that increases (a) the amount and/or activity of the NK cells; and/or (b) the amount of IFN-y.

4. The method of any one of 1-3, wherein the agent comprises NK cells, super-charged NK cells, at least one probiotic bacteria, osteoclasts, IFN-y, IL-2, anti-CD16 antibody, or any combination thereof.

5. The method of 4, wherein the super-charged NK cells are generated by co-culturing the NK cells with osteoclasts, optionally in the presence of at least one probiotic bacteria.

6. The method of 4 or 5, wherein the NK cells are further activated by IL-2, anti-CD16 antibody, at least one probiotic bacteria, or any combination thereof. 7. The method of any one of 1-6, wherein the agent comprises (a) at least one probiotic bacteria and NK cells; or (b) at least one probiotic bacteria and super-charged NK cells.

8. The method of any one of 1-7, wherein the agent comprises (a) at least one probiotic bacteria, NK cells, and osteoclasts; or (b) at least one probiotic bacteria, super-charged NK cells, and osteoclasts.

9. The method of any one of 4-8, wherein the at least one probiotic bacteria comprises the bacteria of the genus Streptococcus (e.g., S. thermophiles'), Bifidobacterium (e.g., B. longum, B. breve, B. infantis), and/ or Lactobacillus (e.g., L. acidophilus, L. helveticus, L. bulgaricus, L. rhamnosus, L. plantarum, and L. easel).

10. The method of any one of 4-9, wherein the at least one probiotic bacteria comprises one or more bacteria selected from Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei, optionally further comprising Lactobacillus bulgaricus.

11. The method of any one of 4-10, wherein the at least one probiotic bacteria comprises AJ2 or a composition comprising Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei, optionally further comprising Lactobacillus bulgaricus.

12. The method of any one of 4-10, wherein the at least one probiotic bacteria comprises AJ4 or a composition comprising Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei.

13. The method of any one of 4-10, wherein the at least one probiotic bacteria comprises a composition comprising about 30% (or about 20% to about 40%) Streptococcus thermophiles, about 20% (or about 10% to about 30%) Lactobacillus acidophilus, about 40% (or about 30% to about 50%) Lactobacillus plantarum, and about 10% (or about 1% to about 20%) Lactobacillus paracasei.

14. The method of 13, wherein the percent bacteria refers to the percentage of the CFU of said bacteria relative to the total CFU of bacteria in the composition.

15. The method of any one of 4-14, wherein the at least one probiotic bacteria and/or osteoclasts are sonicated or intact.

16. The method of any one of 4-15, wherein the NK cells, super-charged NK cells, and/or osteoclasts are allogeneic or autologous to the subject. 17. The method of any one of 1-16, wherein the agent increases trabecular bone formation.

18. The method of any one of 1-17, wherein the agent increases production or secretion of IFN-y by NK cells.

19. The method of 18, wherein the production or secretion of the IFN-y by NK cells is increased by at least, about, or no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, or 500%.

20. The method of any one of 1-19, wherein the agent is administered in a pharmaceutically or nutraceutically acceptable formulation.

21. The method of any one of 1-20, further comprising administering to the subject at least one additional bone loss therapy.

22. The method of 21, wherein the bone loss therapy comprises calcium supplements, estrogen, calcitonin, estradiol, diphosphonates, vitamin D3, parathyroid hormone, or any combination thereof.

23. The method of any one of 1-22, wherein the bone loss condition is selected from osteopenia, osteoporosis, osteolysis, and cancer-associated bone loss.

24. The method of any one of 1-23, wherein the bone loss condition is a cancer- associated bone loss and/or the subject is afflicted with a cancer.

25. The method of any one of 1-24, further comprising administering to the subject at least one cancer therapy.

26. The method of 25, wherein the at least one cancer therapy is chemotherapy, radiotherapy, or immunotherapy.

27. The method of 26, wherein the immunotherapy inhibits an immune checkpoint, optionally wherein the immune checkpoint is selected from CTLA-4, PD-1, VISTA, B7- H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR.

28. The method of 26 or 27, wherein the immunotherapy is selected from: atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, BGB-A317, STI-Al l 10, TSR-042, RG-7446, BMS- 936559, MEDI-4736, MSB-0010718C, AUR-012, and STI-A1010.

29. The method of any one of 2 and 4-28, wherein the cancer is selected from multiple myeloma, prostate cancer, stomach cancer, bladder cancer, esophageal cancer, cervical cancer, liver cancer, kidney cancer, bone cancer, brain cancer, leukemia, head and neck cancer, oral cancer, pancreatic cancer, lung cancer, colon cancer, melanoma, breast cancer, ovarian cancer, and glioblastoma.

30. The method of any one of 2 and 4-29, wherein the cancer is selected from oral cancer, pancreatic cancer, multiple myeloma, lung cancer, colon cancer, melanoma, breast cancer, ovarian cancer, and glioblastoma.

31. The method of any one of 1-30, wherein the agent is administered at least twice to the subject, optionally wherein the agent is administered daily to the subject.

32. The method of any one of 1-31, wherein the method also inhibits proliferation of a cancer cell and/or increases cancer cell differentiation.

33. The method of any one of 1-32, wherein the agent is administered by intravenous, intratumoral, oral, or intramuscular administration.

34. The method of any one of 1-33, wherein the subject is a mammal.

35. The method of 34, wherein the mammal is a mouse or a human.

36. The method of 34 or 35, wherein the mammal is a human.

EXAMPLES

Example 1: Materials and Methods for Examples 2 and 3

Generation ofhu-BLT mice

When human CD34+ progenitor cells are provided with an appropriate thymic microenvironment, they can mature into naive single-positive human T cells. Thus, it was first determined whether CD34+ cells introduced by bone marrow transplantation could systemically repopulate the mouse and sustain thymopoiesis in the implanted human thymic tissue. The NOD/SCID mice were used because they support significantly higher percentages of reconstitution following transplantation with human CD34+ cells. In essence, NOD/SCID mice were first implanted with human fetal liver and thymic tissues, and the mice were allowed to recover from surgery. The implanted mice were preconditioned with a sublethal dose of gamma radiation and were transplanted with autologous CD34+cells obtained from fetal liver. Thy-liv-implanted mice that received a bone marrow transplant with autologous CD34+ cells had readily detectable numbers of human cells in the peripheral blood (49% ± 22% (mean ± s.d.), range 14-82%, n = 20), consisting of B cells, monocytes and macrophages, DCs and, specifically, T cells (FIG. 3).

BLT mice demonstrated a high proportion of human CD45+ cells in bone marrow (48% ± 14% (s.d.), range 23-75%, n = 24), spleen (42% ± 21% (s.d.), range 5-84%, n = 24) and lymph nodes (71% ± 22% (s.d.), range 6-95%, n = 14). Human T cells, B cells, monocytes and macrophages, and DCs were present in all tissues. The mean percentage of CD4+ T cells was 71% (± 11% (s.d.), range 47-91%, n = 73) and the mean percentage of CD8+ T cells was 21% (± 11% (s.d.), range 4-48%, n = 73) for all tissues examined. The differentiation state of the human T cells in the peripheral blood of BLT mice was also determined. BLT mice kept under sterile conditions had a higher percentage of naive T cells (CD45RA+CD27+) than did healthy human controls.

Liver and lung from BLT mice contained substantial numbers of human T cells, B cells, monocytes and macrophages, and both CDl lc+ and CD123+ DCs. Multilineage reconstitution in the gut of BLT mice analyzed by immunohistology was also observed. These data show that BLT mice can generate an extensive state of sustained systemic multilineage reconstitution with human hematopoietic cells (FIG. 4).

Cell Lines, Reagents, and Antibodies

Recombinant human IL-2 was obtained from NIH-BRB. Human TNF-a and IFN-y was obtained from Biolegend (San Diego, CA, USA). Antibody to CD 16 was purchased from Biolegend (San Diego, CA, USA). Fluorochrome-conjugated human and mouse antibodies for flow cytometry were obtained from Biolegend (San Diego, CA, USA). Monoclonal antibodies to TNF-a and IFN-y were prepared in our laboratory and used at 1 : 100 dilutions to block rhTNF-a and rhIFN-y functions. The human NK cell and monocyte purification kits were obtained from Stem Cell Technologies (Vancouver, BC, Canada).

Human immune cells were cultured in RPMI 1640, supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, CA). Oral squamous carcinoma stem cells (OSCSCs) were isolated from oral cancer patient tongue tumors at UCLA School of Medicine and cultured in RPMI 1640, supplemented 10% FBS (Gemini Bio-Products, CA), 1.4% antibiotic antimycotic, 1% sodium pyruvate, 1.4% MEM non-essential amino acids, 1% L-glutamine, 0.2% gentimicin (Gemini Bio-products, CA) and 0.15% sodium bicarbonate (Fisher Scientific, PA). Human monocytes/osteoclasts were cultured in alpha-MEM medium (Life Technologies, CA), supplemented with 10% FBS, and penicillin-streptomycin (Gemini Bio-Products, CA). Human M-CSF (Biolegend, CA) and soluble RANKL (PeproTech, NJ) were dissolved in alpha-MEM and stored at -80°C.

Ethics Approval and Consent to Participate

Written informed consents approved by UCLA Institutional Review Board (IRB) were obtained and all procedures were approved by the UCLA-IRB (IRB#11-000781). Animal research was performed under the written approval of the UCLA Animal Research Committee (ARC) (protocol # 2012-101-13).

Purification of Human NK Cells and Monocytes

NK cells and monocytes were negatively selected from PBMCs using isolation kits from Stem Cell Technologies (Vancouver, BC, Canada). Greater than 96% purity was obtained both for purified NK cells and monocytes based on flow cytometric analysis. Cell Dissociation and Cell Culture of Tissues from hu-BLT Mice

Pancreatic tumors were harvested from hu-BLT mice and cut into 1 mm 3 pieces and placed into a digestion buffer containing 1 mg/mL collagenase IV, 10 U/mL DNAse I, and 1% bovine serum albumin (BSA) in DMEM media for 20 min at 37 °C. The samples were then filtered through a 40 mm cell strainer and centrifuged at 1500 rpm for 10 min at 4 °C. To obtain singlecell suspensions from BM, femurs were flushed using media, and filtered through a 40 pm cell strainer. Spleens were removed and single cell suspensions were prepared and filtered through a 40 pm cell strainer and centrifuged at 1500 rpm for 5 min at 4 °C. The pellets were re-suspended in ACK buffer to remove the red blood cells. Peripheral blood mononuclear cells (PBMCs) were isolated using ficoll-hypaque centrifugation.

Isolations of NK Cells, T Cells and Monocytes from hu-BLT Mice

NK cells and T cells from hu-BLT splenocytes were obtained as described previously by using the human CD56+ and CD3+ selection kits respectively (Stem Cells Technologies, Vancouver, BC, Canada). Monocytes from hu-BLT bone marrow were isolated using human CD14 isolation kit (eBioscience, San Diego, CA, U.S.A.). Generation of Osteoclasts and Expansion of Human and hu-BLT NK Cells — Generation of Super-charged NK cells

Monocytes were purified form human peripheral blood or hu-BLT BM and cultured using alpha-MEM medium containing M-CSF (25 ng/mL) and RANKL (25 ng/mL) for 21 days (medium was refreshed every 3 days). NK cells were activated with rh-IL-2 (1000 U/mL) and anti-CD16 mAh (3pg/mL) for 18-20 h before they were cultured with osteoclasts and sonicated-AJ2 to generate super-charged NK cells. The medium was refreshed every 3 days with RMPI containing rh-IL-2 (1000 U/mL).

Enzyme-Linked Immunosorbent Assays (ELISAs) and Multiplex Cytokine Assay

Human ELISA kits for IFN-y and IL-6 were purchased from Biolegend (San Diego, CA, USA). The assays were conducted as recommended by the manufacturer. For certain experiments multiplex arrays were used to determine the levels of secreted cytokines and chemokines. Analysis was performed using MAGPIX (Millipore, Danvers, MA, USA) and data was analyzed using xPONENT 4.2 (Luminex, Austin, TX, USA).

Surface Staining and Cell Death Assays

Staining was performed by staining the cells with antibodies as described previously, briefly, antibodies were added to 1 x 104 cells in 50 pL of cold-PBS+ 1% BSA and cells were incubated on ice for 30 min. Thereafter cells were washed in cold PBS+ 1% BSA and flow cytometric analysis was performed using Beckman Coulter Epics XL cytometer (Brea, CA, USA) and results were analyzed in FlowJo vX software (Flowjo, Ashland, OR, USA).

Sonicating AJ2

AJ2 is a combination of 7 different strains of gram positive probiotic bacteria which has been cloned to withstand high temperature and also low pH (Streptococcus thermophiles, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei) were used to induce NK activation which in turn increases IFN-y and promotes differentiation of stem cells.

AJ2 was weighed and resuspended in RPMI Medium 1640 containing 10% FBS at a concentration of 10 mg per 1 mL. The bacteria were thoroughly vortexed, then sonicated on ice for 15 seconds, at 6 to 8 amplitude. Sonicated samples were then incubated for 30 seconds on ice. After every five pulses, a sample was taken to observe under the microscope until at least 80 percent of cell walls were lysed. It was determined that approximately 20 rounds of sonication/incubation on ice, were conducted to achieve complete sonication. Finally, the sonicated samples (sAJ2) were aliquoted and stored in a - 80 degrees Celsius freezer. Sonication of bacteria is not required or necessary to render its activities presented herein. Analysis of Human Pancreatic Cancer Cell Growth in Humanized-BLT Mice

Humanized-BLT (hu-BLT; human bone marrow/liver/thymus) mice were generated as previously described. In vivo growth of pancreatic tumors was performed by orthotopic tumor implantation in the pancreas hu-BLT mice. To establish orthotopic tumors, mice were anesthetized using isoflurane, and tumors in a mixture with Matrigel (10 pL) (Corning, NY, USA) were injected in the pancreas using insulin syringe. Mice received 1.5 x 10 ⁶ super-charged NK cells via tail vein injection 7 to 10 days after the tumor implantation. They were also fed AJ2 (5 billion/dose) orally. The first dose of AJ2 was given one or two weeks before tumor implantation, and feeding was continued throughout the experiment at an interval of every 48 h. Mice were euthanized when signs of morbidity were evident. Lumbar vertebrae, pancreas, pancreatic tumors, bone marrow, spleen, and peripheral blood were harvested, and single cell suspensions were prepared from each tissue as described previously and below.

Bone Analysis

The assessment of bone architecture was made by micro computed tomography (micro-CT). Samples were harvested, formalin-fixed and imaged using high-resolution microCT (Skyscan 1275, Skyscan, Belgium) at an image resolution of 10 u pixels and analyzed using Data Viewer, Recon, CTAn and CTVol software provided by the manufacturer.

Specimen preparation for scanning

At the final time point, all mice will be euthanized in a CO2 chamber with the appropriate CO2 concentrations and exposure times. Lumbar vertebrae (L3) were dissected and fixed in 70% ethanol. In this study, vertebras were scanned with Skyscan 1275 (Bruker microCT N. V., Belgium), equipped with a 5-pm focal spot micro-focus x-ray tube at the resolution of 10pm (60 kVp, 166 mA, and 1mm Al Filter). Specimens were aligned with the vertical axis of the scanner, and low-density foam (a non-attenuating material) was used to stabilize the specimens firmly into a 0.25-diameter-tube. Phantom calibration was performed to relate the micro-CT values to a mineral-equivalent value (g/cm3) of calcium hydroxyapatite. Reconstruction

To process images, scanned images were reconstructed with NRecon (Bruker microCT N.V., Belgium) for attenuation correction, ring artifact reduction, and beam hardening. After data acquisition, images were aligned in 3D view for vertical orientation with Data Viewer software for accuracy.

Segmentation of Volume of Interest

Segmentation of the images will be completed manually by comparing the binarized image with the unsegmented image, and a single global threshold of 60 will be applied. An irregular ROI selection will be manually drawn parallel and close to the endocortical surface.

Ex vivo assessment of bone architecture by micro-CT analysis

Length of the ROI was adjusted in proportion to the total vertebral height. For third lumbar vertebrae, transverse micro-CT slices were acquired for the entire vertebral body, and trabecular bone was evaluated within the region of 0.5 mm away from the growth plate. To ensure accuracy, each ROI were drawn manually in a sequential manner for each trans- axial micro-CT slice. Morphometric parameters were computed from the binarized images using a direct threedimensional approach that does not rely on any prior assumptions about the underlying structure. For trabecular morphology, assessment of bone volume fraction (BV/TV %), trabecular thickness (Tb. Th, mm), trabecular number (Tb. N, mm), and trabecular separation (Tb.Sp, mm) were used.

All analyses were performed with CTAn software (Bruker microCT N.V., Belgium). A 3D rendered model of lumbar vertebrae was constructed by CTVol software (Bruker microCT N. V., Belgium). One representative sample was taken from each group. Comparison of the 3D rendered volume was performed to show differences in trabecular structure of the treated group and the control group. FIG. 5 A and FIG. 5B.

Histology and quantitative histomorphometry

Static histomorphometry was carried out on hu-BLT mice. Third lumbar vertebras (L3) were dissected, fixed in 70% ethanol, dehydrated and embedded undecalcified in methyl methacrylate. Frontal sections, 5 pm thick and stained with 0.1% toluidine blue, pH 6.4. Static parameters of bone formation (OB) and resorption (OC) were measured in a defined area between 0.25mm from both growth plates and endochondral bone surfaces. Additional histochemical stain tartrate-resistant acid phosphatase (TRAP) was performed to identify osteoclasts.

Statistical Analysis

For the micro-CT results we used Linear Mixed Effects Models to determine if there were any differences between the comparisons (as difference in means between the groups) in the table. A linear model was chosen because the outcome is on an interval scale (as opposed to a categorical scale) and a mixed effects model was used to account for the correlation between the different outcomes within each mouse.

For the rest of the procedures, an unpaired, two-tailed Student t-test was performed for the statistical analysis. One-way ANOVA using Prism-7 software (Graphpad Prism, San Diego, CA, USA) was used to compare different groups. [1] denotes the number of mice used for each condition in the experiment. The following symbols represent the levels of statistical significance within each analysis, *** (p-value <0.001), ** (p-value 0.001-0.01), value 0.01-0.05). Release Cytotoxicity Assay

⁵¹ Cr was purchased from Perkin Elmer (Santa Clara, CA). Standard ⁵¹ Cr release cytotoxicity assays were used to determine NK cell cytotoxic function in the experimental cultures and the sensitivity of target cells to NK cell mediated lysis. The effector cells (1 x 10 ⁵ NK cells/well) were aliquoted into 96-well round-bottom microwell plates (Fisher Scientific, Pittsburgh, PA) and titrated at four to six serial dilutions. Patient-derived OSCSCs were used as a specific and sensitive NK target to assess NK cell-mediated cytotoxicity. The target cells (5 x 10 ⁵ OSCSCs) were labeled with 50 pCi ⁵¹ Cr (Perkin Elmer, Santa Clara, CA) and chromated for 1 hour. Following incubation, target cells were washed twice to remove excess unbound ⁵¹ Cr. ⁵¹ Cr-labeled target cells were aliquoted into the 96-well round bottom microwell plates containing effector cells at a concentration of 1 x 10 ⁴ cells/well at a top effectortarget (E:T) ratio of 5: 1. Plates were centrifuged and incubated for a period of 4 hours. After a 4-hour incubation period, the supernatants were harvested from each sample and counted for released radioactivity using the gamma counter. Total (containing ⁵¹ Cr-labeled target cells) and spontaneous (supernatants of target cells alone) release values were measured and used to calculate the percentage specific cytotoxicity. The percentage specific cytotoxicity was calculated using the following formula:

% Cytotoxicity = Experimental cpm - spontaneous cpm Total cpm - spontaneous cpm

LU 30/10 ⁶ is calculated by using the inverse of the number of effector cells needed to lyse 30% of target cells xlOO. Example 2: The impact of MP2 tumors, super-charged NK cells, and AJ2 on the secretion of IFN Gamma and its association with bone loss.

We have previously demonstrated that the stage of differentiation of tumors has a profound effect on NK cells' function and that stemlike / poorly differentiated tumors were preferentially targeted by the NK cells. Also, we determined the role of super-charged NK cells in immune mobilization, lysis, and differentiation of stemlike/ undifferentiated tumors implanted in the pancreas of humanized-BLT (hu-BLT) mice fed with or without AJ2 probiotics.

Stem-like/undifferentiated pancreatic tumors grew rapidly and formed large tumors, and exhibited lower expression of above-mentioned differentiation antigens in the pancreas of hu-BLT mice. Unlike stemlike/undifferentiated tumors, NK differentiated MP2 (MiaPaCa-2) tumors or patientderived differentiated tumors could not grow or grew smaller tumors and were unable to metastasize in hu-BLT mice, and they were susceptible to chemotherapeutic drugs. Stemlike/ undifferentiated pancreatic tumors implanted in the pancreas of hu-BLT mice and injected with super-charged NK cells formed much smaller tumors, proliferated less, and exhibited differentiated phenotype. When the differentiation of stemlike tumors by the NK cells was prevented by the addition of antibodies to IFN-y and TNF-a, tumors grew rapidly and metastasized, and they remained resistant to chemotherapeutic drugs.

More significant numbers of immune cells infiltrated the tumors of NK-injected and AJ2-probiotic bacteria fed mice. Moreover, increased IFN-y secretion in the presence of decreased IL-6 was seen in tumors resected and cultured from NK-injected and AJ2 fed mice. Tumor-induced decreases in NK cytotoxicity and IFN-y secretion were restored/increased within PBMCs, spleen, and bone marrow when mice received NK cells and were fed with AJ2. Therefore, we concluded that NK cells prevent the growth of pancreatic tumors through lysis and differentiation, thereby curtailing the growth and metastatic potential of stem-like/undifferentiated-tumors.

Hu-BLT NK cells cultured with osteoclasts, expanded greatly and increased secretion of IFN-y

Hu-BLT NK cells purified from the spleen of mice responded to the activation signals provided by the IL-2 and anti-CD16 mAb treatment and expanded greatly and demonstrated increased secretion of IFN-y when cultured with both autologous and allogeneic osteoclasts in the presence of sAJ2 treatment (FIG. 6A and FIG. 6B), indicating close similarity between hu-BLT and human donor derived NK cell expansion and function by osteoclasts.

A J2 and super-charged NK cells increased the levels of IFN-y in the serum of the hu- BLT mice

Sera from the peripheral blood of either NK injected or NK injected/ AJ2 fed tumorbearing mice exhibited 2.73 and 4.8-fold more IFN-y, respectively, when compared to tumor-bearing mice (FIG. 7 and FIG. 8).

Mice with implantation of the tumor in the absence of any treatment had the least amount of IFN-y in the sera (FIG. 7 and FIG. 8).

Feeding AJ2 alone, or injecting super-charged NK cells in the absence of tumor implantation, or feeding AJ2 with implantation of tumors, or injecting super-charged NK cells and feeding AJ2 all increased the levels of IFN-y in the serum of the hu-BLT mice moderately when compared to control mice in the absence of any treatments. These mice had much less IFN-y in the sera when compared to those which were implanted with the tumor and fed with AJ2 and injected with super-charged NK cells (FIG. 8).

On average, a decrease in IFN-y secretion from the pancreatic cell cultures could be observed in mice implanted with MP2 tumors, when compared to control mice with no tumors.

Injection of NK cells into tumor-bearing mice restored IFN-y secretion in pancreatic cell cultures and the levels exceeded those seen in the control mice with no tumors.

Although slight differences could be seen between NK alone injected or NK- injected and AJ2 fed mice in terms of tumor weight/tumor growth in pancreas, there was, on average, higher secretion of IFN-y by NK injected and AJ2 fed pancreatic cell cultures (FIG. 9).

Blocking MP2 differentiation with anti-IFN-y and anti-TNF-a antibodies resulted in the inhibition of tumor differentiation and generation of tumors with higher tumor weights. (FIG. 10).

Suppression of NK Cell Cytotoxicity and Decreased Secretion of IFN-y in Tumor- Bearing Mice within All Tissue Compartments; Restoration by Super-Charged NK Cells

PBMCs from tumor-bearing mice which were similar to PBMCs and NK cells from pancreatic cancer patients, had significantly lower NK cell-mediated cytotoxicity and exhibited decreased IFN-y secretion, when compared to those from healthy mice or humans, respectively. (FIG. 11A-FIG. 11C). Tumor-bearing mice had much lower cytotoxicity and/or secretion of IFN-y in cells obtained from all tissue compartments, in comparison to those obtained from control mice without tumor, or tumor-bearing mice injected with NK cells, or those implanted with NK- differentiated tumors (FIG. 12).

In previous studies, we have shown that NK cells prevent pancreatic tumors' growth through lysis and differentiation, thereby curtailing the growth and metastatic potential of stemlike/undifferentiated-tumors. Also, we demonstrated that NK cells induced differentiation of MP2 tumors through the functions of IFN-y and TNF-a.

In this study, super-charged NK cells in the presence and absence of feeding AJ2 increased IFN-y secretion in different compartments of hu-BLT mice; like the serum, cell cultures from pancreatic tumors, NK cells purified from hu-BLT splenocytes, PBMCs, splenocytes cell cultures, and bone marrow. On the contrary, MP2 tumor-bearing hu-BLT mice showed decreased IFN-y in the same compartments.

Example 3: Changes in the bone structure of MP2 tumor-bearing mice injected with cultures of NK cells and fed with and without AJ2.

Mice fed with AJ2 presented increased bone formation when compared to the CTRL group.

The AJ2 group presented increased bone formation with increased bone volume and trabecular number when compared to the CTRL group. (FIG. 13A-FIG. 13B).

MP2 tumor-bearing mice injected with cultures of NK cells and fed with AJ2 presented increased bone formation with statistically significant higher trabecular bone volume when compared to MP2 tumor and MP2+AJ2 group, respectively.

MP2 tumor-bearing mice injected with NK cells and fed with AJ2 group showed a statistically significant increase in bone volume fraction (BV/TV), augmented trabecular thickness (Tb.Th), higher trabecular number (Tb.n), and decreased trabecular spacing (Tb.Sp) when compared to the MP2 tumor group. (FIG. 14A-FIG. 14B).

Similarly, MP2 tumor-bearing mice injected with NK cells and fed with AJ2 group showed a statistically significant increase in bone volume fraction (BV/TV), augmented trabecular thickness (Tb.Th), higher trabecular number (Tb.n), and decreased trabecular spacing (Tb.Sp) when compared to the MP2+ AJ2 group. (FIG. 14A-FIG. 14B). Stem-like/undifferentiated tumors implanted in hu-BLT mice injected with cultures of NK cells and fed with or without A J2 presented similar bone formation when compared to the control group.

After six weeks of tumor transplantation, lumbar vertebrae were harvested and scanned with highresolution micro-CT. Stem-like/undifferentiated tumors implanted in hu- BLT mice injected with NK cells' cultures and fed with or without AJ2 presented similar bone formation compared to the control group. (FIG. 15A-FIG. 15B). However, the increase in bone formation was more evident in the lumber microCT than bone parameters (FIG. 15 A), and FIG. 15B clearly showed that there was more bone formation for treatment group than control. It is also evident that hu-BLT mice with MP2 without any treatment showed more significant bone loss.

Histological, immunohistochemical analysis, and static indices assessments at the third lumbar vertebra exhibited an increase of trabecular bone formation in the AJ2 treated group whencompared with the Control and MP2 groups.

To assess the bone remodeling process's underlying cellular mechanisms, histological analysis and static indices assessment at the third lumbar vertebra were performed. Consistent with the micro-CT findings, histological analysis of the AJ2 treatment group exhibited an increase in bone volume fraction (B V/TV), augmented trabecular thickness (Tb.Th), higher trabecular number (Tb.n), and decreased trabecular spacing (Tb.Sp) when compared to the Control and MP2 tumor groups. The histomorphometric values for the MP2+NK +AJ2 sample could not be confirmed due to staining failure (FIG. 17 and Table 2).

For the static parameters of bone resorption (OC), the MP2 tumor sample was positive for TRAP staining. On the contrary, the control, AJ2, and MP2+NK+AJ2 samples were negative for TRAP staining. These results are in accordance with the micro-CT results, suggesting more osteoclast activity and bone resorption in the MP2 tumor sample when compared to the control, AJ2, and MP2+NK+ AJ2 samples (FIG. 18).

We found a remarkable correlation between the 3D images generated from the micro-CT results, the induction and secretion of IFN-y, and bone morphology. In the tumorbearing mice injected with NK and fed with AJ2, there was increased IFN-y secretion in different compartments like the serum, cell cultures from pancreatic tumors, NK cells purified from splenocytes, PBMCs, splenocytes cell cultures, and bone marrow cells. Consistently, the MP2 tumor-bearing mice injected with NK cells' cultures and fed with AJ2 presented increased bone formation with statistically significantly higher trabecular bone volume compared to MP2 tumor-bearing mice group and MP2 tumor-bearing mice fed with AJ2 group, respectively.

On the contrary, MP2 tumor-bearing mice showed reduced bone formation and decreased IFN-y secretion in the same compartments. (FIG. 16 and Table 1). Accordingly, MP2 tumor-bearing mice group presented statistically significant reduced trabecular bone volume compared to the treatment groups.

Consistent with the micro-CT findings, histological analysis of the AJ2 treatment group exhibited increased bone formation when compared to the Control and MP2 tumor groups. TRAP staining indicated more osteoclast activity and bone resorption in the MP2 tumor sample compared to the rest of the samples.

The state of art suggests that IFN-y can either inhibit osteoclast formation or enhance osteoclastogenesis according to the conditions to which the osteoclast precursors are exposed. While IFN-y has been shown to be involved in bone cell differentiation and function with complex effects on skeletal helath, the role of IFN-y in pathological bone diseases has been controversial.

In this study, there was a remarkable correlation between induction and secretion of IFN-y and increase bone formation, demonstrating a curative role of IFN-y in a pathological bone disease. In the tumor-bearing mice injected with NK and fed with AJ2, there was increased IFN-y in different compartments and augmented bone formation. On the contrary, MP2 tumor-bearing mice showed decreased IFN-y and decreased bone formation in the same compartments. These findings indicate that IFN-y (1) induces secretion by NK cells, (2) inhibits tumor growth, and (3) decreases skeletal complications of malignancy by directly acting on host cells to inhibits osteoclast formation and function.

This is a novel report of the role of NK cells and AJ2 in the treatment and prevention of tumor growth and their role in increasing the levels of IFN-y in the hu-BLT mice suppressing tumorinduced bone loss. NK cells prevented the growth of pancreatic tumors through lysis and differentiation, thereby curtailing the growth and metastatic potential of stemlike/undifferentiated-tumors through the functions of IFN-y and TNF-a. Increased IFN-y secretion in the presence of decreased IL-6 was seen in tumors resected and cultured from NK-injected, and AJ2 fed mice.

IFN-y is a multifunctional cytokine produced mainly by NK cells and activated T cells that play a critical role in host immune responses against pathogens and cancer. Hu- BLT NK cells cultured with osteoclasts expanded greatly and increased secretion of IFN-y. Hu-BLT NK cells were purified from the spleen of mice, responded to the IL-2 and antiCD 16 mAh treatment's activation signals, expanded greatly and demonstrated increased secretion IFN-y when cultured with both autologous and allogeneic osteoclasts in the presence of sAJ2 treatment.

AJ2 and super-charged NK cells increased the levels of IFN-y in the serum of the hu-BLT mice. On average, an increase in IFN-y secretion from the pancreatic cell cultures could be observed in mice implanted with MP2 tumors, injected with NK and fed with AJ2 when compared to mice implanted with MP2 tumors and control mice.

We demonstrated the suppression of NK cell cytotoxicity and decreased secretion of IFN-y in tumor-bearing mice within all tissue compartments and restoration by supercharged NK Cells. We found a remarkable correlation between the 3D images originated from the micro-CT analysis results, the induction and secretion of IFN-y, and bone morphology. In the tumor bearing mice, injected with NK and fed with AJ2, we found increased IFN-y in different compartments like the serum, cell cultures from pancreatic tumors, NK cells purified from splenocytes, PBMCs, splenocytes cell cultures, and bone marrow cells. On the contrary, MP2 tumor-bearing mice showed decreased IFN-y in the same compartments. There was also a decreased bone formation and more production of IL-6 in the MP2 group.

In this study, the overall effect of decreased IFN-y secretion significantly reduced bone volume, indicating that deficiency in IFN-y results in a depletion in bone formation. Assessment of bone architecture with 3D micro-CT analysis, histology, and histomorphometry demonstrated that MP2 tumor mice with decreased IFN-g secretion had a significant deficit in trabecular bone. The results provided herein indicate that decreased IFN-y secretion results in lower osteoblast differentiation of MSCs, a significant reduction in bone volume, indicating that deficiency in IFN-y results in a depletion in bone formation. This study outcome also indicates that IFN-y has direct anti-tumor effects and suppresses tumor-induced bone loss by directly targeting host osteoclasts to inhibit osteolysis.

Also, there was correspondence between increased IFN-y induction and secretion, and increased bone formation presented in the 3-D images generated from the micro-CT analysis results. Consistent with the micro-CT findings, histological analysis of the AJ2 treatment group exhibited increased bone formation when compared to the Control and MP2 tumor groups. TRAP staining suggested more osteoclast activity and bone resorption in the MP2 tumor sample compared to the rest of the samples.

In this study, the enhanced osteolytic lesion formation in BLT tumor-bearing mice and NK cells' ability to secrete IFN-y to significantly reduce bone loss in tumor-bearing mice strongly supports a direct anti-osteoclastogenic role for IFN-y in the setting of cancer- induced bone disease.

This study corroborates the close interplay between the immune and skeletal systems. Among the cytokines that have been found to regulate osteoclastogenesis, IFN-y seems to be a critical regulator of bone resorption. In this report, IFN-y decreased tumor growth and prevented tumor-associated bone loss by inhibiting tumor cell growth and osteolysis.

The data presented herein strongly support the physiologic role of IFN-y for skeletal integrity maintenance and indicate that modulation of its signaling pathway may be used advantageously to improve bone strength. Besides, our results indicate that IFN-y suppresses osteoclastogenesis.

Our findings demonstrate the significance of super-charged NK cells and AJ2 in the treatment and prevention of tumor growth and their role in increasing the levels of IFN-y in hu-BLT mice suppressing tumor-induced bone loss.

Example 4: Materials and Methods for Examples 5-9

Cell lines, reagents, and antibodies

RPMI 1640 (Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, CA) was used to culture peripheral blood mononuclear cells (PBMCs), NK cells, and CD8+ T cells. Recombinant IL-2 was obtained from NIH- BRB. Anti-CD16 mAbs, and anti-CD3/CD28 mAbs were obtained from Biolegend (San Diego, CA). Oral squamous carcinoma stem cells (OSCSCs) were isolated from patients with tongue tumors at UCLA. Human ELISA kits for IFN-y were purchased from Biolegend (San Diego, C A). Chroimum-51 radionucleotide was purchased from PerkinElmer, CA, USA.

Isolation of human PBMCs, NK cells, and CD8+ T cells

Written informed consents approved by UCLA Institutional Review Board (IRB) were obtained from healthy individuals, and all procedures were approved by the UCLA- IRB. PBMCs were isolated from peripheral blood as described before. PBMCs were used to isolate NK cells, and CD8+T cells using the EasySep® Human NK cell and EasySep® Human CD8+ T cells, respectively, purchased from stem cell technologies (Vancouver, BC, Canada). Isolated NK cells and CD8+ T cells were stained with anti-CD16, anti-CD3/CD8 antibodies, respectively, to measure the cell purity using flow cytometric analysis.

Enzyme-linked immunosorbent assays (ELISAs), Enzyme-linked immunospot (ELISpot) and multiplex cytokine arrays

Single ELISAs were performed as previously described. To analyze and obtain the cytokine and chemokine concentrations, a standard curve was generated by either two- or three-fold dilution of recombinant cytokines provided by the manufacturer. The ELISpot was conducted according to manufacturer’s instructions. The number of IFN-y secreting cells was determined by using human IFN-y single-color enzymatic ELISpot assay, and analyzed by the ImmunoSpot® S6 UNIVERSAL analyzer and ImmunoSpot® software (all CTL Europe GmbH, Bohn, Germany). For multiple cytokine arrays, the levels of cytokines and chemokines were also determined by multiplex cytokine arrays as recommended by the manufacturer. Analysis was performed using a luminex instrument (MAGPIX, Millipore, Billerica, MA), and data were analyzed using the proprietary software (xPONENT 4.2, Millipore, Billerica, MA).

⁵¹ Cr release cytotoxicity assay

The ⁵¹ Cr release assay was performed as described previously. Briefly, different numbers of effector cells were incubated with ⁵¹ Cr-labeled target cells. After a 4-hour incubation period, the supernatants were harvested from each sample and the released radioactivity was counted using the gamma counter. The percentage specific cytotoxicity was calculated as follows:

% Cytotoxicity = Experimental cpm - spontaneous cpm

Total cpm - spontaneous cpm

Lytic units (LU) 30/10 ⁶ is calculated by using the inverse of the number of effector cells needed to lyse 30% of tumor target cells 400.

Sonication of probiotic bacteria AJ2, AJ3 andAJ4

Gram-positive probiotic bacteria strains for AJ2, AJ3, and AJ4 were weighed and re-suspended in RPMI 1640 medium containing 10% FBS at a concentration of 10 mg/ml. The bacteria were thoroughly vortexed and sonicated on ice for 15 seconds at 6 to 8 amplitudes. Sonicated samples were then incubated for 30 seconds on ice, and the cycle was repeated for five rounds. After every five rounds of sonication, the samples were examined under the microscope until at least 80% of bacterial walls were lysed. It was determined that approximately 20 rounds of sonication/incubation on ice were necessary to achieve complete sonication. Finally, the sonicated probiotic bacteria, sAJ2, sAJ3, and sAJ4 were aliquoted and stored at -80°C until use. Sonication of bacteria is not required or necessary to render its activities presented herein.

Statistical analysis

All statistical analyses were performed using the GraphPad Prism-8 software. An unpaired or paired, two-tailed student’s t-test was performed for the statistical analysis for experiments with two groups. One-way ANOVA with a Bonferroni post-test was used to compare different groups for experiments with more than two groups. Duplicate or triplicate samples were used in the studies, (n) denotes the number of healthy individuals for each experimental condition. The following symbols represent the levels of statistical significance within each analysis: ***(p value <0.001), **(p value 0.001-0.01), *(p value 0.01-0.05).

Example 5: Decreased IFN-Y and increased IL-10 secretions by sAJ3 treated PBMCs in comparison to sAJ4 and sAJ2 treated PBMCs

Examples 5-8 demonstrate how the combination of different strains, sAJ2, sAJ3, and sAJ4 differ in their potential to activate PBMCs, NK cells and CD8+ T cells. In addition, we compared the functional activation of NK cells by sAJ3 and sAJ4 in the presence of monocytes.

We observed increased secretion of IFN-y (Figs. 21A, 21E, and 21F, and Table 6), TNF-a (Fig. 2 IB), and IL-6 (Fig. 21C) in PBMCs when treated with IL-2 and sonicated AJ4 (sAJ4) in comparison to other treatments as shown in figures. Decreased IL-10 secretion was seen in sAJ4 treated PBMCs in comparison to sAJ3 treated PBMCs (Fig. 2 ID). CD 16 receptor plays a significant role in increased secretion of IFN-y in the presence of IL-2 treatment. Since IL-2 or the combination of IL-2 and CD 16 cross-linking increased IFN-y secretion in the presence of negligible IL- 10 secretion, the ratio of IFN-y to IL- 10 remained higher in these treatments, with the highest seen in IL-2 and anti-CD16 mAbs treated PBMCs (Fig. 21G and Table 7A). Ratio of IFN-y to IL-10 was much higher in IL- 2+sAJ4 treated PBMCs in comparison to IL-2+sAJ2 or IL-2+sAJ3 treated PBMCs, IL- 2+sAJ3 having the lowest ratio (Fig. 21G and Table 7B). Even though sAJ4 alone was able to trigger some IFN-y secretion, the synergistic effect was seen when PBMCs were treated with IL-2 and sAJ4. The direct effect of sAJ4 without IL-2 treatment was higher than sAJ3 or sAJ2 on PBMCs (Figs. 21 A, 21E and 21F). Of interest, is the much lower ratio of IFN-y to IL-10 in IL-2+anti-CD3/CD28 mAbs treated PBMCs, when either compared to IL-2 alone or IL-2+anti-CD16mAbs (Fig. 21G).

Example 6: Decreased IFN-Y and increased IL-10 secretion by sAJ3 treated NK cells in comparison to sAJ4 and sAJ2 treated NK cells

NK cells were sorted from the PBMCs and used in the treatments as described above for PBMCs. Similar to PBMCs, we observed significantly increased levels of IFN-y in NK cells when treated with IL-2 and sAJ4 in comparison to IL-2+sAJ3 or IL-2+sAJ2 as shown in figure (Figs. 22A, 22C, 22D, 22E, 22F, and 22G). The ratio of IFN-y to IL- 10 was the highest in IL-2+sAJ4 and the lowest in IL-2+sAJ3 (Figs. 22H and 221, and Table 8). As mentioned above, the combination of IL-2 and CD 16 cross-linking increased IFN-y secretion, and the ratio of IFN-y to IL- 10 was seen the highest in IL-2 and anti-CD16 mAbs treated NK cells (Figs. 22H and 221).

Example 7: Decreased IFN-Y secretion in sAJ3 treated NK cells cultured with monocytes in comparison to sAJ4 treated cells

To assess the effect of probiotics on IFN-y and IL-10 secretion by the NK cells cultured with autologous monocytes, we treated NK and monocytes co-cultures either with IL-2 alone or IL-2 with sAJ3 or sAJ4, or sAJ3 or sAJ4 alone and determined IFN-y and IL- 10 secretion (Fig. 23). Both in the absence or presence of monocytes, NK cells exhibited higher IFN-y secretion when treated with sAJ4 in comparison to sAJ3 (Fig. 23 A). Also, in the absence or presence of monocytes, NK cells exhibited higher IFN-y to IL- 10 ratio when treated with sAJ4 in comparison to sAJ3 (Fig. 23B). Even though sAJ4 alone was able to trigger some IFN-y secretion, the synergistic effect was seen when NK cells were treated with IL-2 and sAJ4. The direct effect of sAJ4 without IL-2 treatment was higher than sAJ3 by NK cells (Fig. 23) Example 8: Unlike NK cells CD8+ T cells secrete considerably lower IFN-Y when treated with IL-2 and probiotic bacteria

CD8+ T cells were sorted from the peripheral blood and treated with IL-2 or IL- 2+anti-CD3/CD28 mAbS or IL-2 with each of sAJ4 and sAJ3. Treatment with IL-2+anti- CD3/CD28 mAbs induced the highest secretion of IFN-y, whereas combination of IL- 2+sAJ4 was considerably lower, but still higher than IL2+sAJ3 in comparison to IL-2+anti- CD3/CD28 mAbs (Fig. 24).

Discussion

In the past several years, there has been a significant interest in the role of microbiome and probiotics in contributing to the health and well-being of humans. Their role in alleviating disease symptoms and prevention has been shown in many disease models, however, our understanding of the mechanisms by which they exert their effect is still in its infancy. Here, we demonstrated disease specific functions of three different formulations of probiotics in auto-immunity, cancer, and disease prevention with emphasis on alleviating ALS symptoms and using them as adjunct to other treatments to significantly delay disease progression. NK-CLK (AJ2), Al-Pro (AJ3) and CA/I-Pro (AJ4) are the three sets of formulations with distinct functions. Since the emphasis is on ALS, we delineated the differences in function of this probiotic formulation in comparison to the other two probiotics. AJ3 probiotic was formulated to augment anti-inflammatory cytokine to counter the aggressive nature of pro-inflammatory cytokine such as IFN-g which is primarily secreted by NK cells and T cells. ALS patients have significantly higher functions of NK and CD8+ T cells and they secrete large amounts of IFN-y upon activation. Indeed, the serum levels of IFN-y in patients is higher in comparison to the healthy controls, and even upon treatment with NAC which blocks most of the other pro-inflammatory cytokines secreted from the immune cells, it is not capable of decreasing IFN-y and TNF-a and IL- 17a. To counter this effect, we pursued the formulation of a probiotic bacteria which is capable of secreting very high levels of anti-inflammatory cytokine IL- 10 to counter the function of IFN-y. As can be observed from the data presented herein, significant increase in IL-10 secretion can be seen by sAJ3 probiotic bacteria when PBMCs, NK cells, and combination of NK cells with monocytes were tested and the results were compared to sAJ2 and sAJ4. It is of significance to note that dynamics of IFN-y, TNF-a, IL-6, and IL-10 secretion are quite different when PBMCs are triggered by sAJ3 and sAJ4 as shown in the present studies (Figs. 21 and 22). Even though there are significant secretion of TNF-a, IL- 6 and IL- 10 in the presence of both sAJ3 and sAJ4, IFN-y secretion mainly occurs when cells are activated with IL-2 or other activators such as anti-CD16 mAbs (Figs. 21 and 22). Indeed, IL-2+anti-CD16 mAbs activated PBMCs and NK cells resulted in a much greater increase in IFN-y/IL-10 ratio, indicating that NK cells when receive activation signals through CD 16 receptor significantly augment the levels of IFN-y in the presence of no or low levels of IL-10 induction (Figs. 21G, 22H and 221). sAJ4, unlike sAJ3, synergistically augment IFN-y in the presence of decreased induction of IL- 10. However, sAJ3 acts on the opposite, increasing IL-10 in the presence of less IFN-y, a scenario which is desirable in auto-immune diseases such as ALS. Of interest, is the observation that T cells when activated through IL-2 and anti-CD3/CD28 mAbs secrete higher IL-10 and the ratio of IFN- y to IL- 10 is much lower than that observed when NK cells are activated through IL-2 and anti-CD16 mAbs. However, when T cells are triggered by the sAJ2, sAJ3 and sAJ4 they secrete relatively much less IFN-y in comparison to NK cells which significant levels of IFN-y is released, and the levels exceed those triggered by IL-2+anti-CD16 mAbs. Whereas, the levels of IL-2 and probiotic in CD8+ T cells is much less than trigger by IL- 2+anti-CD3/CD28 mAbs.

The observation seen in the presence of sAJ3 and sAJ4 on NK cells and synergy with IL-2 and IL-2+anti-CD16 mAbs is surprising and unexpected, and has significant implications for the role of probiotic bacteria in prevention and treatment of disease. If there are no local activation (by e.g., infection, disease) which may not trigger secretion of IL-2 or cross linking of important receptors on immune cells such as NK cells, the default of the effect is increase in anti-inflammatory cytokines, such as those seen in the presence of activation of PBMCs and NK cells by the bacteria alone. Higher induction of IL- 10 in this respect with bacteria should provide an anti-inflammatory environment over pro- inflammatory environment, since increases in IL-10 and IL-6 is evident in the presence of no or very slight induction of IFN-y secretion. The secretion of TNF-a has a similar trend to IFN-y in which there is higher induction of TNF-a in the presence of sAJ4 and lower in the presence of sAJ3. Whereas the release of IL-6 and IL- 10 is much higher by sAJ3 than sAJ4. On the other hand, where there may be local activation of immune cells and secretion of IL- 2, probiotic bacteria will sway the pendulum towards a pro-inflammatory microenvironment where IFN-y will be highly increased. Such finetuning of the environment is elegant and effective since such environment will not only be localized to where the significant aid is needed, but also will ensure that the individual will mount an effective immunity in areas where it is needed.

In conclusion presented herein are three different formulations of probiotic bacteria with different effect on the activation of PBMCs, NK, and CD8+ T cells. AJ3 is effective in increasing IL- 10 and regulating the levels and function of IFN-y, whereas AJ4 triggers higher levels of IFN-y without the increase in IL-10 and therefore, this probiotic will be effective in the treatment of cancer and infections where the increased levels and functions of IFN-y is required for differentiation of tumor cells and prevention of the replication of virus, respectively. On the other hand, AJ3 will be effective in alleviating auto-immunity, in particular in ALS since it will greatly regulate the levels and function of IFN-y, decreasing over activation and death of motor neurons (Fig. 25). AJ2 on the other hand can be used in healthy individuals as a maintenance since it produces IFN-y in the presence of intermediate levels of IL-10, to regulate the IFN-y, and therefore, a balance between the secretion of IFN-y and IL-10 is established.

Table 1 Quantification of induction and secretion of IFN-y and bone morphology shown in FIG. 16.

Table 2 Histological analysis of the AJ2 sample exhibited increased bone formation when compared to the Control and MP2 samples. Table 3 Decreased BV/TV in OSCSC implanted BLT mice and its restoration by either feeding AJ2 or injection with super-charged NK cells and/or sonicated osteoclasts.

Table 4 Decreased bone in MP2 poorly differentiated pancreatic tumor implanted BLT mice and its restoration by either feeding AJ2 and/or injection with super-charged NK cells. Table 5 Decreased BV/TV in MP2 poorly differentiated pancreatic tumor implanted BLT mice and its restoration by either feeding AJ2 and/or injection with super-charged NK cells.

Table 6 Increased IFN-y by sAJ4 treated PBMCs in comparison to sAJ3 and sAJ2 treated PBMCs.

PBMCs were isolated from healthy individuals’ peripheral blood as described in Materials and Methods section in Example 4. PBMCs were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD3/28 antibody (25 pl/ml) or with a combination of IL-2 (1000 U/ml) and sAJ2 (PBMC:sAJ2, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (PBMC:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (PBMC:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y secretion using multiplex assay.

Tables 7A-7B Increased IFN-y and decreased IL- 10 secretions by sAJ4 treated PBMCs in comparison to sAJ3 and sAJ2 treated PBMCs.

Table 7A PBMCs were isolated from healthy individuals’ peripheral blood as described in Materials and Methods section in Example 4. PBMCs were left untreated or treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD3/28 antibody (25 pl/ml) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL- 10 secretion using specific single ELIS As, and ratio of IFN-y to IL- 10 was determined (Table 7A).

Table 7B

PBMCs were left untreated or treated with IL-2 (1000 U/ml) or with sAJ3 (PBMC:sAJ3, 1 :20) or with sAJ4 (PBMC:sAJ4, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (PBMC:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (PBMC:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL- 10 secretion using multiplex assay, and ratio of IFN-y to IL- 10 was determined (Table 7B).

Tables 8A-8B Increased IFN-y and decreased IL- 10 secretions by sAJ4 treated NK cells in comparison to sAJ3 and sAJ2 treated NK cells.

Table 8A

NK cells were isolated from healthy individuals’ PBMCs as described in Materials and Methods section in Example 4. NK cells were treated with IL-2 (1000 U/ml) or with a combination of IL-2 (1000 U/ml) and anti-CD16 mAbs (3 pg/ml) or with a combination of IL-2 (1000 U/ml) and sAJ3 (NK:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (NK:sAJ4, 1 :20) 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL- 10 secretion using specific single ELIS As, and ratio of IFN-y to IL- 10 was determined (Table 8 A).

Table 8B

NK cells were left untreated or treated with IL-2 (1000 U/ml) or with sAJ3 (NK:sAJ3, 1 :20) or with sAJ4 (NK:sAJ4, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ3 (NK:sAJ3, 1 :20) or with a combination of IL-2 (1000 U/ml) and sAJ4 (NK:sAJ4, 1 :20) for 18 hours before the supernatants were harvested from PBMCs to determine IFN-y and IL- 10 secretion using multiplex assay, and ratio of IFN-y to IL- 10 was determined (Table 8B).

Example 9: Exemplary formulations of bacterial compositions

AJ3

The AJ3 bacterial composition comprising Bifidobacterium Longum, Bifidobacterium breve, and Bifidobacterium infantis was effective in inducing secretion of a high level of IL- 10 and G-CSF that are useful for downregulating inflammation in patients afflicted with autoimmune diseases. It is notable that the AJ3 bacterial compositions comprising either intact or sonicated bacteria were equally effective. The effective AJ3 composition comprised: (i) about 40-60% of Bifidobacterium Longum, (ii) about 5-20% of Bifidobacterium breve, and (iii) about 30-50% of Bifidobacterium infantis. An exemplary formulation for AJ3 is shown in Table 9.

Table 9. Exemplary formulation of AJ3 AJ4

The AJ4 bacterial composition comprising Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus paracasei was effective in inducing secretion of IFN-g and MCP-1, proinflammatory cytokines that increase the immune response to cancer. It is notable that the AJ4 bacterial compositions comprising either intact or sonicated bacteria were equally effective. The effective AJ4 composition comprised: (i) about 20-40% of Streptococcus thermophiles, (ii) about 10-30% of Lactobacillus acidophilus, (iii) about 30-50% Lactobacillus plantarum, and (iv) about 5- 20% Lactobacillus paracasei. An exemplary formulation for AJ4 is shown in Table 10.

Table 10. Exemplary formulation of AJ4

References

1. Bradley, J.P., et al., Antley-Bixler syndrome: correction of facial deformities and longterm survival. Plast Reconstr Surg, 2003. 111(4): p. 1454-60.

2. Med, T.H.J.M., Mechanistic insight into osteoclast differentiation in osteoim-munology. 2005;83: 170-9., J Mol Med.

3. Chambers, T.J., et al., The effects of calcium regulating hormones on bone resorption by isolated human osteoclastoma cells., in J Pathol. 1985. p. 145(4): p. 297-305.

4. Suda, T., N. Takahashi, and T.J. Martin, , Modulation of osteoclast differentiation. , in Endocr Rev. 1992. p. 13(1): p. 66-80.

5. Karsenty, G.a.E.F.W., Reaching a genetic and molecular understanding of skeletal development. , in Dev Cell. 2002. p. 2(4): p. 389-406.

6. Bai, S., et al., NOTCH1 regulates osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblast lineage cells., in J Biol Chem. 2008. p. 283(10): p. 6509-18. 7. Pernow, Y., et al., Osteoblast dysfunction in male idiopathic osteoporosis., in Calcif Tissue Int. 2006. p. 78(2): p. 90-7.

8. Arai, F., et al., Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors. , in J Exp Med. 1999. p. 190(12): p. 1741-54.

9. Hattersley, G., et al., Macrophage colony stimulating factor (M-CSF) is essential for osteoclast formation in vitro., in Biochem Biophys Res Commun. 1991. p. 177(1): p. 526- 31.

10. Kong, Y.Y., W.J. Boyle, and J.M. Penninger, Osteoprotegerin ligand: a common link between osteoclastogenesis, lymph node formation and lymphocyte development. , in Immunol Cell Biol. 1999. p. 77(2): p. 188-93.

11. Nakagawa, N., et al., RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis, in Biochem Biophys Res Commun. 1998. p. 253(2): p. 395-400.

12. Li, J., et al., RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. , in Proc Natl AcadSci USA. 2000. p. 97(4): p. 1566-71.

13. Simonet, W.S., et al., Osteoprotegerin: a novel secreted protein involved in the regulation of bone density., in Cell. 1997. p. 89(2): p. 309-19.

14. Burgess, T.L., et al., The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. , in J Cell Biol. 1999. p. 145(3): p. 527-38.

15. Boyle, W.J., W.S. Simonet, and D.L. Lacey, , Osteoclast differentiation and activation. , in Nature. 2003. p. 423(6937): p. 337-42.

16. Ruggeri, L.C., M.; Urbani, E.; Perruccio, K.; Shlomchik, W.D.; Tosti, A.; Posati, S.; Rogaia, D.; Frassoni, F.; Aversa, F.; et al. , Effectiveness of Donor Natural Killer Cell Allor eactivity in Mismatched Hematopoietic Transplants. Science 2002, 295, 2097- -2100.

17. Suda, T., et al., Regulation of osteoclast function., in J Bone Miner Res. 1997. p. 12(6): p. 869- 79.

18. Takayanagi, H., J, Mechanistic insight into osteoclast differentiation in osteoimmunology. Mol Med (Berl). 2005. p. 83(3): p. 170-9.

19. Zupan, J., M. Jeras, and J. Marc, Osteoimmunology and the influence of pro- inflammatory cytokines on osteoclasts. , in Biochem Med (Zagreb). 2013. p. 23(1): p. 43-63. 20. Popoff, S.N., et al., Coexistence of reduced function of natural killer cells and osteoclasts in two distinct osteopetrotic mutations in the rat. , in J Bone Miner Res. 1991. p. 6(3): p. 263-71.

21. Takayanagi, H., et al., T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma. , in nature. 2000. p. 408(6812): p. 600-5.

22. Gao, Y., et al., IFN-gamma stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation., in The Journal of clinical investigation. 2007. p. 117(1): p. 122-32.

23. Cenci, S., et al., Estrogen deficiency induces bone loss by increasing T cell proliferation and lifespan through IFN-gamma-induced class II transactivator., Proceedings of the National Academy of Sciences of the United States of America. 2003. p. 100(18): p. 10405- 10.

24. Hu, M., et al, Activated invariant NKT cells regulate osteoclast development and function., in J Immunol. 2011. p. 186(5): p. 2910-7.

25. Delves, P.J.a.I.M.R., The immune system. Second of two parts. . 2000, N Engl J Med. p. 343(2): p. 108-17.

26. Hayashi, M., et al., Ly49Q, an ITIM-bearing NK receptor, positively regulates osteoclast differentiation. , in Biochem Biophys Res Commun. 2010. p. 393(3): p. 432-8.

27. Kong YY, Y.H., Sarosi I, et al., OPGL is a key regulator of osteoclast-ogenesis, lymphocyte development and lymph-node organogenesis . 1999; 397:315-23., Nature.

28. Abraham AK, R.M., Weinstock-Guttman B, et al. , Mecha-nisms of interferon-beta effects on bone homeostasis. Biochem Pharmaco2009;77: 1757-62.

29. Gough DJ, L.D., Johnstone RW, et al. , IFNgamma signaling-does it meanJAK-STAT? Cytokine Growth Factor Rev 2008;19:383-94.

30. Reinhardt RL, L.H., Bao K, et al. , A novel model for IFN- gammamediatedautoinflammatory syndromes. J Immunol 2015; 194:2358-68.

31. Mermut S, B. A., Akin E, et al. , Effects of interferon-gamma on bone remod-eling during experimental tooth movement. Angle Orthod 2007; 77: 135-41.

32. Xu Z, H.M., Deng H, et al. , Interferon-gamma targets cancer cells andosteoclasts to prevent tumor-associated bone loss and bone metastases. J BiolChem 2009;284:4658-66.

33. 2005;40:287-93., T.H.I.b.d.a.o.J.P.-o.R., Inflammatory bone destruction and osteoimmunology. J Peri-odontal Res 2005;40:287-93.

34. Li H, L.Y., Qian J, et al. , Human osteoclasts are inducible immunosuppressivecells in response to T cell-derived IFN-gamma and CD 40 ligand in vitro. J BoneMiner Res 2014;29:2666-75.

35. Ji ID, P.-M.K., Shen Z, et al. , Inhibition of RANK expression and osteoclast-ogenesis by TLRs and IFN-gamma in human osteoclast precursors. J Immunol2009; 183:7223-33.

36. Duque G, H.D., Macoritto M et al. , Autocrine regulation of interferon gamma in mesenchymal stem cells plays a role in early osteoblastogenesis. Stem Cells 2009;27:550- 558.

37. Duque G, H.D., Dion N et al. , Interferon g plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice. J Bone Miner Res 2011 26:1472-1483.

38. FP., R., Osteoclast biology and bone resorption. In: Rosen CJ, Compston JE, Lian JB, eds. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 7 th ed. Washington: American Society for Bone and Mineral Research, 2008:16-22.

39. Ferrari Lacraz S, F.S., Is IFN-c involved in bone loss or protection? Nothing is simple with cytokines. Bonekey Osteovision 2007;4:83-87.

40. H., T., Osteoimmunology and the effects of the immune system on bone. Nat Rev Rheumatol 2009;5:667-76.

41. Key LL Jr., R.R., Willi SM et al. , Long-term treatment of osteopetrosis with recombinant human interferon gamma. N Engl J Med 1995;332:1594-1599.

42. Takayanagi H, S.K., Takaoka A et al. , Interplay between interferon and other cytokine systems in bone metabolism. Immunol Rev 2005; 208: 181-193.

43. Gao Y, G.F., Ryan MR et al., IFN-gamma stimulates osteoclast formation and bone loss in vivo via antigen-driven T cell activation. J Clin Invest 2007;117:122-132.

44. Ayon Haro ER, U.T., Yokoyama M et al. , Locally administered interferon-c accelerates lipopolysaccharide-induced osteoclastogenesis independent of immunohistological RANKL upregulation. J Periodontal Res 2011;46:361-373.

45. Farag, S.S.a.M.A.C., Human natural killer cell development and biology., in Blood Reviews. 2003. p. 20(3): p. 123-137.

46. board, I.-E., Publication Info. , in Molecular Immunology. 2005. p. 42(4): p. IFC.

47. Moretta, A., et al., NK cells at the interface between innate and adaptive immunity. , in Cell Death Differ . 2008. p. 15(2): p. 226-33.

48. Vivier, E., et al., Innate or adaptive immunity? The example of natural killer cells., in Science. 2011. p. 331(6013): p. 44-9.

49. Jewett, A., Y.G. Man, and H.C. Tseng, Dual functions of natural killer cells in selection and differentiation of stem cells; role in regulation of inflammation and regeneration of tissues. , in J Cancer. 2013. p. 4(1): p. 12-24.

50. Colucci, F., M. A. Caligiuri, and J.P. Di Santo, What does it take to make a natural killer?, in Nat Rev Immunol. 2003. p. 3(5): p. 413-25.

51. Cooper, M.A., T.A. Fehniger, and M.A. Caligiuri, The biology of human natural killercell subsets., in Trends Immunol . 2001. p. 22(11): p. 633-40.

52. Sun, H., et al., NK cells in immunotolerant organs., in Cell Mol Immunol. 2013. p. 10(3): p. 202- 212.

53. Palmer, J.M., et al., Clinical relevance of natural killer cells following hematopoietic stem cell transplantation., in J Cancer . 2013. p. 4(1): p. 25-35.

54. Fildes, J.E., N. Yonan, and C.T. Leonard, , Natural killer cells and lung transplantation, roles in rejection, infection, and tolerance., in Transpl Immunol. 2008. p. 19(1): p. 1-11.

55. Farag, S.S.a.M.A.C., Human natural killer cell development and biology., in Blood Rev. 2006. p. 20(3): p. 123-37.

56. Trinchieri, Biology of natural killer cells., in G., Adv Immunol. 1989. p. 47: p. 187-376.

57. Lanier, L.L., NK cell recognition., in Annu Rev Immunol . 2005. p. 23: p. 225-74.

58. Tseng, H.C., et al., Increased lysis of stem cells but not their differentiated cells by natural killer cells; de-differentiation or reprogramming activates NK cells. , in PLoS One. 2010. p. 5(7): p. el 1590.

59. Soderstrom, K., et al., Natural killer cells trigger osteoclastogenesis and bone destruction in arthritis. , in Proceedings of the National Academy of Sciences of the United States of America. 2010. p. 107(29): p. 13028-33.

60. Aparicio-Pages, M.N.V., H.W.; Pena, A.S.; Larners, C.B. , Natural killer cell activity in patients with adenocarcinoma in the upper gastrointestinal tract. J. Clin. Lab. Immunol. 1991, 35, 27-32.

61. Duan, X.D., L.; Chen, X.; Lu, Y.; Zhang, Q.; Zhang, K.; Hu, Y .; Zeng, J.; Sun, W. , Clinical significance of the immunostimulatory MHC class I chain-related molecule A and NKG2D receptor on NK cells in pancreatic cancer. Med. Oncol. 2011, 28, 466-474.

62. Peng, Y.P.Z., Y.; Zhang, J.J.; Xu, Z.K.; Qian, Z.Y.; Dai, C.C.; Jiang, K.R.; Wu, J.L.; Gao, W.T.; Li, Q.; et al., Comprehensive analysis of the percentage of surface receptors and cytotoxic granules positive natural killer cells in patients with pancreatic cancer, gastric cancer, and colorectal cancer. J. Transl. Med. 2013, 11, 262.

63. Kaur, K.C., J.; Park, S.H.; Topchyan, P.; Kozlowska, A.; Ohanian, N.; Fang, C.; Nishimura, I.; Jewett, A., Novel Strategy to Expand Super-Charged NK Cells with Significant Potential to Lyse and Differentiate Cancer Stem Cells: Differences in NK Expansion and Function between Healthy and Cancer Patients. Front. Immunol. 2017, 8, 297.

64. Kaur, K.C., H.H.; Cook, J.; Eibl, G.; Jewett, A. , Suppression of Gingival NK Cells in Precancerous and Cancerous Stages of Pancreatic Cancer in KC and BLT-Humanized Mice. Front. Immunol. 2017, 8, 1606.

65. Kaur, K.C., H.H.; Topchyan, P.; Cook, J.M.; Barkhordarian, A.; Eibl, G.; Jewett, A. , Deficiencies in Natural Killer Cell Numbers, Expansion, and Function at the PreNeoplastic Stage of Pancreatic Cancer by KRAS Mutation in the Pancreas of Obese Mice. Front. Immunol. 2018, 9, 1229.

66. Venstrom, J.M.P., G.; Gooley, T.A.; Chewning, J.; Spellman, S.R.; Haagenson, M.D.; Gallagher, M.M.; and M.P. Malkki, E.W.; Dupont, B.; et al. , HLA-C-dependent prevention of leukemia relapse by donor activating KIR2DS1. N. Engl. J. Med. 2012, 367, 805 816.

67. Iliopoulou, E.G.K., P.; Karamouzis, M.V.; Doufexis, D.; Ardavanis, A.; Baxevanis, C.N.; and G.P. Rigatos, M.; Perez, S.A. , A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer. Cancer Immunol. Immunother. 2010, 59, 1781-1789.

68. Miller, J.S.S., Y.; Panoskaltsis-Mortari, A.; McNearney, S.A.; Yun, G.H.; Fautsch, S.K.; McKenna, D.; and C.D. Le, T.E.; Bums, L.J.; et al. , Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005, 105, 3051-3057.

69. Re, F.S., C.; Zamai, L.; Vecchio, V.; Bregni, M. , Killer cell Ig-like receptors ligandmismatched, alloreactive natural killer cells lyse primary solid tumors. Cancer 2006, 107, 640-648.

70. Geller, M.A.C., S.; Judson, P.L.; Ghebre, R.; Carson, L.F.; Argenta, P.A.; Jonson, A.L.; Panoskaltsis- and A.C. Mortari, J.; McKenna, D.; et al. , A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer. Cytotherapy 2011, 13, 98-107.

71. Bui, V.T.T., H.C.; Kozlowska, A.; Maung, P.O.; Kaur, K.; Topchyan, P.; Jewett, A. , Augmented IFNgamma and TNF-alpha Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL- 10. Front. Immunol. 2015, 6, 576. 72. Ryschich, E.N., T.; Hinz, U.; Autschbach, F.; Ferguson, J.; Simon, I.; Weitz, J.;

Frohlich,

B.; Klar, E.; Buehler, M.W.; et al. , Control of T-C ell-mediated immune response by HLA class I in human pancreatic carcinoma. Clin. Cancer Res. 2005, 11, 498- -504.

73. al., K.K.e., Probiotic-Treated Super-Charged NK Cells Efficiently Clear Poorly Differentiated Pancreatic Tumors in Hu-BLT Mice.

74. Pandha, H.R., A.; John, J.; Lemoine, N. , Loss of expression of antigen-presenting molecules in human pancreatic cancer and pancreatic cancer cell lines. Clin. Exp. Immunol. 2007, 148, 127-135.

75. Wu, Y., Tian, Z., & Wei, H. , Developmental and Functional Control of Natural Killer Cells by Cytokines. Frontiers in Immunology. 2017 ;8: 930.

76. Floros, T., & Tarhini, A. A. , Anticancer Cytokines: Biology and Clinical Effects of IFN-a2, IL-2, IL-15, IL-21, and IL-12. Seminars in Oncology. 2015; 42(4): 539-548.

77. Kaur, K., Nanut, M. P., Ko, M., Safaie, T., Kos, J., & Jewett, , A. Natural killer cells target and differentiate cancer stem-like cells/undifferentiated tumors: Strategies to optimize their growth and expansion for effective cancer immunotherapy. Current Opinion in Immunology. 2018.51: 170-180.

78. Beldi-Ferchiou, A., & Caillat-Zucman, S. , Control ofNK Cell Activation by Immune Checkpoint Molecules. International Journal of Molecular Sciences. 2017; 18(10): 2129.

79. Lapteva, N., Duret, A. G., Sub, J., Rollins, L. A., Huye, L. L., Fang, J. Rooney, C. M. , Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications. Cytotherapy. 2012; 14(9): 1131 1143.

80. Deng, X., Terunuma, H., Nieda, M., Xiao, W ., & Nicol, , A. Synergistic cytotoxicity of ex vivo expanded natural killer cells in combination with monoclonal antibody drugs against cancer cells. International Immunopharmacology. 2012; 14(4): 593-605.

81. Romee, R., Rosario, M., Berrien-Elliott, M. M., Wagner, J. A., Jewell, B. A., Schappe, T., Fehniger, T. A. , Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia. Science Translational Medicine. 2016;8(357): 357.

82. Parkhurst, M.R., Riley, J. P., Dudley, M. E., & Rosenberg, S. A. , Adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression. Clinical Cancer Research : An Official Journal of the American Association for Cancer Research. 2011; 17(19): 6287-6297. 83. E. Ishikawa, K.T., K. Saijo, H. Harada, S. Takano, T. Nose, T. Ohno. , Autologous natural killer cell therapy for human recurrent malignant glioma Anticancer Res. 2004; 24(3BO: 1861-1871.

84. Woll, P.S., Grzywacz, B., Tian, X., Marcus, R. K., Knorr, D. A., Verneris, M. R., & Kaufman, D. S. , Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity. Blood. 2009; 113(24): 6094 6101.

85. Hermanson, D.L., Bendzick, L., Pribyl, L., McCullar, V., Vogel, R. I., Miller, J. S., Kaufman, D. S. , Induced pluripotent stem cell-derived natural killer cells for treatment of ovarian cancer. Stem Cells (Dayton, Ohio). 2016; 34(1): 93 101.

86. Li, Y ., Hermanson, D. L., Moriarity, B. S., & Kaufman, D. S. (2018). , Human iPSCDerived Natural Killer Cells Engineered with Chimeric Antigen Receptors Enhance Antitumor Activity. Cell Stem Cell. 201823(2).

87. Shah, N., Martin-Antonio, B., Yang, H., Ku, S., Lee, D. A., Cooper, L. J. N., Shpall, E. J., Antigen Presenting Cell-Mediated Expansion of Human Umbilical Cord Blood Yields Log-Scale Expansion of Natural Killer Cells with Anti-Myeloma Activity. PLoS ONE.

2013; 8(10): e76781.

88. Ayello, J., Hochberg, J., Flower, A., Chu, Y., Baxi, L. V., Quish, W ., Cairo, M. S. , Genetically re-engineered K562 cells significantly expand and functionally activate cord blood natural killer cells: Potential for adoptive cellular immunotherapy. Experimental Hematology. 2017; 46: 38-47.

89. Veluchamy, J.P., Heeren, A. M., Spanholtz, J., van Eendenburg, J. D. H., Heideman, D. A. M., Kenter, G. G., de Gruijl, T. D., High-efficiency lysis of cervical cancer by allogeneic NK cells derived from umbilical cord progenitors is independent ofHLA status. Cancer Immunology, Immunotherapy. 2017: 66(1), 51 61.

90. Li, L., Liu, L. N., Feller, S., Allen, C., Shivakumar, R., Fratantoni, J., Peshwa, M., Expression of chimeric antigen receptors in natural killer cells with a regulatorycompliant non-viral method. Cancer Gene Therapy. 2010; 17(3): 147.

91. Chu, Y., Hochberg, J., Yahr, A., Ayello, J., Ven, C. V., Barth, M., Cairo, M. S.

Targeting CD20 Aggressive B-cell Non-Hodgkin Lymphoma by Anti-CD20 CAR mRNA- Modified Expanded Natural Killer Cells In Vitro and in NSG Mice. Cancer Immunology Research. 2014; 3(4): 333-344.

92. Kruschinski, A., Moosmann, A., Poschke, I., Norell, H., Chmielewski, M., Seliger, B., Charo, J. , Engineering antigen-specific primary human NK cells against HER-2 positive carcinomas. Proceedings of the National Academy of Sciences of the United States of America. 2008; 105(45): 17481-17486.

93. Akdis M, B.S., Crameri R et al., Interleukins, from 1 to 37, and interfer on- [gamma] : Receptors, functions, and roles in diseases. J Allergy Clin Immunol 2011; 127:701-72 Lei e70. 94. H., T., Osteoimmunology and the effects of the immune system on bone. Nat Rev Rheumatol 2009;5:667-76.

95. Ferrari Lacraz S, F.S.I., Is IFN-c involved in bone loss or protection? Nothing is simple with cytokines. Bonekey Osteovision 2007;4:83-87.

96. Ayon Haro ER, U.T., Yokoyama M et al. , Locally administered interferon-c accelerates lipopolysaccharide-induced osteoclastogenesis independent of immunohistological RANKL upregulation. J Periodontal Res 2011;46:361-373.

97. WB., v.d.B., Anti-cytokine therapy in chronic destructive arthritis. Arthritis Res. 2001;3:18-26.

98. Bemiller LS, R.D., Starko KM, Curnutte JT., Safety and effectiveness of long-term interferon gamma therapy in patients with chronic granulomatous disease. Blood Cells Mol Dis. 1995;21:239-247.

99. Yang S, M.P., Ries W, et al. , Characterization of interferon gamma recep-tors on osteoclasts: effect of interferon gamma on osteoclastic superoxidegeneration. J Cell Biochem 2002;84:645-54.

100. Key LL Jr, R.R., Willi SM, et al. , Long-term treatment of osteopetrosis with recombinant human interferon gamma. N Engl J Med. 1995;332:1594-1599.

101. Grewal TS, G.P., Brabbs AC, Birch M, Skerry MT., Best5: a novel interferon- inducible gene expressed during bone formation. FASEB J. 2000;14:523-531.

102. Segal JG, L.N., Tsung YL, Norton JA, Tsung K., The role of IFN-gamma in rejection of established tumors by IL-12: source of production and target. Cancer Res. 2002 ; 62: 4696-4703.

103. Steinmuller C, F.-U.G., Lohmann-Matthes ML, Emmendorffer A., Local activation of nonspecific defense against a respiratory model infection by application of interferongamma: comparison between rat alveolar and interstitial lung macrophages. Am J Respir Cell Mol Biol. 2000; 22:481-490.

104. Kohara H, K.H., Fujimura Y, et al. , IFN-gamma directly inhibits TNF -alpha-induced osteoclastogenesis in vitro and in vivo and induces apoptosis mediatedby Fas/Fas ligand interactions. Immunol Lett 2011; 137:53 61.

105. De Klerck, B., Carpentier, I., Lories, R. J., Habraken, Y., Piette, J., Carmeliet, G., and R. Beyaert, Billiau, A., and Matthys, P. (2004) Arthritis Res Ther 6(3), R220-231, Arthritis Res Ther 6(3), R220-231.

106. Vermeire, K., Heremans, H., Vandeputte, M., Huang, S., Billiau, A., and Matthys, P., J Immunol 158(11), 5507-5513-1997.

107. Iliopoulou, E.G.K., P.; Karamouzis, M.V.; Doufexis, D.; Ardavanis, A.; Baxevanis, C.N.; and Z.X.e. al., INTERFERON -GAMMA TARGETS CANCER CELLSAND OSTEOCLASTS TO PREVENT TUMOR ASSOCIATED BONE LOSS AND BONE METASTASES. The American Society for Biochemistry and Molecular Biology. 2008.

108. Dunn, G.P., Sheehan, K. C., Old, L. J., and Schreiber, R. D. , Cancer Res 65(8), 3447-3453. 2005.

109. Lee, K.Y., Geng, H., Ng, K. M., Yu, J., van Hasselt, A., Cao, Y., Zeng, Y. X., Wong, A., W. H., X., Ying, J., Srivastava, G., Lung, M. L., Wang, L. D., Kwok, T. T., Levi, B., and C. Z., A. T., Sung, J. J., and Tao, Q. , Oncogene 27(39), 5267-5276. 2008.

110. Kaplan, D.H., Shankaran, V., Dighe, A. S., Stockert, E., Aguet, M., Old, L. J., and and R.D. Schreiber, Proc Natl Acad Sci USA 95(13), 7556-7561.1998.

111. Fox, S.W., and Chambers, T. J. , Biochem Biophys Res Commun 276(3), 868-872. 2000.

112. De Klerck, B., Carpentier, I., Lories, R. J., Habraken, Y., Piette, J., Carmeliet, G., and R. Beyaert, Billiau, A., and Matthys, P. , Arthritis Res Ther 6(3), R220-23. 2004.

113. Chu, C.Q., Song, Z., Mayton, L., Wu, B., and Wooley, P. H. , Ann Rheum Dis 62(10), 983-990. 2003.

114. Vermeire, K., Heremans, H., Vandeputte, M., Huang, S., Billiau, A., and Matthys, P., J Immunol 158(11), 5507-5513. 1997.

115. Toh, M.L., and Miossec, P. , Curr Opin Rheumatol 19(3), 284-288. 2007.

116. Zhu, L., Ji, F., Wang, Y., Zhang, Y., Liu, Q., Zhang, J. Z., Matsushima, K., Cao, Q., and and Y. Zhang, J Immunol 177(11), 8226-8233. 2006.

117. Wan, B., Nie, H., Liu, A., Feng, G., He, D., Xu, R., Zhang, Q., Dong, C., and Zhang, J. Z., J Immunol 177(12), 8844-8850. 2006.

118. Cochran, A.J., Wen, D. R., Farzad, Z., Stene, M. A., McBride, W., Lana, A. M., Hoon, and a.M. D. S., D. L. , Anticancer Res 9(4), 859-864.1989.

119. Kim, R., Emi, M., and Tanabe, K. , Immunology 121(1), 1-14. 2007. 120. Merchant, M.S., Melchionda, F., Sinha, M., Khanna, C., Helman, L., and Mackall, C. L., Cancer Immunol Immunother 56(7), 1037-1046. 2007.

121. Xaus, J., Cardo, M., Valledor, A. F., Soler, C., Lloberas, J., and Celada, A. , Immunity 11(1), 103-113. 1999.

122. Cornish J, G.M., Callon KE, Horwood NJ, Moseley JM, Reid IR. , Interleukin- 18 is a novel mitogen of osteogenic and chondrogenic cells. Endocrinology. 2003; 144: 1194 1201.

123. Koide M, M.H., Roccisana JL, Kawanabe N, Reddy SV., Cytokine regulation and the signaling mechanism of osteoclast inhibitory peptide- 1 (OIP-1/hSca) to inhibit osteoclast formation. J Bone Miner Res. 2003; 18:458 465.

124. Mogi M, K.K., Kondo A, Togari A. , Involvement of nitric oxide and biopterin in proinflammatory cytokine-induced apoptotic cell death in mouse osteoblastic cell line MC3T3-E1. Biochem Pharmacol. 1999; 58: 649 654.

125. Ozeki N, M.M., Nakamura H, Togari A. , Differential expression of the Fas-Fas ligand system on cytokine-induced apoptotic cell death in mouse osteoblastic cells. Arch Oral Biol. 2002;47:511-517.

126. Aguirre J, B.L., O’Shaughnessy M, et al. , Endothelial nitric oxide synthase genedeficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity. Am J Pathol. 2001;158:247-257.

127. Armour KE, A.K., Gallagher ME, et al. , Defective bone formation and anabolic response to exogenous estrogen in mice with targeted disruption of endothelial nitric oxide synthase. Endocrinology. 2001;142:760-766.

128. Ralston SH, T.D., Helfrich M, Benjamin N, Grabowski PS. , Human osteoblast-like cells produce nitric oxide and express inducible nitric oxide synthase. Endocrinology. 1994;135:330-336.

129. Van’t Hof RJ, R.S., Nitric oxide and bone. Immunology. 2001; 103:255-261.

Additional references

1. Vivi er E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, et al. Innate or adaptive immunity? The example of natural killer cells. Science (New York, NY).

2011;331(6013):44-9.

2. Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killer-cell subsets. Trends in immunology. 2001;22(l l):633-40.

3. Sun JC, Lanier LL. Is There Natural Killer Cell Memory and Can It Be Harnessed by Vaccination? NK Cell Memory and Immunization Strategies against Infectious Diseases and Cancer. Cold Spring Harb Perspect Biol. 2018;10(10).

4. Shaw SY, Tran K, Castoreno AB, Peloquin JM, Lassen KG, Khor B, et al. Selective modulation of autophagy, innate immunity, and adaptive immunity by small molecules. ACS chemical biology. 2013;8(12):2724-33.

5. Pant H, Hughes A, Miljkovic D, Schembri M, Wormaid P, Macardle P, et al. Accumulation of effector memory CD8+ T cells in nasal polyps. American journal of rhinology & allergy. 2013;27(5):el 17-26.

6. Tomala J, Chmelova H, Mrkvan T, Rihova B, Kovar M. In vivo expansion of activated naive CD8+ T cells and NK cells driven by complexes of IL-2 and anti-IL-2 monoclonal antibody as novel approach of cancer immunotherapy. J Immunol. 2009;183(8):4904-12.

7. Tanaka J, Toubai T, Miura Y, Tsutsumi Y, Kato N, Umehara S, et al. Differential expression of natural killer cell receptors (CD94/NKG2A) on T cells by the stimulation of G-CSF-mobilized peripheral blood mononuclear cells with anti-CD3 monoclonal antibody and cytokines: a study in stem cell donors. Transplantation proceedings. 2004;36(8):2511- 2.

8. Burke S, Lakshmikanth T, Colucci F, Carbone E. New views on natural killer cellbased immunotherapy for melanoma treatment. Trends in immunology. 2010;31(9):339-45.

9. Larsen SK, Gao Y, Basse PH. NK cells in the tumor microenvironment. Critical reviews in oncogenesis. 2014; 19(l-2):91-105.

10. Nolibe D, Poupon MF. Enhancement of pulmonary metastases induced by decreased lung natural killer cell activity. Journal of the National Cancer Institute. 1986;77(l):99-103. 11. Imai K, Matsuyama S, Miyake S, Suga K, Nakachi K. Natural cytotoxic activity of peripheral -blood lymphocytes and cancer incidence: an 11 -year follow-up study of a general population. Lancet (London, England). 2000;356(9244): 1795-9.

12. Harning R, Koo GC, Szalay J. Regulation of the metastasis of murine ocular melanoma by natural killer cells. Investigative ophthalmology & visual science. 1989;30(9): 1909-15.

13. Coca S, Perez-Piqueras J, Martinez D, Colmenarejo A, Saez MA, Vallejo C, et al. The prognostic significance of intratumoral natural killer cells in patients with colorectal carcinoma. Cancer. 1997;79(12):2320-8.

14. Zanetti M. Tapping CD4 T cells for cancer immunotherapy: the choice of personalized genomics. J Immunol. 2015;194(5):2049-56.

15. Gerosa F, Baldani-Guerra B, Nisii C, Marchesini V, Carra G, Trinchieri G. Reciprocal activating interaction between natural killer cells and dendritic cells. J Exp Med. 2002;195(3):327-33.

16. Assarsson E, Kambayashi T, Schatzle JD, Cramer SO, von Bonin A, Jensen PE, et al. NK cells stimulate proliferation of T and NK cells through 2B4/CD48 interactions. J Immunol. 2004; 173(1): 174-80.

17. Zingoni A, Somasse T, Cocks BG, Tanaka Y, Santoni A, Lanier LL. Cross-talk between activated human NK cells and CD4+ T cells via 0X40-0X40 ligand interactions. J Immunol. 2004;173(6):3716-24.

18. Crouse J, Xu HC, Lang PA, Oxenius A. NK cells regulating T cell responses: mechanisms and outcome. Trends Immunol. 2015;36(l):49-58.

19. Crome SQ, Lang PA, Lang KS, Ohashi PS. Natural killer cells regulate diverse T cell responses. Trends Immunol. 2013;34(7):342-9.

20. Zingoni A, Ardolino M, Santoni A, Cerboni C. NKG2D and DNAM-1 activating receptors and their ligands in NK-T cell interactions: role in the NK cell-mediated negative regulation of T cell responses. Front Immunol. 2012;3:408.

21. Pallmer K, Oxenius A. Recognition and Regulation of T Cells by NK Cells. Front Immunol. 2016;7:251.

22. Nielsen N, Odum N, Urso B, Lanier LL, Spee P. Cytotoxicity of CD56(bright) NK cells towards autologous activated CD4+ T cells is mediated through NKG2D, LFA-1 and TRAIL and dampened via CD94/NKG2A. PLoS One. 2012;7(2):e31959-e. 23. Noval Rivas M, Hazzan M, Weatherly K, Gaudray F, Salmon I, Braun MY. NK cell regulation of CD4 T cell-mediated graft-versus-host disease. J Immunol.

2010;184(12):6790-8.

24. Seo AN, Lee HJ, Kim EJ, Kim HJ, Jang MH, Lee HE, et al. Tumour-infiltrating CD8+ lymphocytes as an independent predictive factor for pathological complete response to primary systemic therapy in breast cancer. Br J Cancer. 2013;109(10):2705-13.

25. Hadrup S, Donia M, Thor Straten P. Effector CD4 and CD8 T cells and their role in the tumor microenvironment. Cancer microenvironment : official journal of the International Cancer Microenvironment Society. 2013;6(2): 123-33.

26. Kim ST, Jeong H, Woo OH, Seo JH, Kim A, Lee ES, et al. Tumor-infiltrating lymphocytes, tumor characteristics, and recurrence in patients with early breast cancer. American journal of clinical oncology. 2013;36(3):224-31.

27. Robbins SH, Bessou G, Cornillon A, Zucchini N, Rupp B, Ruzsics Z, et al. Natural killer cells promote early CD8 T cell responses against cytomegalovirus. PLoS pathogens. 2007;3(8):el23.

28. Wodarz D, Sierro S, Klenerman P. Dynamics of killer T cell inflation in viral infections. Journal of the Royal Society, Interface. 2007;4(14):533-43.

29. Bui VT, Tseng HC, Kozlowska A, Maung PO, Kaur K, Topchyan P, et al. Augmented IFN-gamma and TNF-alpha Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL- 10. Frontiers in immunology. 2015;6:576.

30. Kaur K, Topchyan P, Kozlowska AK, Ohanian N, Chiang J, Maung PO, et al. Super-charged NK cells inhibit growth and progression of stem-like/poorly differentiated oral tumors in vivo in humanized BLT mice; effect on tumor differentiation and response to chemotherapeutic drugs. Oncoimmunology. 2018;7(5):el426518.

31. Kaur K, Cook J, Park SH, Topchyan P, Kozlowska A, Ohanian N, et al. Novel Strategy to Expand Super-Charged NK Cells with Significant Potential to Lyse and Differentiate Cancer Stem Cells: Differences in NK Expansion and Function between Healthy and Cancer Patients. Front Immunol. 2017;8:297.

32. Kabelitz D. Expression and function of Toll-like receptors in T lymphocytes. Curr Opin Immunol. 2007;19(l):39-45. 33. Nouri Y, Weinkove R, Perret R. T-cell intrinsic Toll-like receptor signaling: implications for cancer immunotherapy and CAR T-cells. Journal for ImmunoTherapy of Cancer. 2021;9(l l):e003065.

34. Reynolds JM, Pappu BP, Peng J, Martinez GJ, Zhang Y, Chung Y, et al. Toll-like Receptor 2 Signaling in CD4+ T Lymphocytes Promotes T Helper 17 Responses and Regulates the Pathogenesis of Autoimmune Disease. Immunity. 2010;32(5):692-702.

35. Lauzon NM, Mian F, MacKenzie R, Ashkar AA. The direct effects of Toll-like receptor ligands on human NK cell cytokine production and cytotoxicity. Cell Immunol. 2006;241(2): 102-12.

36. Adib-Conquy M, Scott-Algara D, Cavaillon JM, Souza-Fonseca-Guimaraes F. TLR- mediated activation of NK cells and their role in bacterial/viral immune responses in mammals. Immunol Cell Biol. 2014;92(3):256-62.

37. Chalifour A, Jeannin P, Gauchat JF, Blaecke A, Malissard M, N'Guyen T, et al. Direct bacterial protein PAMP recognition by human NK cells involves TLRs and triggers alpha-defensin production. Blood. 2004;104(6): 1778-83.

38. Pandey S, Kawai T, Akira S. Microbial sensing by Toll-like receptors and intracellular nucleic acid sensors. Cold Spring Harb Perspect Biol. 2014;7(l):a016246.

39. Vabulas RM, Ahmad-Nejad P, da Costa C, Miethke T, Kirschning CJ, Hacker H, et al. Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. J Biol Chem. 2001;276(33):31332-9.

40. Ohashi K, Burkart V, Flohe S, Kolb H. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol.

2000;164(2):558-61.

41. Okamura Y, Watari M, Jerud ES, Young DW, Ishizaka ST, Rose J, et al. The extra domain A of fibronectin activates Toll-like receptor 4. J Biol Chem. 2001;276(13): 10229- 33.

42. Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, et al. Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med. 2002;195(l):99-l 11.

43. Rahman AH, Taylor DK, Turka LA. The contribution of direct TLR signaling to T cell responses. Immunol Res. 2009;45(l):25-36. 44. Yeap WH, Wong KL, Shimasaki N, Teo ECY, Quek JKS, Yong HX, et al. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes. Scientific Reports. 2016;6(l):34310.

45. Bhatnagar N, Ahmad F, Hong HS, Eberhard J, Lu IN, Ballmaier M, et al. FcgammaRIII (CD16)-mediated ADCC by NK cells is regulated by monocytes and FcgammaRII (CD32). Eur J Immunol. 2014;44(l l):3368-79.

46. Oboshi W, Watanabe T, Matsuyama Y, Kobara A, Yukimasa N, Ueno I, et al. The influence of NK cell-mediated ADCC: Structure and expression of the CD 16 molecule differ among FcgammaRIIIa-V158F genotypes in healthy Japanese subjects. Hum Immunol. 2016;77(2): 165-71.

47. Nimmerjahn F, Ravetch JV. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol. 2008;8(l):34-47.

48. Kaur K, Chen PC, Ko MW, Mei A, Chovatiya N, Huerta-Yepez S, et al. The Potential Role of Cytotoxic Immune Effectors in Induction, Progression and Pathogenesis of Amyotrophic Lateral Sclerosis (ALS). Cells. 2022; 11(21).

49. Bui VT, Tseng HC, Kozlowska A, Maung PO, Kaur K, Topchyan P, et al. Augmented IFN-y and TNF-a Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL-10. Front Immunol. 2015;6:576.

50. Tseng HC, Arasteh A, Paranjpe A, Teruel A, Yang W, Behel A, et al. Increased lysis of stem cells but not their differentiated cells by natural killer cells; de-differentiation or reprogramming activates NK cells. PloS one. 2010;5(7):el 1590.

51. Tseng HC, Bui V, Man YG, Cacalano N, Jewett A. Induction of Split Anergy Conditions Natural Killer Cells to Promote Differentiation of Stem Cells through Cell-Cell Contact and Secreted Factors. Frontiers in immunology. 2014;5:269.

52. Tseng HC, Inagaki A, Bui VT, Cacalano N, Kasahara N, Man YG, et al. Differential Targeting of Stem Cells and Differentiated Glioblastomas by NK Cells. Journal of Cancer. 2015;6(9):866-76.

53. Bui VT, Tseng H-C, Maung PO, Kozlowska A, Mann K, Topchyan P, et al. Augmented IFN-y and TNF-a Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL- 10. Frontiers in immunology. 2015;6.

54. Jewett A, Bonavida B. Target-induced inactivation and cell death by apoptosis in a subset of human NK cells. J Immunol. 1996;156(3):907-15.

55. Jewett A, Wang MY, Teruel A, Poupak Z, Bostanian Z, Park NH. Cytokine dependent inverse regulation of CD54 (ICAM1) and major histocompatibility complex class I antigens by nuclear factor kappaB in HEp2 tumor cell line: effect on the function of natural killer cells. Hum Immunol. 2003;64(5):505-20.

Incorporation by Reference

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web at tigr.org and/or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.