MYELOID-DERIVED SUPPRESSOR CELL CLUSTER ANALYSIS IN VITRO

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.

62/581,438, filed on November 3, 2017, the disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under contract numbers

CA79765 and AI082039 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Human health depends on an orchestrated balance between T cell-based adaptive immunity and an immune-suppressive network. Preclinical and clinical studies have established that a subset of immunosuppressive cells termed rayeloid-rferived suppressor cells (MDSC) accumulate in abnormally large numbers within the bone marrow, blood, spleen, and tumors of cancer patients and contribute to immune escape by tumor cells. MDSC populations are also prevalent in a setting of severe infection, sepsis, trauma, and autoimmunity and MDSC-based cellular therapeutics are in development to dampen immune reactions directed against normal tissues post-transplantation and in autoimmune disorders. MDSC thwart the local T cell arm of the immune response through multiple mechanisms including chemical modification of the antigen-recognition receptor on T cells, production of immunosuppressive soluble factors, support of immunosuppressive regulatory T cells and monocytes, and expression of immune checkpoint molecules such as programmed cell-death ligand-1 (PD-L1). In a cancer setting, MDSC further drive tumor progression and resistance to chemotherapy and radiation therapy by promoting angiogenesis, survival of cancer stem cells, and metastasis Preclinical studies in mouse tumor models showing that depletion of MDSC rescues the T cell arm of antitumor immunity during checkpoint inhibitor immunotherapy serve as the basis for clinical trials that explore the merit of depleting MDSC prior to standard therapy or cancer immunotherapy. Success of MDSC-targeted therapies hinges on accurate and reliable measurement of functional immunosuppressive MDSC within patient biospecimens. Routine measurement of MDSC has proven problematic, however, since complex phenotypic analysis involving 6 or more surface markers are required to distinguish MDSC from normal polymorphonuclear cells (i.e., neutrophils) and myeloid cells, leading to inter-laboratory discrepancies about MDSC burdens in patients Moreover, MDSC burdens are often underestimated since these cells are unstable during routine specimen processing and storage of biospecimens while immunosuppression bioassays present technical difficulties for clinical laboratories. Thus, there is a need for novel approaches to characterize and quantify the immunosuppressive MDSC burden in patient biospecimens so that appropriate treatment regimens can be prescribed.

SUMMARY OF THE DISCLOSURE

[0004] The present disclosure is based on the identification of the presence of MDSC clusters in circulation, and demonstration of an immunosuppressive function associated with the presence of MDSC clusters in blood. The presence or level of MDSC clusters in blood can be determined in fresh blood samples by histologic analysis of blood smears or by microfiltration and analysis of the filtered out cells for presence of MDSC clusters. The presence or level of MDSC clusters in blood can also be determined by flow cytometry in blood samples that are processed after collection or after storage.

[0005] The presence of MDSCs in circulating blood is indicative of the immune status of an individual. For example, circulating MDSC clusters provide a readout of the immunosuppressive function of MDSC, e.g., suppression of T-cells. Based on their presence in the blood of an individual, therapeutic approaches can be identified, ongoing approaches can be modified or augmented in the treatment of cancers, and other immune related conditions.

[0006] In as aspect, this disclosure provides a method for identifying the presence of

MDSC clusters in circulating blood of an individual comprising obtaining a sample of blood from in individual, preparing a blood smear and performing histologic analysis (such as visualization under a microscope) to identify the presence of MDSC cell clusters, and/or quantifying the number of such clusters In an embodiment, the MDSC cell clusters may be separated by microfiltration techniques and then identified by detecting the presence of clusters in the separated cells by microscopic examination. In an embodiment, the MDSC cell clusters may be identified by flow cytometric analysis.

[0007] In an aspect, this disclosure provides a method for identifying T-cell suppression in an individual comprising identifying the formation of MDSC cluster formation in a blood sample obtained from an individual. The MDCS clusters may comprises at least two MDSCs, or at least one MDSC and one or more lymphocytes. The MDSCs may be homotypic PMN-MDCSs, homotypic M-MDCSs, heterotypic PMN-MDSC + M-MDSC, or heterotypic PMN- and/or M-MDSC + T-cell, B-cell, and/or NK cell. The presence of MDSCs in blood samples may be identified by visualization of fresh blood smear (histologic examination under a microscope), isolation and identification of cells using microfiltration techniques, which may be used in conjunction with staining for specific cell markers, or by flow cytometry of processed blood samples.

[0008] In an aspect, this disclosure provides a method for treatment of an individual who has been identified as having T-cell suppression (such as by the identification of MDSC clusters in blood) comprising administering to the individual MDSC-depleting treatment regimen.

[0009] In an aspect, this disclosure provides a method for treatment of an individual afflicted with cancer, comprising conducting analysis of a blood sample from the individual for the presence of MDSC clusters, and if the presence of such clusters is detected (or if the level of such clusters in higher than normal), then identifying that individual as not being suitable for immunotherapy without prior or concomitant reduction of circulating MDSC clusters. The individual can be treated with chemotherapy instead, or can be administered agents to reduce circulating MDSC cluster levels prior to, together with, or after initiation of immunotherapy.

BRIEF DESCRIPTION OF THE FIGURES

[0010] Figure 1. L-selectin downregulation on naive T cells is restricted to specific anatomical compartments associated with MDSC accumulation. (A) Flow cytometric analysis demonstrating CDl lb ⁺ Gr-l ⁺ MDSC accumulation (% CD45 ⁺ leukocytes, top) and L-selectin expression on CD4 ⁺ CD44 ^l0 and CD8 ⁺ CD44 ^l0 naive T cells (below) in the indicated lymphoid organs (thymus, peripheral lymph node (pLN), inguinal (Ing) LN, tumor- draining Ing LN, spleen, and/or blood) of non-tumor bearing (NTB) mice or 4T1 -bearing mice (tumor volume 1,150±150 mm ³ ). (B) MDSC-T cell suppression assay. Splenic CDl lb ⁺ cells (>85% CDl lb ⁺ Gr-l ⁺ ) either from NTB mice or 4Tl-bearing mice (tumor volume 2,600±380 mm ³ ) were co-cultured with CFSE-labeled target splenocytes from NTB mice at the indicated splenocyte:myeloid cell ratios. Proliferation (based on CFSE dilution) in T cell subsets was measured 72 hours after addition of anti-CD3/CD28 antibody-conjugated activation beads. Percent suppression is for one experiment (mean±s.e.m, «=3 replicates per condition) and is representative of three independent experiments. (C) Total numbers of viable naive CD4 ⁺ CD44 ^l0 and CD8 ⁺ CD4 ¹⁰ T cell subsets (left) and percentages of annexin V ⁺ early apoptotic T cells (right) were quantified by flow cytometric analysis from peripheral lymph nodes and spleens of NTB or 4Tl-bearing mice (tumor volume 1,340±242 mm ³ ) Data (mean±s.e.m.) are of one experiment (n=3 mice per group) and are representative of two independent experiments. (B,C) *P<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student's Mest. (D) Splenic cryosections from NTB and 4T1 -bearing mice stained for B220 ⁺ , Gr-1 ⁺ and CD3 ⁺ cells; parallel fluorocytometric analysis (as in A) confirmed that >90% of splenic Gr-1 ⁺ cells co-expressed CDl lb. Scale bar, 50 μιη. (E) L-selectin expression on splenic B220 ⁺ cells of NTB and 4Tl-bearing mice. (A,E) Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity.

(A,B,D-E) Data are for one experiment and are representative of > three independent experiments (n=3 replicates or mice per group). pLN, peripheral lymph node; Ing LN, inguinal lymph node; NTB, non-tumor bearing.

[0011] Figure 2. MDSC express low levels of L-selectin. Flow cytometric analysis of L-selectin expression on splenic CD3 ⁺ CD44 ^l0 naive T cells from non-tumor bearing (NTB) mice and CD1 lb ⁺ Gr-l ⁺ MDSC from 4T1 -bearing mice (tumor volume 1,050±150 mm ³ ). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity (MFI). Data (mean±s.e.m.) are of one experiment (n=3 mice per group) and are representative of two independent experiments. *P<0.05; data were analyzed by unpaired two-tailed Student's i-test. NTB, non-tumor bearing; MFI, mean fluorescence intensity.

[0012] Figure 3. Inverse correlation between MDSC expansion and L-selectin expression on naive CD4 ⁺ and CD8 ⁺ T cells during 4T1 tumor progression. (A)

Fluorocytometric analysis of CDl lb ⁺ Gr-l ⁺ MDSC accumulation (% CD45 ⁺ leukocytes, top) and L-selectin expression on splenic CD4 ⁺ CD44 ^l0 and CD8 ⁺ CD44 ^l0 naive T cells (below) of non-tumor bearing (NTB) or 4T1 -bearing male mice (tumor volume -2,000 mm ³ ). (B) Tumor volume (top left) and accumulation of splenic CD1 lb ⁺ Gr-l ⁺ MDSC (bottom left) in 4T1- tumor-bearing female mice were measured over time. Representative flow cytometric analysis is shown for CD1 lb ⁺ Gr-l ⁺ cell frequency (% CD45 ⁺ leukocytes) of NTB controls compared to 28 days post-4Tl implantation (right). (C) L-selectin on splenic CD4 ⁺ CD44 ^l0 and CD8 ⁺ CD44 ^l0 naive T cells in 4Tl-bearing female mice was assessed during tumor progression and compared with NTB mice (left). Representative L-selectin profiles are shown for NTB and 4T1 -bearing mice at 28 days post-tumor implantation (right). (B,C) Data (mean±s.e.m.) are for one representative experiment (n=3 mice per group); *P<0.05; data were analyzed by unpaired two-tailed Student' s i-test. (D) Flow cytometric analysis of CDl lb ⁺ Gr-l ⁺ MDSC (% CD45 ⁺ leukocytes, lefi) and L-selectin expression profiles on CD4 ⁺ and CD8 ⁺ T cells (right) in spleens of female mice implanted with orthotopic 4T1 tumors in the mammary fat pad (MFP; tumor volume >1,500 mm ³ ). (A,C-D) Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity (MFI). (A-D) Data are of one experiment («=3 mice per group) and are representative of > three independent experiments. NTB, non-tumor bearing; MFP, mammary fat pad; MFI, mean fluorescence intensity.

[0013] Figure 4. MDSC expansion coincides with L-selectin downregulation on naive CD4 ⁺ and CD8 ⁺ T cells during AT-3 tumor progression. (A) Tumor volume (top left) and accumulation of splenic CD1 lb ⁺ Gr-l ⁺ MDSC (bottom left) in AT-3-tumor-bearing mice were measured over time Representative flow cytometric analysis is shown (right) for CD1 lb ⁺ Gr-l ⁺ cell frequency (% CD45 ⁺ leukocytes) in the spleens of non-tumor bearing (NTB) controls compared with AT-3 tumors-bearing mice (28 days post-tumor implantation). (B) L-selectin on splenic CD4 ⁺ CD44 ^l0 and CD8 ⁺ CD44 ^l0 naive T cells in AT3-bearing mice was compared to L-selectin levels on T cells from NTB mice (left). Representative L-selectin profiles of NTB and AT-3 -bearing mice are shown (right). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity (MFI). (A,B) Data (mean±s.e.m.) are from one experiment (n=3 mice per group) and are representative of > three independent experiments. *P<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student's Mest. NTB, non-tumor bearing, MFI, mean fluorescence intensity.

[0014] Figure 5. L-selectin down-modulation is associated with MDSC expansion in different tumor types. Flow cytometric analysis of CDl lb ⁺ Gr-l ⁺ MDSC (% CD45 ⁺ leukocytes, top) and L-selectin expression profiles on CD4 ⁺ and CD8 ⁺ T cells (bottom) in spleens of mice implanted with subcutaneous B 16 melanoma and CT26 colorectal tumors (in C57BL/6 mice or BALB/c mice, respectively). Tumor volume >1,500 mm ³ for all tumor models. CD1 lb ⁺ Gr-l ⁺ cells in non-tumor bearing (NTB) controls were <3% of CD45 ⁺ leukocytes (data not shown). Comparative analysis of L-selectin expression is shown for tumor-bearing mice and NTB controls; horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. Data are representative of > three independent experiments. NTB, non-tumor bearing.

[0015] Figure 6. L-selectin loss occurs on na'ive T cells within the MDSC- enriched blood compartment of splenectomized mice. (A) Experimental design in which splenectomy or sham surgery was performed in non-tumor bearing (NTB) mice. Mice were then inoculated with 4T1 tumor or maintained as NTB controls. Fluorescently-labeled L- selectin CD8 ⁺ T cells isolated from NTB mice (input) were used for intravenous adoptive cell transfer (ACT) into tumor-bearing mice or in NTB controls (B) 4T1 tumor volume of sham and splenectomized mice at 21 days post-4Tl implantation. Data (mean±s.e.m.) are for a single representative experiment (n=3 mice per group); ns, not significant; data were analyzed by unpaired two-tailed Student's Mest. (C) Representative flow cytometric analysis showing accumulation of CD1 lb ⁺ Gr-l ⁺ cells (% CD45 ⁺ leukocytes, top) and L-selectin expression (bottom) on CD8 ⁺ CD44 ^l0 T cells before ACT (input) and 24 h post-ACT in the blood of sham or splenectomized NTB and 4T1 -bearing recipient mice. Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. (A-C) Data are representative of three independent experiments (n=3 mice per group). NTB, non- tumor bearing; ACT, adoptive cell transfer.

[0016] Figure 7. L-selectin downregulation on na'ive CD4 ⁺ CD44'° T cells occurs in the MDSC-enriched peripheral blood compartment of splenectomized mice. Mice underwent sham surgery or splenectomy 10 days prior to 4T1 tumor inoculation as described for Figure 2. L-selectin expression was then analyzed 22 days after tumor inoculation on endogenous naive CD4 ⁺ CD44 ^l0 T cells in the spleen or blood of non-tumor bearing (NTB) and 4Tl-bearing sham and splenectomized mice. Data are from one experiment (n=3 mice per group) and are representative of three independent experiments. Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing.

[0017] Figure 8. L-selectin downregulation on B cells occurs exclusively in the peripheral blood. Splenectomy or sham surgery was performed 10 days prior to 4T1 tumor inoculation and tissues were evaluated for MDSC expansion and L-selectin expression 22 days after tumor inoculation. Flow cytometric analysis of CDl lb ⁺ Gr-l ⁺ cell burden (% CD45 ⁺ leukocytes; top) and L-selectin expression on endogenous B220 ⁺ B cell populations (bottom) in the indicated organs (BM, bone marrow; spleen; blood) of sham or

splenectomized non-tumor bearing (NTB) and 4T1 -tumor bearing mice. Horizontal lines on histograms indicate positively stained cells; numbers are mean fluorescence intensity. Data are representative of > three independent experiments (n=3 mice per group), NTB, non-tumor bearing; BM, bone marrow.

[0018] Figure 9. L-selectin downregulation on T and B cells occurs in the MDSC- enriched peripheral blood compartment of splenectomized mice. Schematic is shown for experimental design in which 4Tl-bearing mice underwent splenectomy or sham surgery 14 days-post tumor implantation. Fluorescently-labeled CD8 ⁺ T cells isolated from NTB mice (input) were intravenously transferred 21 days post-4Tl inoculation (tumor volume

1,499±449 mm ³ , n=3 mice). Representative flow cytometric dot plots demonstrating

CD1 lb ⁺ Gr-l ⁺ MDSC accumulation in blood (% of CD45 ⁺ leukocytes, top) and histograms (below) for L-selectin expression on CD8 ⁺ CD44 ^l0 T cells before adoptive cell transfer (ACT) (input) and on circulating transferred T cells 24 hours post-ACT in sham and splenectomized NTB recipients or 4Tl-bearing recipients. L-selectin profiles are also shown for endogenous B220 ⁺ B cells in spleen and blood of sham and splenectomized NTB mice and 4Tl-tumor bearing mice (bottom histograms). Data are from one experiment (n=3 mice per group). Horizontal lines in histograms indicate positively stained cells; numbers are mean

fluorescence intensity. NTB, non-tumor bearing; ACT, adoptive cell transfer.

[0019] Figure 10. L-selectin loss on naive T and B lymphocytes occurs rapidly in the blood of tumor-bearing mice. Schematic is shown for experiment design in which fluorescently-labeled, L-selectin ^M splenocytes isolated from non-tumor bearing donors (input) were adoptively transferred into 4T1 -bearing recipient mice (tumor volume 1,026±54 mm ³ , n = 2 mice). Representative flow cytometric dot plots demonstrating CD1 lb ⁺ Gr-l ⁺ MDSC accumulation in blood (% CD45+ leukocytes, left) and histograms for L-selectin expression (right) on CD8 ⁺ CD44 ¹⁰ , CD4 ⁺ CD44 ^l0 , and B220 ⁺ splenocytes before adoptive cell transfer (ACT) (input) and on circulating transferred lymphocyte populations 2 hours post-ACT into 4Tl-bearing recipient mice. Data are from one experiment (n=2 mice per group). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing; ACT, adoptive cell transfer.

[0020] Figure 11. MDSC-associated downregulation of L-selectin on na ^' ive T and

B lymphocytes occurs in autochthonous mammary carcinoma MTAG mice. Blood was collected from individual transgenic MTAG mice (with the indicated total tumor volume) and from age-matched non-tumor bearing (NTB) wildtype littermates. Flow cytometric analysis is shown for CD1 lb ⁺ Gr-l ⁺ cell accumulation (% CD45 ⁺ leukocyte, above) and L-selectin expression on naive CD4 ⁺ CD44 ^l0 and CD8 ⁺ CD44 ¹⁰ T cells and B220 ⁺ B cells (below). Data are from one experiment (n>3 mice per group) and are representative of three independent experiments. Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing.

[0021] Figure 12. L-selectin on human na ^' ive T cells is downregulated following transfer into 4T1 tumor-bearing mice. Normal human-donor peripheral blood lymphocytes were adoptively transferred into non-tumor bearing (NTB) severe-combined immunodeficient (SCTD) mice or 4Tl-bearing SCID mice at 21 days-post tumor implantation (average tumor volume for 3 experiments, 1,190±197 mm ³ ). MDSC burden in the blood of NTB and 4T1- bearing SCID mice was measured 24 hours after adoptive cell transfer (top). MDSC burden was quantified based on blood volume for T- and B-cell deficient SCID mice. Data

(mean±s.e.m.) are from three independent experiments for 3 different lymphocyte donors (n=3 mice per group in each experiment). * <0.05; data were analyzed by unpaired two- tailed Student's i-test. Flow histograms depict L-selectin expression of individual donor CD3 ⁺ CD45RA ⁺ T cells at 24 hours post-ACT in NTB or 4T1 tumor-bearing recipient SCID mice (bottom). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing.

[0022] Figure 13. MDSC induce L-selectin loss on T and B lymphocytes in 4T1 tumor-bearing mice via a contact-dependent mechanism. (A) 4T1 -tumor-bearing mice were treated with anti-Gr-1 antibodies (a-Gr-1 Ab) or isotype control antibodies (Iso Ab) every 3 days for 3 weeks starting at 3 days post-tumor implantation. Endpoint tumor volumes are shown. (B) CDl lb ⁺ Gr-l ⁺ MDSC burden (% CD45 ⁺ leukocytes, left) and L-selectin expression (mean fluorescence intensity, MFI) on endogenous CD4 ⁺ CD44 ^l0 , CD8 ⁺ CD44 ^l0 , and B220 ⁺ lymphocytes (right) were measured in the blood of NTB or in 4T1 -bearing mice treated with Iso Ab or anti-Gr-1 Ab. (C) Splenocytes from NTB or 4T1 -bearing mice were depleted of CD1 lb ⁺ cells by magnetic bead isolation (94.8±1.8% depletion, n=3 mice). These cell populations were then fluorescently-labeled with different tracking dyes, co-mixed at a 1 : 1 ratio, and cultured in vitro or adoptively transferred into NTB recipients. Representative flow cytometric L-selectin profiles are shown for naive CD4 ⁺ CD44 ^l0 and CD8 ⁺ CD44 ^l0 T cells before culture or adoptive cell transfer (ACT) (input) and 4 days after in culture (in vitro) or for cells recovered from blood and spleen post-ACT (left). Quantification of L-selectin modulation (right) is based on a ratio of the MFI for T cells from 4Tl-bearing mice relative to NTB mice; dashed lines indicate NTB control. (D) MDSC or CD1 lb ⁺ control cells were isolated from 4Tl-tumor bearing mice (tumor volume > 1,000 mm ³ ) or NTB mice, respectively. Myeloid cells were then co-cultured with fluorescently-labeled splenocytes from NTB mice (10: 1 ratio) in media alone or with IFN-γ (20 U/mL) and LPS (100 ng/mL).

MDSC and splenocytes were separated by 0.4 μιη transwell inserts in the indicated co- cultures. After 24 hours, L-selectin expression on viable naive CD8 ⁺ CD44 ^l0 T cells was analyzed by flow cytometry; representative profiles are shown (left). Relative changes in L- selectin expression were normalized to untreated CD8 ⁺ CD44 ^l0 T cells (indicated by dashed lines; right). (A-D) Data (mean±s.e.m.) are from one experiment (n=3 mice per group or >3 replicates per group) and are representative of > two independent experiments. TO.05; ns, not significant; data were analyzed by unpaired two-tailed Student's t-test. (C,D) Horizontal lines in histograms indicate positively stained cells; numbers are MFI. NTB, non-tumor bearing; Ab, antibody; Iso, isotype; MFI, mean fluorescence intensity.

[0023] Figure 14. MDSC-induced L-selectin downregulation is post- transcriptional and does not depend on the ADAM17 metalloprotease. (A) Flow cytometric analysis of surface L-selectin (mean fluorescence intensity; MFI) on splenic naive CD4 ⁺ CD44 ^l0 and CD8 ⁺ CD44 T cells of non-tumor bearing (NTB) and 4Tl-bearing mice is shown (left). L-selectin mRNA expression in splenic CD4 ⁺ and CD8 ⁺ T cells from NTB and 4T1 -bearing mice was determined by qRT-PCR with fold-change normalized with β-actin (right). (B) Soluble (s)L-selectin in serum of individual NTB and 4Tl-bearing mice was assessed by ELISA. (A,B) Data are from three independent experiments (ri>2 mice per group in each experiment; tumor volume >1,500 mm ³ ; average frequency of splenic CD1 lb ⁺ Gr-l ⁺ cells (% CD45 ⁺ leukocytes) in tumor-bearing mice was -40%). (C) Splenocytes were isolated from NTB wildtype (WT) C57BL/6 mice, L(E)-selectin transgenic mice, Adaml 7^ ^ox/ ^ ^ox /Vavl- Cre mice (Adaml 7 ^~A ), or age-matched WT littermate controls. WT splenocytes were pretreated for 30 minutes with the ADAM17-specific inhibitor PF-5480090 (10 μΜ) or the ADAM 17/10-specific inhibitor INCB7839 (20 μΜ) (left). WT, L(E), and Adaml 7 - splenocytes were then cultured 2 hours with or without phorbol myristate acetate (PMA, 100 ng/mL). Surface L-selectin (MFI) on viable naive CD8 ⁺ CD44 ^l0 T cells relative to untreated controls was determined by fluorocytometric analysis. (D) Splenic CD1 lb ⁺ Gr-l ⁺ MDSC were purified from 4T1 -bearing mice; splenocytes were from various NTB mice as described in (C). MDSC and WT splenocytes were both pretreated for 30 minutes with or without PF- 5480090 or INC7839 (left). MDSC and splenocytes were then co-cultured at a 10: 1 ratio for 24 hours in media containing IFN-γ (20 U/mL) and LPS (100 ng/mL). L-selectin on viable naive CD8 ⁺ CD44 ^l0 T cells was assessed by flow cytometric analysis. (E) Fluorescently- labeled WT, L(E), and Adaml 7 ^"A splenocytes (i.e., from NTB mice) were adoptively transferred into NTB severe-combined immunodeficient (SCID) mice or 4T1 -bearing SCID mice at 21 days-post tumor implantation (average tumor volume for all experiments, 1 , 102± 191 mm ³ ; average circulating CDl lb ⁺ Gr-l ⁺ frequencies in NTB SCID recipients, 75±8 cells^L blood, and 4Tl-bearing SCID recipients, 4,081±876 cells^L blood). After 24 hours post- ACT, L-selectin was assessed by flow cytometry on transferred splenocytes recovered from the blood of NTB and 4T1 -bearing SCID mice. Representative flow histograms depict L-selectin expression on naive CD8 ⁺ CD44 ^l0 T cells (above); horizontal lines indicate positively stained cells, numbers are mean fluorescence intensity. Normalized data for L-selectin expression on B220 ⁺ , CD4 ⁺ CD44 ^l0 , CD8 ⁺ CD44 ^lc cells 24 hours post- adoptive transfer are for one representative experiment (n>2 mice per group) (below). (A-E) * <0.05; ns, not significant; all data (mean±s.e.m.) were analyzed by unpaired two-tailed Student' s i-test. (C-E) Data are representative of > two independent experiments (n>2 replicates or mice per group) and are normalized to untreated or NTB controls (indicated by dashed lines). NTB, non-tumor bearing; WT, wildtype; MFI, mean fluorescence intensity; sL-selectin, soluble L-selectin; ACT, adoptive cell transfer.

[0024] Figure 15. PMA-induced loss of L-selectin depends on c s-acting

ADAM17. Wildtype (WT) and Adam 17 ^'1' splenocytes were labeled with different fluorescent dyes prior to co-culture at a 10: 1 ratio for 2 hours with or without phorbol myristate acetate (PMA, 100 ng/mL). L-selectin on viable WT and Adaml?^ CD8 ⁺ CD44 ^l0 T cells was assessed by flow cytometric analysis and compared to untreated controls (indicated by dashed line). Data (mean±s.e.m.) are from one experiment (n=2 replicates per group). *P<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student's West. WT, wildtype.

[0025] Figure 16. L-selectin-deficient CD8 ⁺ T cells from AT-3-bearing mice exhibit reduced firm adhesion and faster rolling velocity in LN HEV. (A) Flow cytometric analysis of L-selectin expression prior to intravenous adoptive transfer of CD8 ⁺ T cells from non-tumor bearing mice (NTB CD8 ⁺ ) or CD8 ⁺ T cells from AT-3-bearing mice (AT-3 CD8 ⁺ ). Horizontal line in histogram indicates positively stained cells; numbers are mean fluorescence intensity. (B) Representative photomicrograph (left) and schematic (right) of the postcapillary vascular tree visualized by epifluorescence intravital microscopy in inguinal LN of NTB recipient mice. Hierarchical branches of venular orders I and II (low- order venules, LOV) and III-V (high-order venules corresponding to HEV) are labeled. Direction of blood flow in post-capillary venules is from order V to order I venules which directly empty into collecting veins. Scale bar, 100 μηι. (C) Rolling fraction and sticking fraction of fluorescently-labeled CD8 ⁺ T cells isolated from NTB or AT-3-bearing mice following adoptive transfer into NTB recipients. Data (mean±s.e.m.) are from three independent experiments; ri>3 mice per group. TO.05; ns, not significant; data were analyzed by unpaired two-tailed Student's i-test. (D) Cumulative rolling velocity curve was generated by measuring the velocities of transferred CD8 ⁺ T cells in order V venules of inguinal LN in three independent experiments. Comparison of cumulative rolling velocity plot data was performed by unpaired two-tailed Student' s t-test; L-selectin NTB CD8 ⁺ T cells versus L-selectin ^im/1 ° AT-3 CD8 ⁺ T cells, *P<0.01. (E) Distributions of rolling velocities in velocity histograms in order V venules were evaluated by a nonparametric Mann-Whitney U test; L-selectin ⁺ NTB CD8 ⁺ T cells versus L-selectin ^int/1 ° AT-3 CD8 ⁺ T cells, * P<0.01. NTB, non-tumor bearing.

[0026] Figure 17. Adhesion molecule expression and function on CD4 ⁺ and CD8 ⁺

T lymphocytes. (A) Single parameter fluorocytometric histograms {left) depicting expression of L-selectin, CCR7, and CD1 la (i.e., the OIL subunit of LFA-1) on CD8 ⁺ CD44 ¹⁰ splenic T cells derived from non-tumor bearing (NTB) mice or AT-3-bearing mice (3 weeks post-AT-3 implantation, tumor volume -4,000 mm ³ ). Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity (MFI); trafficking molecule expression (MFI) is quantified for n=3 mice (right). (B) Transwell chemotaxis assays compared migration of purified splenic CD8 ⁺ T cells from NTB and AT-3-bearing mice in response to recombinant CCL21 (70 nM). (C) LFA-1 -dependent homotypic aggregation of CD8 ⁺ T cells was induced by phorbol myristate acetate (PMA). Purified CD8 ⁺ splenic T cells from NTB and AT-3-bearing mice were pretreated with or without a-CDl la antibody (10 ug/mL) for 15 minutes. Cells were then cultured with and without PMA (50 ng/mL) for 18 hours. Percent aggregation was quantified based on total input cells (left); representative photomicrographs of untreated and PMA-treated CD8 ⁺ T cells are shown (right). (A-C) Data (mean±s.e.m.) are representative of > two independent experiments (ri>2 mice per treatment group with >3 replicates per condition). TO.05; ns, not significant; data were analyzed by unpaired two-tailed Student's i-test. NTB, non-tumor bearing; MFI, mean fluorescence intensity.

[0027] Figure 18. L-selectin down-modulation on CD8 ⁺ T cells of AT-3-bearing mice inhibits trafficking across LN HEV. (A) Competitive homing studies used a 1 : 1 ratio of splenic CD8 ⁺ T cells isolated from non-tumor bearing mice (NTB CD8 ⁺ , green (+)) and AT-3-bearing mice (AT-3 CD8 ⁺ , red (*)). One hour after intravenous adoptive transfer of CD8 ⁺ T cells, the extent of homing of fluorescently-tagged transferred cells was examined in LN or spleens either of NTB controls (i.e., homeostatic trafficking) or in an inflammatory model in which the core body temperature of tumor-free recipient mice was elevated prior to T cell transfer (39.5±0.5°C for 6 hours; whole body hyperthermia, WBH). Representative photomicrographs of fluorescently-labeled homed cells in histological LN and splenic cryosections; counterstaining with CD31 antibody identified cuboidal high endothelial venules (HEV, denoted by white arrows, left) in LN. Scale bar, 50 μιτι. Note the majority of T cells detected in images extravasated across HEV and were located in the LN parenchyma. Data (mean±s.e.m.; right) are from 1 experiment (n=3 mice per group) and are representative of three independent experiments. *P<0.05; ns, not significant; data were analyzed by unpaired two-tailed Student's t-test. (B) L-selectin expression profiles of CD8 ⁺ T cells (originating from NTB mice or AT-3-bearing mice) that were recovered 1 hour after adoptive transfer in LN or spleen of NTB recipients. Horizontal lines in histograms indicate positively stained cells; numbers are mean fluorescence intensity. NTB, non-tumor bearing; WBH, whole body hyperthermia.

[0028] Figure 19. Adhesion molecule expression on LN HEV. Lymph node cryosections of NTB mice were stained for peripheral lymph node addressin (PNAd), CCL21, or intercellular adhesion molecule-1 (ICAM-1) in non-tumor bearing mice maintained under homeostatic control conditions or in inflamed high endothelial venules (HEV) of mice pre-treated with whole body hyperthermia (WBH). White arrows denote position of cuboidal HEV determined by counterstaining tissues with PNAd or CD31 -specific Ab {not shown). Trafficking molecule expression on HEV was assessed by quantitative image analysis for immunofluorescence staining intensity in cuboidal HEV and depicted by histograms. The x-axis indicates fluorescence intensity and the y-axis indicates the number of pixels with each intensity; numbers in histograms denote mean fluorescence intensity. Data are representative of > three independent experiments (n>2 mice per treatment group). Scale bar, 50 μιη. PNAd, peripheral lymph node addressin; WBH, whole body hyperthermia.

[0029] Figure 20. Antigen-driven activation of CD8 ⁺ OT-I T cells is compromised by poor L-selectin dependent trafficking in lymph nodes. (A) Schematic of competitive activation assays. CD8 ⁺ T cell populations (>95 % CD8 ⁺ ) were isolated from non-tumor bearing OT-I mice (NTB OT-I, CD8 ⁺ L-selectin ^M ) and from AT-3 -bearing OT-1 mice (AT-3 OT-I, CD8 ⁺ L-selectin intermediate-to-low; L-selectin ^int/1 °; tumor volume 3,825±123 mm ³ for n=4 mice). T cells (depleted of CD1 lb ⁺ MDSC) from NTB or tumor-bearing mice were then labeled ex vivo with different proliferation dyes (CellTrace Violet or CellTrace CFSE, respectively), co-mixed at a 1 : 1 ratio, and assessed for functional responses to cognate antigen (SIINFEKL (SEQ ID NO: 1)) after 4 days in competitive activation assays in vitro and in vivo. (B) Flow cytometric analysis of proliferation of NTB OT-I CD8 ⁺ and AT-3 OT-I CD8 ⁺ T cells after activation by SIINFEKL (SEQ ID NO: l)-loaded dendritic cells (DC) for 4 days in vitro. Horizontal lines on histograms indicate percent proliferating cells. (C)

Competitive in vivo activation assay in which NTB recipient mice were vaccinated (via footpad) with SIINFEKL (SEQ ID NO: l)-loaded DC 6 hours before adoptive transfer of a 1 : 1 mixture of L-selectin ¹¹¹ NTB OT-I and L-selectin ^int/1 ° AT-3 OT-I CD8 ⁺ T cells. After 4 days, the ratios of the adoptively transferred cells were assessed in the following lymphoid compartments: spleen (Spl), contralateral popliteal lymph node (cLN), and draining popliteal lymph node (dLN). Data (mean±s.e.m.) are from one experiment (n=4 mice per group) and are representative of 2 independent experiments. * <0.05; ns, not significant; data were analyzed by unpaired two-tailed Student's t-test (D) IFN-γ expression profiles for endogenous CD8 ⁺ T cells and adoptively transferred NTB OT-I CD8 ⁺ and AT-3 OT-I CD8 ⁺ T cells recovered in dLN of DC-vaccinated mice. Horizontal lines on histograms indicate positively stained cells; data are representative of 2 mice per group. NTB, non-tumor bearing; DC, dendritic cell.

[0030] Figure 21. Preconditioning of antigen-inexperienced TcR-transgenic

CD8 ⁺ and CD4 ⁺ T cells with MDSC in vitro suppresses responsiveness to subsequent antigen challenge. (A) Schematic of MDSC preconditioning protocol. MDSC were isolated from the blood of 4ΊΊ -bearing mice (>93% CD1 lb ⁺ Gr-l ⁺ ) and co-cultured with splenocytes from non-tumor bearing (NTB) OT-I and DO11.10 T cell receptor (TcR) transgenic mice. After 16 hours, MDSC were depleted by magnetic separation using biotinylated anti-Gr-1 antibody and Streptavidin beads. Remaining splenocyte populations contained 0.4-4% CD1 lb ⁺ Gr-l ⁺ cells. Cognate peptide was then added to each culture. After 3 days in culture, splenocytes were pulsed with ³ H-thymidine and harvested 16 hours later. (B) Proliferative responses are shown for OT-I CD8 ⁺ {left) and DO11.10 CD4 ⁺ {right) T cells based on ³ H- thymidine incorporation (CPM). Ratios denote the relative proportion of purified MDSC OT- I or MDSC:DO11.10 splenocytes used in the preconditioning phase. Data (mean±s.d.) are of six replicate cultures in a single experiment and are representative of three independent experiments. * <0.002; unpaired two-tailed Student's i-test. CPM, counts per minute; TcR, T cell receptor; NTB, non-tumor bearing.

[0031] Figure 22. Model for MDSC actions at remote sites that compromise adaptive immunity in the LN compartment. (A) Restricted localization of MDSC in the splenic marginal zone leads to preferential, downregulation of L-selectin (i.e., intermediate- to-low phenotype, L-selectin ^int/l0 ), on naive CD4 ⁺ and CD8 ⁺ T cells, but not B220 ⁺ B cells. MDSC in the splenic marginal zone also precondition CD4 ⁺ and CD8 ⁺ T cells which leads to suppressed responsiveness to antigen outside the splenic environment. L-selectin on circulating T and B cells can be independently targeted by MDSC within the blood compartment, leading to significantly elevated levels of circulating soluble L-selectin.

MDSC-mediated downregulation of L-selectin is contact-dependent and occurs post- transcriptionally, but is independent of the major L-selectin sheddase, ADAM17. (B) Diminished L-selectin expression reduces trafficking of blood-borne lymphocytes across high endothelial venules (HEV) in the lymph node compartment. Boxed region is shown in more detail in inset. (C) Inset of lymph node region showing that moderate L-selectin loss (L- selectin ^int/1 ° phenotype) results in faster rolling of T cells on lymph node HEV which, in turn, reduces the transition to firm arrest and subsequent transendothelial migration into the underlying parenchyma. Diminished trafficking in HEV, in combination with sustained immunosuppression caused by MDSC preconditioning in the spleen, profoundly

compromises the generation of effector T cells in response to cognate antigen presented by dendritic cells (DC). L-selectin ^mt/1 °, L-selectin intermediate-to-low expression; PNAd, peripheral lymph node addressin; LFA-1, leukocyte function-adhesion molecule- 1 ; ICAM- 1/2, intercellular adhesion molecule- 1 and -2.

[0032] Figure 23. Illustration of MDSC clusters.

[0033] Figure 24. (A) Experimental design for 4T1 metastatic liver model. (B)

Representative bioluminescence images of indicated mouse groups.

[0034] Figure 25. (A) Flow cytometric analysis for CD1 lb ⁺ Gr-l ⁺ myeloid cells and

L-selectin on naive CD3 ⁺ CD44 ^b T cells of 1° 4T1, M0, Ml, M2, and non-tumor bearing (NTB) mice {left), dashed lines, NTB controls. MDSC flow plots and L-selectin histograms (right). Serum soluble (s)L-selectin by ELISA. (B) Blood CD1 lb ⁺ Gr-l ⁺ MDSC (1° 4T1) or normal CD1 lb ⁺ myeloid cells (NTB) were co-cultured 3 days with CFSE-labeled splenocytes in the presence of anti-CD3/CD28 T cell-activation beads. % T cell suppression assessed by flow cytometry. Data (mean ± SEM) representative of >3 experiments (n>2 mice/group); *P<0.05.

[0035] Figure 26. (A) Representative images of blood smears stained by Diff-Quik

(left). Arrowheads, lymphocytes; arrows, myeloid cells. Cluster frequency was quantified from blood smears (right). Data representative of > 4 independent experiments, n>2 mice/group. *P<0.05. (B) Representative immunofluorescence images of blood-borne cellular clusters from microfiltration assays for 4T1 model and ER ⁺ PR ⁺ Her2 ^' breast cancer patient. White arrow, PMN-MDSC; arrowhead, M-MDSC.

[0036] Figure 27. Splenic L-selectin ¹¹¹ naive T cells from normal donor mice were labeled with CFSE. Cells were pretreated 20' with Ab for CDl la (a integrin subunit of LFA- 1), and ICAM-1 and then adoptively transferred i.v. into non-tumor bearing (NTB) or 1° 4T1- bearing mice. L-selectin was determined by flow cytometry on transferred cells recovered from blood after 30' . Representative histograms shown for L-selectin; numbers denote MFI (left). Fold-change of L-selectin MFI relative to mean L-selectin on T cells prior to transfer for each group. Data (mean ± SEM); n = 3 mice/ group. * <0.05. [0037] Figure 28. (A) Tumor progression in murine Pan02 pancreatic tumor model is

+ +

associated with (B) expansion of CD1 lb Gr-1 MDSC and (C) L-selectin loss on naive T cells and CDl lb Gr-1 MDSC. Data are mean ± S.E.M; ns, not significant. MFI, mean fluorescence intensity.

[0038] Figure 29. L-selectin on blood-borne CD3 ⁺ CD44 ^l0 naive T cells was evaluated by flow cytometry at the indicated intervals in NTB mice or post-4Tl inoculation. Data for NTB mice for weeks 1-4 was averaged. Clusters were quantified for high powered fields

2

(HPF; unit area=0.34 mm ) in 10 ul blood smears (+EDTA) stained by Diff-Quik. Blood smear images: arrowheads, myeloid cell; arrows, lymphocyte. Data represent > 4 independent experiments, n=3-5 mice/group; * <0.04, 4T1 versus NTB; ns, not significant.

[0039] Figure 30. (A) Non-tumor bearing mice were treated for 6 h with whole-body hyperthermia (WBH, core temperature elevated to 39.5 ± 0.5°C); control mice at room temperature maintained normal body temperature (~37°C). Neutrophil counts were elevated following WBH treatment compared to untreated controls (Untx). (B) Peripheral blood smears were evaluated for cell clusters (statistics for untreated versus WBH treated mice). (C)

L-selectin expression was determined by flow cytometry on naive T cells (CD3 CD44 ¹⁰ ) or

(D) neutrophils (CDl lb Gr-1 ); Data are mean ± S.E.M. (n=3-5 mice/group) and are representative of 2 independent experiments; ns, not significant.

[0040] Figure 31. (A) Representative data from 4T1 -bearing mouse analyzed by ImageStream. (B) ImageStream photomicrographs; quantification for clusters per μΐ of heparinized blood using Flow-Count Fluorospheres. Cells were visually confirmed for all images to exclude non-cellular debris from the analysis. (C) Cluster composition is shown. Data represent > 6 independent experiments, n=3-5 mice/group; *P<0.05, 4T1 versus NTB.

[0041] Figure 32. Mice were injected with fluorescent-labeled Gr-1 antibody and blood vessels were immediately visualized by single-photon epifluorescence microscopy;

+

fluorescent images were collected with B/W camera. The percent Gr-1 cells in clusters in blood of 4Tl-bearing mice was determined by comparing the aspect ratio for the widest dimension to values obtained for NTB (non-tumor-bearing) control mice.

[0042] Figure 33. (A) Peripheral blood from 4T1 -bearing mice (n=5) was dually stained with Ly6G and Ly6C-specific Ab prior to analysis by flow cytometry -based

ImageStream. (B) Peripheral blood from 4Tl-bearing mice (n=4) was stained with antibodies specific for Gr-1, CD44, and CD3, and then analyzed by ImageStream flow cytometry. [0043] Figure 34. Events evaluated from peripheral blood specimens from 4T1- bearing model were gated in ImageStream analysis based on size in the brightfield channel (CH 01). Events were initially scored visually as clusters (>1 cell detected in brightfield images) and then examined for the presence of CD41 CD49 platelets; arrows demark position of platelets within clusters.

[0044] Figure 35. (A) Schematic for experimental design for adoptive cell transfer

(ACT) experiment. L-selectin leukocytes isolated from NTB mice were labeled with CFSE- tracking dye immediately prior to adoptive transfer (i.v.) into NTB or 4T1 -bearing recipient mice. (B) L-selectin expression is shown for adoptively transferred CFSE cells recovered from blood 30 min post-ACT. (C) Blood specimens were evaluated for clusters by flow cytometry-based ImageStream analysis. Arrows in representative photomicrographs demark position of CSFE cells in clusters. NTB, non-tumor-bearing.

[0045] Figure 36. Splenic leukocytes were isolated from non-tumor bearing (NTB) mice, labeled with CFSE-tracking dye, and then transferred into 4Tl-bearing mice. L-selectin expression was analyzed on CFSE cells recovered from the blood. L-selectin is strongly downregulated on normal naive T cells (CD3 CD44 ¹⁰ ), neutrophils (CDl lb Gr-1 ) and monocytes (CDl lb Gr-l^) at 30 min after adoptive transfer into MDSC^ 4Tl-bearing mice. Numbers are mean fluorescence intensity (MFI).

[0046] Figure 37. Peripheral blood from a 4T1 -bearing mouse was collected into EDTA and RBC were depleted by lysis (3-times). L-selectin ^M target leukocytes isolated from spleens of non-tumor-bearing mice (NTB) mice were collected through a 70 μιτι mesh, treated with lysis buffer twice and stained using CellTrace Violet (CTV; Ch 7). Cells were mixed at a 10: 1 ratio (i.e., 200,000 4Tl-derived blood cells [largely MDSC effectors] mixed with 20,000 NTB-derived target cells [largely T and B cells]. Cells were cultured for the indicated time in a 96-well round-bottom plate Alternately, NTB-derived target cells

(220,000per well) were incubated without 4Tl-derived effector cells. All cells were cultured in MLM supplemented with IFN-γ and LPS. At the indicated timepoints, aliquots were fixed and analyzed by ImageStream flow cytometry. Total events were gated based on CTV and visually inspected for inclusion in clusters. Arrows in photomicrographs demark position of CTV-labeled target cells within newly formed clusters.

[0047] Figure 38. (A) Representative images and quantification of blood-borne clusters from microfiltration assays for 4T1 model and Stage IV luminal breast cancer patient. White arrow, PMN-MDSC; arrowhead, M-MDSC. (B) The breakdown for PMN- MDSC versus M-MDSC constituents in blood clusters is shown for 3 patients. 'X' indicates nucleated cell type other than myeloid cell.

DESCRIPTION OF THE DISCLOSURE

[0048] This invention exploits observations that MDSCs have the ability of execute immune-suppressive functions by forming stable multicellular clusters with other leukocytes (including, but not limited to other MDSC, T lymphocytes, B lymphocytes, neutrophils, monocytes, and natural killer cells). While such clusters are known to occur in splenic compartments, such clusters have not been demonstrated in the blood compartment. In the present disclosure, the presence of such clusters in blood samples was identified and their presence in circulation was correlated to immunosuppressive function. Based on these observations, therapeutic approaches can be identified or current approaches augmented in the treatment of cancers, and other immune related conditions.

[0049] The present disclosure provides a method for in vitro analysis of myeloid derived suppressor cell clusters. The method comprises obtaining a blood sample from an individual, and determining if MDSC clusters are present in the sample. Analysis for the presence of the clusters can be done by microscopy, microfiltration, or by flow cytometry - based techniques. For example, for flow cytometry, blood cells can be suspended, stained with specific antibodies to detect leukocyte subsets and fixed. Small clusters will remain intact by this procedure. Cell clusters of varying sizes can be analyzed by imaging flow cytometry system and visualized to confirm the presence of 2 or more cells. An advantage of using histological analysis of blood smears or analysis via microfiltration is that identification of circulating myeloid cell clusters (CMCs) can be made in fresh blood within a period of minutes, without the process and wait time required for flow cytometry. For example, CMC presence can be detected in blood smears by histological examination. Clusters of cells that contain MDCSs (such as cells with segmented nuclei), for example, can be carried out within and up to about 4 hours of obtaining the blood sample. Typically, a blood smear can be examined immediately after collection, such as within about 2 to 15 minutes, or up to 20, 30, 45, 60, 90, 120, 180, or 240 minutes. In the case of microfiltration, fresh blood can be immediately, or within a short period of time (such as a couple of hours), subjected to microfiltration and then the filtered out cells can be stained with antibodies for specific markers to identify CMCs. If CMCs are detected by histologic examination of blood smears, or by microfiltration, this can be followed up by flow cytometric analysis where phenotypic characters of the cells within clusters can be confirmed.

[0050] MDSC-containing clusters in blood (termed herein as circulating myeloid cell clusters or CMCs) provides a metric for monitoring the immunosuppressive status of patients, stratification of patients for therapy, prediction and monitoring therapeutic effectiveness, screening of immune-boosting therapeutics, and isolation of enriched MDSC populations for further analysis. The presence of MDSC-containing clusters in blood may contribute to or perform immunosuppressive function by causing loss of L-selectin homing receptor on target leukocyte. For example, measurements of CMC can provide a metric for the immunological fitness of patients. CMC can be used as a blood-based indicator of patient prognosis or whether a patient is likely to be resistant or refractory to immunotherapies. Elevated CMC can be a measure or indicator of an immune-suppressed status and used to inform decisions about whether a cancer patient should be treated with a T cell-based immunotherapy or if a cancer patient should be treated with a myelodepleting agent to reduce MDSC numbers prior to or together with treatment with the immunotherapy. In an embodiment, patients treated with T cell-based immunotherapeutics (including, but not limited to, immune checkpoint inhibitors, adoptive T cell transfer therapy, or cancer vaccines) can be monitored for changes in CMC during their course of treatment. For instance, if CMC become elevated, this can provide the basis for combined treatment with MDSC-depleting regimens. CMC can also be used as a research tool to study the mechanisms of MDSC-mediated immune suppression.

[0051] The present disclosure provides a method for quantifying a population of immunosuppressive myeloid-derived suppressor cells (MDSC) in patient biological samples for monitoring immunosuppressive status, stratification for therapeutic regimens, prediction and monitoring therapeutic effectiveness, screening of immune-boosting therapeutics, and isolation of enriched MDSC populations for research.

[0052] The disclosure also provides a method for determining L-selectin levels in blood and using the levels as a measure of immunosuppressive status, stratification for therapeutic regimens, prediction and monitoring therapeutic effectiveness, and screening of immune-boosting therapeutics. L-selectin levels can be determined in blood, serum or plasma or may be determined in any other biological fluid. L-selectin levels may be measured by immune based assays such as ELISA and the like. The L-selectin levels can be compared to levels from normal individuals, or with values equivalent thereof. [0053] The present disclosure provides an in vitro method for identification of MDSC clusters after collection of blood from an individual. The method comprises obtaining a blood sample and determining if MDSC clusters are present in the sample.

[0054] A blood sample may be collected from an individual in any routine manner. The individual may be a human or a non-human animal. For example blood may be conveniently drawn from a vein of an individual (such as the external jugular vein, antecubital vein, existing central lines, the wrist area, to name a few). The drawn blood may be directly collected in a container (generally referred to herein as a "tube") in which it is to be processed or may be collected in any standard collection tube and then transferred to a processing tube. The collected whole blood can be exposed to an anti-coagulant as soon as collected. For example, the collection tube may pre-contain an amount of anti-coagulant sufficient to inhibit coagulation or the anticoagulant may be added quickly into the collection tube after blood has been added to it or collected in it. A commonly used anticoagulant is heparin. The collected blood may be exposed to divalent ion chelator such as

ethylenediaminetetraacetic acid (EDTA) during collection so as to prevent coagulation without inducing separation of phases of the whole blood. In an embodiment, heparin or EDTA is not used.

[0055] The MDSC clusters may be identified in a blood smear (such as on a glass slide) under a microscope. The clusters can be identified as a two cell clusters or as multicell clusters. Analysis of the clusters can involve the size of the clusters, the number of cells in the clusters, the type of cellular constituents in clusters, or other features of the clusters including any physicochemical properties or biochemical properties, such as expression of surface proteins and the like. These parameters may be compared to a reference value obtained from an individual (or a group of individuals) who does/do not have the particular indication (such as cancer), or individuals who are deemed to be healthy, or in some instances the comparison may be made with the same individual' s numbers over a period of time.

[0056] The clusters may be formed by two or more MDSCs, or one or more MDSCs with one or more of the following cells: T- cells, B-cells, NK cells, or other leukocytes or platelets. The clusters generally do not include tumor cells. In one embodiment, they are free of tumor cells.

[0057] In the CMC population, in one embodiment, the cells are predominantly

PMN-MDSCs. For example, clusters may be predominantly PMN-MDSC pairs (homotypic cells in the cluster). In an embodiment, the percentage of PMN-MDSCs homotypic clusters in the CMCs in a blood sample is at least 90%, the percentage of M-MDSC homotypic clusters in less than 10%, and the percentage of heterotypic PMN-MDSC/M-MDSC clusters is less than 1%.

[0058] The clusters may be visualized using light microscopy, filtration techniques which can filter out any cell clusters, and flow cytometry-based fluorescence cell detection techniques, or any other techniques that can detect the presence of such clusters.

[0059] Identification and analysis of the clusters can be carried our immediately after collection of blood or may be carried out later. In one embodiment, it is carried out in a time period from 30 seconds to 1 hour after blood collection. For example, it can be carried out within 1 to 30 minutes of collection. The analysis can be carried out in without any processing of the blood, i.e., blood smears can be prepared or the blood processed for flow cytometry analysis without processing. For visualization of blood smears, an anti-coagulant is not needed. However, EDTA can be used for blood smears, microfiltration and flow cytometry techniques.

[0060] Circulating L-selectin can be measured by clinically-validated commercial ELISA techniques.

[0061] MDSC clusters can be enumerated and characterized in blood smears, in cellular materials obtained by microfiltration, or by flow cytometry. Further analysis of MDSC populations, including quantification of sub-populations can be carried out.

Microfiltration techniques described in Zhou et al. (Scientific Reports, 4:7392 DOI:

10.1038/srep07392, December 2014), Ferreira et al., (2016), Molecular Oncology, 10(2016) 374-394; Harouaka et al., 2013, J. Lab Automation 18(6) 455-468, the relevant methods from all incorporated herein by reference) can be used for isolating MDSC clusters.

[0062] Human PM -MDSCs are also known as granulocytic MDSCs. In an example, human PMN-MDSC are characterized as CD1 lb ⁺ CD33 ⁺ CD15 ⁺ CD14-HLA-DR ^ne and human M-MDSC (also known as monocytic MDCSs) are characterized as CD1 lb ⁺ CD33 ⁺ CD15 ^" CD14 ⁺ HLA-DR ^low/ne . The most immature human MDSC subset is CD1 lb ⁺ CD33 ⁺ CD15 ^" CD 14 ^" HLA-DR ^neg . Mouse PMN-MDSC are characterized as CDl lb ⁺ Gr- l ^Mgh or CD1 lb ⁺ Ly6G ⁺ Ly6C ^l0 . Mouse M-MDSC are characterized as CD1 lb ⁺ Gr- l ^low or CD1 lb ⁺ Ly6G ^neg Ly6C ^high .

[0063] Specific markers for T-cells, B-cells, NK cells, and platelets are known in the art and these cells can be identified by these markers. For example, in one embodiment the markers can be, T-cells (human/mouse: CD3 ⁺ , CD4 ⁺ , CD8 ⁺ ), B-cells (human: CD19 ⁺ ; mouse: B220 ⁺ , CD19 ⁺ ), NK cells (human: CD56 ⁺ ; mouse: NKG2D ⁺ ), platelets (human/mouse:

CD41 ⁺ CD49 ⁺ ), and tumor cells (pan-cytokeratin ⁺ ). [0064] As an example, the presence of the specific markers can be identified by using specific antibodies against these proteins. Such antibodies are commercially available. For example, specific antibodies to cell markers for MDSCs, T-cells, B-cells, NK cells, and platelets can be obtained from Biolegend, San Diego, CA, BD Biosciences, San Jose, CA, R&D Systems, Minneapolis, MN, BioXCell, West Labanon, NH, and eBiosciences, San Diego, CA, and others. Identification can be carried out by multiparameter phenotypic analysis. Identification of specific cell types in a cluster can be performed by detection of specific markers in those clusters. Clusters (including a two cell cluster) can be distinguished over a single cells. For example, a two MDSC cell cluster can be distinguished from a single MDSC based on size and visual analysis of the cells within clusters that are stained with markers for MDSCs or by flow cytometric analysis.

[0065] In an embodiment, the present method comprises obtaining a blood sample from an individual, contacting the blood sample with binding agents to identify a plurality of markers and based on the presence or absence of the markers, identifying the presence of homotypic PMN-MDSC or M-MDSC clusters. A cluster is identified as being homotypic if all the cells within the cluster express the same markers. For example if all the cells within a cluster express markers for PMN-MDSC the cluster would be considered a homotypic PMN- MDSC cluster, and if all the cells within a cluster express markers for M-MDSC, the cluster would be considered a homotypic M-MDSC cluster. If one or more cells within a cluster expressed markers for PMN-MDCS and one or more cells within the same cluster expressed markers for M-MDSC, the cluster would be considered a heterotypic MDSC cluster.

[0066] In an embodiment, the present method comprises obtaining a blood sample from an individual, contacting the blood sample with binding agents to identify a plurality of markers and based on the presence or absence of the markers, identifying the presence of homotypic M-MDSC clusters.

[0067] In an embodiment, the present method comprises obtaining a blood sample from an individual, contacting the blood sample with binding agents to identify a plurality of markers and based on the presence or absence of the markers, identifying the presence of heterotypic PMN-MDSC/M-MDSC clusters.

[0068] In an embodiment, the present method comprises obtaining a blood sample from an individual, contacting the blood sample with binding agents to identify a plurality of markers and based on the presence or absence of the markers, identifying the presence of homotypic PMN-MDSC clusters, homotypic M-MDSC clusters, and heterotypic PMN- MDSC/M-MDSC clusters. [0069] The present observation of MDSC-T-cell cluster formation in vitro in blood samples can be used as a screening method for identification of agents that are able to disrupt such clusters. The method comprises obtaining a blood sample from an individual, exposing aliquots of the blood sample to candidate agents, determining if the agents result in disruption of the clusters or if the agents affect any other features of the clusters. Promising agents can then be processed for in vivo effects on the treatment of cancer.

[0070] In an embodiment, the present method comprises obtaining a blood sample from an individual, and within a period of up to 1 to 2 hours, preparing a blood smear and carrying out a histological analysis of the prepared blood smear to detect and quantify CMCs, which contain two or more myeloid cells, which can be identified by neutrophil-like features such as segmented nuclei, and/or in some cases lymphocytes that are identified by a high nucleancytoplasmic ratio. The MDSC containing clusters are typically distinguishable from normal neutrophils because the neutrophils from healthy individuals do not tend to form clusters.

[0071] The present method can be used for detecting the presence of CMCs in cancers or other conditions. For example, the present method can be used to identify the status or metric of an individual' s immunological status in patients with chronic infections, such as tuberculosis, Staphylococcus aureus, hepatitis viruses (hepatitis B virus (HBV), hepatitis C virus (HCV)), and human immunodeficiency viruses (HIV) and fungal and parasitic infections, or patients with autoimmune diseases, such as Type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease.

[0072] If CMCs are identified, the individuals can be subjected to additional therapeutic regimens to bring the CMCs to a desired status. This may involve administrations aimed at increasing or decreasing CMCs.

[0073] For example, in cases of persistent chronic infections, the presence of CMC could suggest using more aggressive therapeutics/antibiotics or potentially using therapies to decrease CMCs. As another example, in the case of autoimmune disease, suboptimal levels of CMC may contribute to the severity of the disease. Thus, in autoimmune patients where measurements for CMC are low-to-non-detectable, strategies might be warranted to elevate the CMC levels by treating patients with cytokine therapy (G-CSF or GM-CSF) or by adoptive transfer of MDSC-based therapeutics. Thus, in an embodiment, this disclosure provides a method of treatment of an individual afflicted with an autoimmune disease comprising prior to administration of an anti-immune therapy or during such therapy, a regimen of cytokine therapy (e.g., G-CSF or GM-CSF) or adoptive cell transfer therapy (e.g., MDSC-based therapeutics).

[0074] In an embodiment of the disclosure, measurements of CMC provide a metric for the immunological fitness of patients. For example, the presence of CMCs can be used for patient prognosis or whether a patient is likely to be resistant or refractory to

immunotherapies. A level of CMCs over a control level established from normal individuals (also referred to herein as "normal level", "control level" or "control") can be a measure or indicator of an immune-suppressed status and could be used to inform decisions about whether a cancer patient should be treated with a T cell-based immunotherapy or if a cancer patient should be treated with a myelodepleting agent to reduce MDSC numbers prior to treatment with the immunotherapy. A control level may be established from an individual or set of individuals who are known to not have an immunological abnormal condition, such as, as judged by clinical evaluation. As an example, in mice, a value above 0.5 CMC per microliter of blood is considered above normal (determined by flow cytometry) or 0.05 CMC per mm ² is considered above normal (determined by histology in blood smears). Similarly, analysis of normal human blood samples can be carried out to determine normal CMC values in humans. In an embodiment, elevated levels of CMC are observed that can be from 2X to 1000X (and all values therebetween) relative to control levels. For example, elevated levels of CMCs can be 10, 50, 100, 250, 500, 750 times that of control levels.

[0075] In an embodiment, if elevated CMCs are detected compared to control values, then the individual may be considered suitable for, and therefore administered, chemotherapy rather than immunotherapy, or the individual can be treated with agents to reduce the CMC level before immunotherapy treatment. Chemotherapeutic approaches to treat cancer are well-known and selection of the particular chemotherapeutic agent depends upon the type and stage of cancer, and the specifics of the individual. Examples of chemotherapeutic agents include paclitaxel, cyclophosphamide, gemcitabine and many others. In an embodiment, if CMCs become elevated during the course of immunotherapy treatment, that this would warrant closer monitoring of patients. Patients may be subjected to follow up further analysis to obtain definitive evidence of acquired resistance to immunotherapy or cancer

recurrence/progression. The follow-up analysis may include flow cytometry analysis, or other investigative analyses.

[0076] In an embodiment, patients treated with T cell-based immunotherapeutics

(including, but not limited to, immune checkpoint inhibitors, adoptive T cell transfer therapy, or cancer vaccines) can be monitored for changes in CMC during their course of treatment; for instance, if CMC become elevated, this could provide the basis for combined treatment with MDSC-depleting regimens. Examples of T cell-based immunotherapies include adoptive cell transfer therapies in which patients are infused with their own immune cells (e.g., T cells include enriched populations of tumor-reactive T cells, genetically-engineered CAR-T cells (chimeric antigen receptor T cells) or T cell receptor- engineered T cells, and natural killer cells ( K cells; FATE-NKIOO)); cancer vaccines including dendritic cell (DC)-based vaccines; or antibody therapies directed against immune checkpoints (PD-1 (nivolumab, pembrolizumab, PDR001, MEDI4736/duralumab, ABBVI- 181), PD-L1, CTLA-4 (ipilimumab, tremelimumab ), LAG-3 (TSR-033), Tim-1 (TSR-022)) or immune-activating antibodies (e.g., directed against 41BB (utomilumab);Ox40 (PF- 04518600, ABBV-368 ); and CD122 (NKTR-262, NKTR-214).

[0077] In an embodiment, the present disclosure provides a method for administering an immunotherapeutic treatment to an individual in need of treatment comprising conducting a test for detecting the presence of CMCs, and if such presence is identified, then identifying that the individual will be resistant or refractory to immunotherapy, and treating the individual by MDSC depleting regimens prior to, or concurrent with immunotherapeutic treatment.

[0078] In an embodiment, the disclosure provides a method of treatment of an individual with cancer comprising detecting the presence and/or levels of CMCs in the individual, and if the CMC levels are considered to be elevated, then administering MDSC depleting regimens to the individual, and following the administration of the MDSC- depleting regimen or subsequent to it, administering a cancer therapeutic regiment, such as immunotherapeutic regimen. Examples of MDSC-depleting regiment includes administration of 5-fluorouricil/5-FU, histone deacetylase (HDAC) inhibitor (entinostat), hypomethylating agents (decitabine), and liver-X nuclear hormone receptor agonist (RGX-104).

[0079] In an embodiment, the disclosure provides a method for treatment of an individual with chronic infections, such as M. tuberculosis, S. aureus, hepatitis viruses (hepatitis B virus (HBV), hepatitis C virus (HCV)), and human immunodeficiency viruses (HIV) and fungal and parasitic infections, comprising conducting a test for detecting the presence of CMCs, and if such presence is detected, then treating the individual with MDSC depleting therapy in conjunction with treatment of the chronic infection.

[0080] In an embodiment, this disclosure provides a method for in vitro analysis of

MDSC cell cluster formation in blood removed from an individual comprising collecting a blood sample from an individual and determining the formation of cell clusters comprising at least two MDSCs, or at least one MDSC and one or more lymphocytes. The lymphocyte can be T-cell, B-cell, and NK cell or other leukocytes or can be platelets. In an embodiment, the cluster is free of tumor cells. The formation of cell clusters can be done on a blood smear and visualized under a microscope. The number and size of clusters can be determined, such as by flow cytometry.

[0081] The following examples are provided as illustrative examples and are not intended to be restrictive in any way.

EXAMPLE 1

[0082] Results

[0083] Spatiotemporal correlation between L-selectin loss and MDSC co- localization with na ^' ive T cells in the splenic compartment

[0084] To gain insight into the anatomical location where L-selectin downregulation occurs in vivo we mapped MDSC expansion and L-selectin density on na ^' ive T cell subsets in lymphoid organs where expression of this LN homing receptor is known to be under tight control. In this regard, it is considered that during normal development T cell precursors leave the bone marrow and emigrate to the thymus where they differentiate into L-selectin ⁺ mature T cells. After exiting the thymus, na ^' ive CD4 ⁺ and CD8 ⁺ T cells use L-selectin to traffic directly into LN via gatekeeper HEV, or recirculate through spleen which is devoid of HEV and does not require L-selectin for entry. In order to maximize the potential for detecting MDSC functions in various lymphoid organs we opted to use the 4T1 mammary tumor model in which tumor-produced granulocyte-colony stimulating factor drives robust expansion of MDSC. We excluded non-lymphoid organs and tumor tissues from this analysis since na ^' ive T cells do not recirculate at a high frequency at these sites.

[0085] We found the thymus was devoid of myeloid cells co-expressing the canonical murine MDSC markers CD1 lb and Gr-1 despite high systemic MDSC burdens in 4T1 tumor- bearing BALB/c female mice {Figure 1A). Moreover, L-selectin was not altered on thymic na ^' ive CD4 ⁺ CD44 ^l0 and CD8 ⁺ CD44 ^l0 T cells when compared to non-tumor bearing controls (NTB). MDSC were also rare in peripheral LN (pLN) of 4Tl-bearing mice, including tumor- draining inguinal LN, which correlated with the absence of L-selectin modulation on naive T cells at these sites (Figure 1A). MDSC exclusion from LN is likely explained by their low L- selectin expression as compared to normal naive T cells, (Figure 2). The uniformly high L- selectin density detected on intranodal T cells in both control and tumor-bearing mice is suggestive of a stringent requirement for a high L-selectin threshold for entry of blood-borne T cells across HEV.

[0086] In contrast, we detected profound L-selectin downregulation in naive CD4 ⁺ and CD8 ⁺ T cells that was associated with substantial MDSC elevation in the blood and splenic compartment of female and male 4T1 -bearing mice {Figure I A and Figure 3 A). Splenic CD1 lb ⁺ Gr-1 ⁺ cells from 4Tl-bearing mice were confirmed to exhibit prototypical MDSC suppressor function defined by potent inhibition of CD3/CD28-driven proliferation of CD4 ⁺ and CD8 ⁺ T cells (from non-tumor bearing mice) in vitro {Figure IB). Conversely, control CD 1 lb ⁺ cells from non-tumor bearing mice were not immunosuppressive. Data showing an overall decrease in naive T cells in LN, together with an increase in spleens of 4T1 -bearing mice without changes in the apoptotic index {Figure 1C), support the notion that suboptimal L-selectin expression reduces T cell access to LN and causes compensatory redistribution to the spleen.

[0087] We further determined that L-selectin loss on splenic CD4 ⁺ and CD8 ⁺ T cells correlated temporally with the extent of MDSC expansion which varied among different murine tumor types. Thus, while MDSC expansion early during tumor progression (i.e., 7 days post-4Tl implantation) coincided with significant L-selectin downregulation on naive CD4 ⁺ and CD8 ⁺ T cell subsets, even greater L-selectin loss occurred at later time-points with higher 4T1 tumor burdens at subcutaneous sites or the mammary fat pad {Figure 1 -Figure 3B-D). Compared to the 4T1 system, MDSC expansion was delayed in C57BL/6 mice implanted with AT-3 mammary tumor cells derived from genetically-engineered MMTV- PyMT/B6 transgenic mice (MTAG) (Waight et al ., J Clin Invest 123(10): 4464- 4478.10.1172/JCI68189), corresponding with moderate but significant L-selectin

downregulation on naive T cell subsets at >21 days post-tumor implantation {Figure 1- Figure 4A and 4B). L-selectin downregulation also occurred on splenic T cells in other tumor models including B 16 melanoma and CT26 colorectal tumor, but only in rare individual mice with abundant MDSC {Figure 1-Figure 5).

[0088] Clues about the spatial regulation of L-selectin emerged from

immunohistological staining of the spleen that revealed dense focal accumulations of Gr-1 ⁺ cells congregating with CD3 ⁺ T cells in the marginal zone that were sharply segregated from intrafollicular B220 ⁺ B cells in 4Tl-bearing mice {Figure ID). Additional insight into the MDSC mechanism of action came from profiling of splenic B cells that showed that while these cells express relatively low L-selectin compared to T cells, there was no change in L- selectin density on splenic B cells of tumor-bearing mice {Figure IE). This differential regulation of L-selectin in splenic T and B cells suggests a model in which close physical contact with MDSC is a prerequisite for L-selectin loss.

[0089] L-selectin downregulation occurs on both T and B cells in the blood compartment. To determine if L-selectin loss in T cells is restricted to the spleen or also occurs outside organized lymphoid organs, we performed proof-of-principle experiments in splenectomized mice. Mice were sham-surgically treated or splenectomized 10 days prior to implantation of 4T1 tumors {Figure 6A). We validated that 4T1 tumor growth and MDSC expansion in the blood was equivalent in sham and splenectomized mice at 21 days post- tumor implantation (Figure 6B and C), offsetting any concern that splenectomy might reduce the circulating MDSC burden. We then performed adoptive cell transfer (ACT) of L- splenic T cells derived from non-tumor bearing mice. L-selectin fate was assessed on transferred cells recovered from the blood after 24 hours.

[0090] High L-selectin expression was maintained following transfer of CD8 ⁺ T cells

(input cells) into sham, non-tumor bearing controls whereas substantial L-selectin loss occurred within a 24 hour window after transfer into sham-treated 4T 1 -bearing mice with high MDSC burdens (Figure 6C). We surprisingly found that the extent of L-selectin downregulation on adoptively-transferred CD8 ⁺ T cells was indistinguishable whether tumor- bearing recipient mice underwent sham surgery or splenectomy. These results suggested that L-selectin down-modulation on CD8 ⁺ T cells can occur in the intravascular space and does not require structural scaffolds provided by organized tissue compartments.

[0091] Additional studies examined L-selectin regulation on endogenous circulating

CD4 ⁺ T cells and B220 ⁺ B cells in the context of splenectomy. Like CD8 ⁺ T cells, we found that L-selectin was strongly downregulated on CD4 ⁺ T cells of tumor-bearing mice regardless of whether there was an intact splenic microenvironment (Figure 7). In the case of B220 ⁺ B cells, the varying levels of baseline L-selectin detected in lymphoid organs of tumor-free mice was expected since L-selectin is known to fluctuate after B cells exit the bone marrow and recirculate through the blood and spleen. Moreover, we did not detect L-selectin loss in the bone marrow and splenic compartments despite increased MDSC burden in sham or splenectomized tumor-bearing mice (Figure 8). In sharp contrast, L-selectin was nearly completely downregulated on blood-borne B220 ⁺ B cells in both sham and splenectomized tumor-bearing mice (Figure 8). These observations allowed us to pinpoint the blood compartment as the preferential site of L-selectin modulation for B cells in tumor-bearing mice. Similar results were obtained for L-selectin downregulation on circulating T and B lymphocytes if the timing sequence was reversed by allowing tumor-induced MDSC to accrue prior to splenectomy (Figure 9). The conclusion that blood is a prominent site of L- selectin loss was further supported by the strong L-selectin downregulation observed only 2 hours after adoptive transfer of naive CD8 ⁺ and CD4 ⁺ T cells and B220 ⁺ B cell into the MDSC-rich vascular compartment of 4Tl-bearing mice (Figure 10). Labeled cells detected in the blood at this short time-point mainly represent transferred populations retained in the vascular compartment since 2 hours is not sufficient for lymphocytes to recirculate from blood, through tissues, and back to the blood (e.g., transit times for T cells through lymph nodes, spleen, and peripheral tissues are -8-12 , 5, and 24 hours, respectively, and -24 hours for B cells at these tissue sites) (Ford et al., 1979, J Clin Pathol Suppl (R Coll Pathol) 13: 63- 69; Issekutz et al., 1982, Immunology 46(1): 59-66; Girard et al., 2012, Nat Rev Immunol 12(1 1): 762-773.10.1038/nri3298).

[0092] L-selectin loss was also detected in circulating T and B cells in MTAG mice with a high cumulative mammary tumor burden (-9,000 mm ³ ) and moderate MDSC expansion (14% of CD45 ⁺ peripheral blood cells), but not in MTAG mice with low MDSC (7% of CD45 ⁺ cells) and tumor burdens (-2,500 mm ³ ) (Figure 11). L-selectin on

CD3 ⁺ CD45RA ⁺ naive human T cells was additionally shown to be subject to downregulation following adoptive transfer of normal donor-derived human peripheral blood lymphocytes into MDSC ^M 4T1 -bearing severe-combined immunodeficient (SCID) mice (Figure 12), indicating that a non-species restricted mechanism was operative in vivo. Collectively, these findings provide evidence that the MDSC-enriched blood compartment of tumor-bearing mice is a major site of L-selectin downregulation and that both T and B lymphocytes are targeted.

[0093] MDSC cause L-selectin loss through a contact-mediated mechanism independent of ADAM17. We took several approaches to explore MDSC contributions to reducing L-selectin in vivo. Partial MDSC depletion (-50%) by administration of anti-Gr-1 depleting antibody at 3 day intervals after 4T1 implantation had no impact on tumor growth but significantly rescued L-selectin on circulating naive T and B cells (Figure ISA andB). Complementary studies showed that L-selectin loss was reversible in an environment devoid of MDSC. In this regard, when CD1 lb ⁺ MDSC were depleted from L-selectin ¹⁰ splenic CD4 ⁺ and CD8 ⁺ T cells (from 4Tl-bearing mice) we observed complete L-selectin recovery within 4 days after culture or post-adoptive transfer into non-tumor bearing recipient mice (Figure 13C), demonstrating that continued exposure to MDSC is required to maintain L-selectin down-modulation. Finally, MDSC from tumor-bearing mice (>95% CDl lb ⁺ Gr-l ⁺ ) but not CD1 lb ⁺ cells from non-tumor bearing controls were shown to cause moderate but significant L-selectin loss during co-culture with splenic CD8 ⁺ T cells for 24 hours (Figure J 3D).

Moreover, MDSC acted through a contact-dependent mechanism that was abrogated if MDSC were physically separated from target lymphocytes by cell-impermeable transwell inserts (Figure J 3D). Taken together, these results establish that MDSC directly target lymphocytes for L-selectin loss.

[0094] Further investigation into the mechanisms underlying MDSC activity showed that L-selectin mRNA levels detected by quantitative RT-PCR were unchanged in splenic CD4 ⁺ and CD8 ⁺ T cells of 4Tl-bearing mice compared to non-tumor bearing controls (Figure 14A), indicating that L-selectin loss does not involve transcriptional repression in vivo.

MDSC-mediated L-selectin downmodulation was instead accompanied by a 2.5-fold increase in soluble (s)L-selectin in the serum which was in line with a sheddase-dependent mechanism operative in vivo (Figure 14E). L-selectin is a well-known target of the AD AMI 7 ecto- protease which operates in cis to cleave substrates on the same membrane surface. We investigated the role of ADAM 17 in MDSC-induced L-selectin loss.

[0095] Head-to-head comparison between the well-established phorbol myristate acetate (PMA)-induced ADAM17 pathway versus MDSC-directed L-selectin loss on the surface of CD8 ⁺ T cells (Figure 14C and 14D) or CD4 ⁺ T cells and B220 ⁺ B cells (data not shown) revealed a sharp demarcation in their AD AMI 7 requirements in vitro. Thus, in agreement with the obligate role of ADAM 17 reported for PMA-induced L-selectin shedding, we found that PMA-induced loss of lymphocyte L-selectin was abrogated (1) by an

ADAM17-specific inhibitor (PF-5480090) and by a dual AD AMI 7/ AD AMI 0 inhibitor (INCB7839); (2) in cells from L(E)-selectin mice expressing a mutated ADAM cleavage site due to substitution of the L-selectin membrane-proximal extracellular domain with the shorter E-selectin homologous domain; or (3) in ADAM17-deficient lymphocytes from Adaml7fi ^ox/ fi ^ox /Vavl-Cre mice cultured alone (Figure 14C) or co-mixed with wildtype cells (Figure 15). Together these findings confirm reports of a strict requirement for czs-acting ADAM17 for PMA-induced L-selectin down-modulation. In contrast, MDSC-induced L- selectin downregulation in vitro was unaffected by inhibitors of AD AMI 7 or

ADAM 17/ AD AM 10; L(E)-selectin mutation; or lymphocyte-intrinsic ADAM17 deficiency (Figure 14D). Further, while elevated constitutive L-selectin expression in mutant L(E)- selectin lymphocytes or on ADAM17- ⁷ - cells was indicative of an AD AMI 7 mechanism in vivo , this pathway was dispensable for MDSC-induced L-selectin downregulation in mutant L(E)-selectin-expressing T and B cells or in ADAM17 ^7' cells following their adoptive transfer into MDSC 4T1 -bearing SCID mice (Figure 14E). Collectively, these data exclude a role for ADAM17 or ADAM10 in either a cis or trans orientation for MDSC-induced L- selectin loss and are suggestive of the involvement of another ecto-protease.

[0096] L-selectin loss reduces murine CD8 ⁺ T cell trafficking across LN HEV.

Observations that early tumor development is associated with moderate L-selectin loss raised the question of whether this would be sufficient to compromise trafficking, particularly since L-selectin is present in such excess on leukocyte surface membranes. To address the functional consequence of moderate L-selectin loss we isolated L-selectin ¹¹¹ CD8 ⁺ T cells (>90% purity) from spleens of non-tumor bearing controls (NTB CD8 ⁺ ) or L-selectin intermediate-to-low (L-selectin ^ini/l0 ) CD8 ⁺ cells from AT-3-bearing mice (AT-3 CD8 ⁺ ) {Figure 16A). Cells were then labeled ex vivo with tracking dye and their adhesive behavior was visualized in real-time by epifluorescence intravital microscopy in LN venules of non- tumor bearing recipients. For L-selectin CD8 ⁺ T cells, tethering and rolling interactions and firm sticking occurred primarily in high-order (III-V) postcapillary venules that constitute the HEV (Figure 16B and C;). L-selectin-mediated tethering and slow rolling on HEV ligands termed peripheral LN addressin (PNAd) is a prerequisite for CC-chemokine receptor-7

(CCR7) engagement of CCL21 which, in turn, triggers stable binding of LFA-1 integrin to endothelial ICAM-1/2. Minimal adhesion of L-selectin ¹¹¹ CD8 ⁺ T cells occurred in low-order venules (LOV; e.g., order II venules) lacking PNAd or CCL21 (Figure 16B and C).

[0097] L-selectin ^{mt lo} CD8 ⁺ T cells from AT-3-bearing mice surprisingly exhibited a normal frequency of tethering and rolling interactions in HEV (Figure 16C). However, we detected a significant decrease in their transition from rolling to firm arrest in order IV and V venules (Figure 16C) despite normal expression and function of CCR7 and LFA-1, as determined by flow cytometric profiling, CCL21-driven chemotaxis assays, and LFA-1- dependent homotypic aggregation assays {Figure 17A-C). An explanation for these paradoxical findings was revealed by data showing defective rolling behavior of L- selectin ^int/1 ° CD8 ⁺ T cells, as evidenced by their significantly faster median rolling velocity when compared to L-selectin ¹¹¹ CD8 ⁺ T cells in order V venular segments (65.0 versus 37.2 μπι/sec, respectively; Figure 6D andE).

[0098] Competitive short-term homing assays further identified a defect in the ability of L-selectin ^int/1 ° cells to extravasate across LN HEV and enter the underlying parenchyma. For these studies, enriched populations of L-selectin ^M and L-selectin ^int/1 ° CD8 ⁺ T cells from non-tumor bearing mice and AT-3-bearing mice, respectively, were labeled with different tracking dyes ex vivo, co-mixed at a 1 : 1 ratio, and transferred intravenously into tumor-free recipients. The impact of L-selectin deficits on trafficking was assessed 1 hour later in peripheral LN under homeostatic (control) conditions and under conditions of heightened HEV function as encountered in inflamed lymph nodes. Homeostatic trafficking of L- selectin ^int/1 ° CD8 ⁺ T cells across LN HEV was strongly inhibited (-80%) when compared to L-selectin ¹¹¹ T cells {Figure 18A). Transferred CD8 ⁺ T cells that successfully extravasated across LN HEV during homeostatic trafficking were uniformly L-selectin ¹¹¹ regardless of whether they originated from non-tumor bearing mice or AT-3-bearing mice {Figure 18B), suggesting that a high L-selectin density is necessary to stabilize T cell adhesion during extravasation. In contrast, differential L-selectin expression was maintained on transferred CD8 ⁺ T cells recovered from the spleen where trafficking is not dictated by L-selectin status {Figure 18A and E). Reduced CD8 ⁺ T cell trafficking due to moderate L-selectin loss was also observed in an inflammatory model in which HEV express elevated CCL21 and ICAM-1 in response to whole body hyperthermia (WBH) {Figure 18 A; Figure 19). Taken together, these results establish the biological significance of MDSC-induced L-selectin loss in limiting T cell access to LN via HEV portals.

[0099] Reduced L-selectin-dependent trafficking compromises antigen-driven activation in LN. We formally tested the prediction that MDSC-directed downregulation of L-selectin-dependent trafficking diminishes T cell responses to cognate antigen within the LN compartment using CD8 ⁺ OT-I transgenic mice expressing T cell receptors (TcR) specific for OVA257-264 peptide (SIINFEKL (SEQ ID NO: l)). L-selectin ¹¹¹ and L-selectin ^int/1 ° CD8 ⁺ T cells were purified from spleens of non-tumor bearing OT-I mice and AT-3-bearing OT-I mice, respectively (e.g., as in Figure 16A). These OT-I cells were then labeled ex vivo with different proliferation dyes and co-mixed at a 1 : 1 ratio to assess functional responses to antigen in competitive activation assays in vitro and in vivo {Figure 20A).

[00100] Since prior studies reported that T cells isolated from tumor-bearing mice and cancer patients have intrinsically diminished antigen responsiveness (Alexander et al., 1993, Cancer Res 53(6): 1380-1387, Jiang et al., 2015, Cell Death Dis 6:

el792.10.1038/cddis.2015.162), we first established a relative baseline level of function for CD8 ⁺ OT-I T cells from tumor-bearing mice under in vitro conditions where access to antigen is L-selectin-independent {Figure 20A andB). Co-cultures of OT-I cells from non- tumor bearing mice and AT-3-bearing transgenic mice (i.e., 1 : 1 ratio) were stimulated in vitro for 4 days with SIINFEKL (SEQ ID NO: l)-loaded bone marrow-derived dendritic cells (DC). OT-I T cells from non-tumor bearing mice were >2 times more responsive to antigen- driven proliferation than OT-I cells from AT-3-bearing mice {Figure 20B and C). We considered that these results might be explained by T cell preconditioning (i.e., before antigen exposure) by MDSC within tumor-bearing mice. Thus, we set up parallel in vitro culture systems to model the high splenic MDSC concentrations that T cells would encounter in tumor-bearing mice {Figure 21A). These studies revealed that transient co-culture of MDSC with naive OT-I CD8 ⁺ or DO 1 1.10 CD4 ⁺ TcR-transgenic cells (i.e., 16 hour 'preconditioning' phase), followed by removal of MDSC, markedly suppressed T cell proliferation during subsequent challenge with cognate peptide antigens {Figure 21B).

[00101] We next examined the impact of L-selectin deficits on antigen-responsiveness in an in vivo model that depends on L-selectin-dependent trafficking for access to Ag. In this regard, a 1 : 1 ratio of L-selectin ^M OT-I cells and L-selectin ^{int 1} ° OT-I cells was adoptively transferred into non-tumor bearing recipients that were pre-vaccinated in the footpad with SIINFEKL (SEQ ID NO: l)-pulsed DC {Figure 2 OA). We used tumor-free recipients to interrogate the causal relationship between L-selectin loss and impaired adaptive immunity which would be difficult to assess in tumor-bearing mice because of the additional immunosuppressive mechanisms operative in LN (e.g., tolerogenic DC, Treg). Flow cytometric analysis of transferred cells recovered from the draining popliteal LN (dLN) 4 days after adoptive transfer revealed that activated OT-I T cells from tumor-free donors outnumbered OT-I cells from AT-3-bearing donors by -70: 1 {Figure 20C). These OT-I cells were largely differentiated effectors based on interferon-γ production {Figure 20D). This biased response by L-selectin OT-I T cells was not observed outside the primary site of antigen exposure including contralateral LN (cLN) or spleen {Figure 20C). Taken together, the profound discrepancy between antigen responsiveness of L-selectin ^M versus L-selectin ^mt/1 ° OT-I T cells in vitro and in vivo (2: 1 versus 70: 1 ratio, respectively; Figure 20B and 20C) supports a model in which MDSC operate at distal sites (i.e., blood and spleen compartments) to subvert adaptive immunity by restricting L-selectin-directed access of naive CD8 ⁺ T cells to cognate antigens within the LN microenvironment {Figure 22).

[00102] Discussion

[00103] Regional LN are major lines of defense against cancer, serving as hubs for the generation of acute antitumor adaptive immunity and durable memory. A rate-limiting step for immune surveillance involves trafficking at HEV which ensures that DC present cognate antigens to a sufficiently diverse repertoire of naive T cells in order to drive expansion of CD4 ⁺ and CD8 ⁺ effector T cell pools and B cell antibody production. Here we provide evidence that tumor-induced MDSC act from remote sites through two independent mechanisms to suppress systemic adaptive immunity in widely dispersed LN. The major findings of the current study establish that MDSC located in anatomically discrete sub- compartments of the spleen and, unexpectedly in the blood, downregulate expression of the L-selectin LN homing receptor in murine CD4 ⁺ and CD8 ⁺ naive T cell subsets and on blood- borne murine B cells. In the case of naive CD8 ⁺ T cells, even moderate L-selectin loss severely limits trafficking across HEV, causing a profound reduction in antigen-driven expansion within the LN stroma. Thus, MDSC-induced L-selectin down-modulation could significantly impair immune surveillance during the early phases of tumor escape as well as compromise immunotherapy regimens dependent on immune cell access to LN. Additionally, our results reveal that T cells can be preconditioned by MDSC in the spleen or blood, resulting in diminished responsiveness to subsequent challenge with cognate antigen.

Collectively, these findings lead us to propose that LN are important sites of MDSC suppression which could not be predicted from routine profiling of immune constituents since MDSC are largely excluded from this lymphoid compartment.

[00104] Results of the current study expand our understanding of the biological role of splenic and circulating MDSC in repressing immune function in a setting of tumor progression. The spleen is already considered a major reservoir for peripheral MDSC with direct immunosuppressive activity, while the blood is not known to be an active site of MDSC function in situ. We found discrete compartmentalization of MDSC within the splenic marginal zone of tumor-bearing mice. This localization was linked to specific downregulation of L-selectin on T cells but not B cells, consistent with MDSC exclusion from B cell follicles. Whether this restricted spatial distribution of MDSC is a byproduct of their profuse systemic accumulation or reflects preferential trafficking and/or retention is an open question. We initially considered that splenic MDSC act directly on naive T cells only within the splenic stroma, which is then reflected in the blood as L-selectin ^int/1 ° T cells exit the spleen. However, subsequent analysis of splenectomized tumor-bearing mice revealed that the blood is a major site for MDSC-targeted L-selectin downregulation for both T and B cells. Evidence that L- selectin down-modulation is restricted to specific organs as well as sub-anatomical compartments supports a scenario in which MDSC-to-target cell contact is a prerequisite for L-selectin down-modulation, as validated by MDSC-T cell co-cultures in the present study. Regardless of whether the loss occurs in spleen or blood, L-selectin status is a critical determinant of homing potential since lymphocytes emigrating from each of these sites have nearly immediate access to LN HEV.

[00105] The extent of the homing defect in HEV is remarkable considering the modest change in overall L-selectin surface density observed for T cells in the AT-3 tumor model. During leukocyte rolling it is estimated that ~5 microvillous tips are in simultaneous contact with endothelial surfaces, with L-selectin concentration exceeding 90,000 molecules per μηι ² at a single microvillous tip (Shao et al., 1999, Biophys J 77(1): 587-596.10.1016/S0006- 3495(99)76915-8). Our results suggest that relatively subtle decreases in L-selectin density within these multivalent focal patches increase rolling speed, thus destabilizing adhesion within HEV. Our intravital data demonstrate that L-selectin modulation by MDSC is of primary importance during adhesion on the lumenal surface of HEV prior to extravasation.

[00106] The current results establish an unprecedented correlation between MDSC expansion, L-selectin loss by T and B lymphocytes, and a >2-fold increase in circulating sL- selectin in tumor-bearing mice. Emerging clinical studies have reported that serum sL- selectin levels are also increased in patients with solid tumors (i.e., bladder and thyroid cancer). Lymphocytes are the primary source of the circulating sL-selectin pool under normal conditions. Thus, increased serum sL-selectin in tumor-bearing mice likely reflects MDSC- induced cleavage from lymphocyte surface membranes although MDSC could be an additional source of sL-selectin. Accordingly, two independent but complementary mechanisms are proposed to reduce LN trafficking in cancer: (1) MDSC-directed

downregulation of lymphocyte L-selectin which we showed negatively impacts homing in HEV, and (2) elevated circulating sL-selectin which, at the concentrations detected in tumor- bearing mice (1 μg/mL in tumor-bearing mice compared to -0.4 g mL in tumor-free controls), reportedly functions as a competitive antagonist of L-selectin-directed lymphocyte homing in vivo.

[00107] Our results have broad translational implications for cancer immunotherapy including vaccines that aim to stimulate antitumor antibody production. The nearly complete loss of L-selectin detected on circulating B cells in response to MDSC in our study strongly suggests that B cell homing at HEV would be effectively blocked. Thus, antibody responses would be expected to be severely diminished as a result of MDSC-induced suboptimal trafficking of both naive B cells and CD4 ⁺ precursors of the follicular helper T cells required for T-dependent antibody production in LN. Collectively, our findings could provide an explanation for observations that patients diagnosed with premalignant advanced colonic adenomas who have elevated circulating MDSC failed to produce antibodies in response to vaccination with the MUC-1 tumor-associated antigen (Kimura et al., 2013, Cancer Prev Res (Phila) 6(1): 18-26.10.1158/1940-6207.CAPR-12-0275).

[00108] Emerging data also highlight the importance of LN trafficking for durable responses to T cell-based ACT cancer immunotherapy. Presently, there is strong interest in developing ex vivo activation protocols that maintain expression of LN homing receptors on T cell immunotherapeutics. Our findings suggest that an unexpected outcome of clinical ACT immunotherapy in patients with high MDSC burdens is that a high proportion of transferred T cells will undergo two major MDSC-mediated events that impair T cell responses in LN: (1) reduced LN homing due to rapid L-selectin loss within 24 hours, and (2) immune suppression stemming from T cell preconditioning prior to direct tumor-antigen exposure. These mechanisms would necessitate the transfer of excessive numbers of ex vivo- expanded T cells to achieve therapeutic efficacy.

[00109] In summary, here we uncovered combinatorial mechanisms of systemic immune suppression whereby tumor-induced MDSC localized outside LN shape the magnitude of adaptive immune responses in the intranodal compartment. Findings that L- selectin loss is not indelibly imprinted on T cells suggest that neoadjuvant treatments that target MDSC can provide immune-based cancer therapies that depend on T and B cell trafficking to lymph nodes. Additionally, these results expand general concepts about the immunobiology underlying the suppressive mechanisms of MDSC that could be operative during chronic infections and inflammatory disorders such as autoimmunity.

[00110] Materials and methods

[00111] Animals. Female, age-matched BALB/c and C57BL/6 mice (8-12 weeks) were from the National Cancer Institute (Fredrick, MD), Charles River (Wilmington, MA), or Taconic (Hudson, NY). Male BALB/c mice (8-12 weeks) were from Charles River.

Transgenic male and female OT-I mice (B6.12 S6-Rag2tmlFwa Tg(TcraTcrb)l lOOMjb or C57BL/6-Tg(TcraTcrab)l lOOMjb/J) were from Taconic or Jackson Laboratory (Bar Harbor, ME); DO11.10 mice (C.Cg-Tg(DOl l . l l)10Dlo/J) were from Jackson Laboratory. Female severe combined immunodeficiency mice (SCID; C.B Igh-lb Icr Tac Prkdc scid) were bred in-house by the Roswell Park Department of Laboratory Animal Resources, Buffalo, NY. MMTV-PyMT/B6 transgenic female mice (MTAG; >150 days of age with multifocal mammary tumors) expressing the polyomavirus middle T antigen controlled by the MMTV- LTR promoter were originally a gift from S. Gendler (Mayo Clinic, Scottsdale, AZ). Female and male Adam 17^ ^ox/ ^ ^ox /Vavl-Cre conditional knockout mice (6-14 weeks) in which all leukocytes lack functional ADAM17 were generated as described (Mishra et al., 2016, J Leukoc Biol AO A 189/jlb.3VMAB l 115-496RR). L(E)-selectin transgenic female and male mice (8 weeks) in which substitution of the membrane-proximal L-selectin extracellular domain with E-selectin prevents L-selectin shedding by ADAM17 were described previously (Venturi et al., 2003, Immunity 19(5): 713-724), and kindly provided by T. Tedder (Duke University School of Medicine). Sex- and age-matched wildtype littermates were included as controls. All mice were maintained under specific pathogen-free conditions in accordance with approved Institutional Animal Care and Use Committees protocols at participating institutions.

[00112] Tumor models. 4T1, B 16, and CT26 cells were cultured in complete media (RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 U ml ^"1 penicillin, 50 μg mΓ ¹ streptomycin, and 50 μΜ β-mercaptoethanol [Therm oFisher, Waltham, MA]). AT-3 were cultured in complete media supplemented with 1 mM sodium pyruvate, 1% MEM non-essential amino acids, and 25 mM HEPES (Therm oFisher). Tumor cells (10 ⁶ ) were injected s.c. into the left flank or into the mammary fat pad as described (Fisher et al., 2011, J Clin Invest 121(10): 3846-3859.10.1 172/JCI44952; Waight et al., 2011, PLoS One 6(1 1): e27690.10.1371/journal.pone.0027690; Waight et al., 2013, J Clin Invest 123(10): 4464-4478.10.1172/JCI68189. Peripheral lymph nodes included the left/right axillary, left/right brachial and left/right inguinal lymph nodes; the tumor-draining lymph node was identified as the left inguinal lymph node using Evans blue dye. Tumor short (I) and long (L) diameters were measured, and tumor volume was calculated as P x L/2. Studies were completed before tumors exceeded 20 mm in any diameter or mice became moribund.

MTAG mice developed up to 10 discrete mammary gland tumors and no single tumor mass was allowed to exceed 20 mm in any diameter; total tumor burden was between 1,000-12,000 mm ³ in individual mice.

[00113] Flow cytometry. Analysis of single cell suspensions was performed as described (Chen et al., Nat Immunol 7(12): 1299-1308.10.1038/nil406; Hanson et al., 2009, J Immunol 183(2): 937-944.10.4049/jimmunol.0804253; Fisher et al., 201 1, J Clin Invest 121(10): 3846-3859.10.1 172/JCI44952; Waight et al., 201 1, PLoS One 6(1 1):

e27690.10.1371/journal.pone.0027690; Waight et al., 2013, J Clin Invest 123(10): 4464- 4478.10.1172/JCI68189) using multiparameter flow cytometry with monoclonal antibodies. Annexin V (FITC) and viability reagents (Zombie UV and aqua) (Biolegend, San Diego, CA) were used to assess the percentage of early apoptotic cells (annexin V ⁺ Zombie UV ^'/dim ) within specific tissue compartments. Flow-count fluorospheres (Beckman Coulter, Brea, CA) were used to calculate absolute numbers of viable cells. A LSR Fortessa or LSR2 flow cytometer (BD Biosciences, San Jose, CA) was used for flow cytometric analysis;

compensation and analysis were performed using Winlist 7.1 or 8.0 (Verity Software House, Inc., Topsham, ME).

[00114] Immunofluorescence histology. Spleens were frozen in optimum cutting temperature compound (Sakura Finetek, Torrance, CA) and 9 μπι cryosections were fixed at room temperature in 4% formaldehyde for 10 min. Sections were blocked 1 hour with 5% FBS and 5% rat gamma globulin (Jackson ImmunoResearch, West Grove, PA) and stained overnight at 4°C with fluorochrome-labled anti-Gr-1, anti-B220, and anti-CD3 antibodies. Quantification of relative trafficking molecule expression on LN HEV was performed as described (Chen et al., Nat Immunol 7(12): 1299-1308.10.1038/nil406; Fisher et al., 2011, J Clin Invest 121(10): 3846-3859.10.1 172/JCI44952) in mice injected via tail vein with anti- CCL21 or anti-ICAM-1 antibodies 20 minutes before tissue harvest. Tissue cryosections were counter-stained with primary monoclonal anti-PNAd or anti-CD31 antibodies and fluorochrome-labeled secondary antibodies (i.e., goat anti-rat IgG, goat anti-Armenian hamster IgG, and goat anti-rat IgM). Digital images of >10 randomly selected fields per section (unit area of each field, 0.34 mm ² ) were captured by observers blinded to specimen identity using an Olympus BX50 upright fluorescence microscope (Olympus Optical, Miami, FL) equipped with a SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI);

identical exposure times and image settings were used within each experiment. ImageJ software (http://rsb.info.nih.gov/ij) was used to quantify the relative fluorescence intensity for FIEV staining as described (Abramoff et al., 2004, Biophotonics International 11(7): 36-42; Chen et al., 2006, Nat Immunol 7(12): 1299-1308.10.1038/nil406; Fisher et al., 2011, J Clin Invest 121(10): 3846-3859.10.1 172/JCI44952).

[00115] T cell suppression assays. To evaluate T cell suppression during continuous exposure to MDSC, splenic CD1 lb ⁺ Gr-l ⁺ cells were purified from non-tumor bearing (NTB) and 4Tl-bearing BALB/c mice (tumor volume >2,000 mm ³ ) using anti-CDl lb ⁺ magnetic beads (Miltenyi Biotec, San Diego, CA) as described (Waight et al., 201 1, PLoS One 6(11): e27690.10.1371/journal.pone.0027690). Isolated MDSC populations were >95% CDl lb ⁺ Gr- 1 ⁺ while >80% of the enriched NTB CD1 lb ⁺ cells coexpressed Gr-1 ⁺ . CD1 lb ⁺ Gr-l ⁺ cells were combined at the indicated ratios with CFSE-labeled (ThermoFisher) splenocytes from tumor-free BALB/c mice and cultured for 72 hours with anti-CD3/CD28 antibody-conjugated beads (1 μΐ per 100 μΐ culture, ThermoFisher) and IL-2 (30 U/mL; Peprotech, Rocky Hill, NJ). T cell proliferation was measured based on CFSE-dilution determined by flow cytometry: % suppression=[l - (proliferation with MDSC)/(mean proliferation without MDSC)] x 100.

[00116] To assess the suppressive effects of MDSC on T cells prior to exposure to antigen, splenocytes from OT-I and DO11.10 transgenic mice were 'preconditioned' by incubation overnight with MDSC from the blood of 4T1 -tumor-bearing BALB/c mice (>92% CDl lb ⁺ Grl ⁺ cells) (2xl0 ⁶ OT1 splenocytes±2-6xl0 ⁶ MDSC/4ml; MDSC:splenocyte ratios of 1 : 1 or 3 : 1). Alternatively transgenic T cells were cultured overnight in serum-free medium (HL-1; Lonza Scientific, Walkersville, MD) without MDSC. Six replicates per sample were then pooled and resuspended in 250 μΐ PBS-2% FCS, and incubated at room temperature for 10 min with biotinylated antibody to Gr-1. MDSC were then depleted using Rapidsphere streptavidin magnetic beads according to the manufacturer' s protocol (31.25 μΐ beads;

StemCell Inc., Newark, CA). Depleted populations ranged from 0.4-4% CDl lb ⁺ Gr-l ⁺ cells. Resulting splenocytes were then cultured for 3 days with or without cognate peptide (5 μg of OVA257-264 [SIINFEKL] (SEQ ID NO: l) or OVA323-339 [ISQAVHAAHAEINEAGR] (SEQ ID NO:2) for OT-I and DO 1 1.10, respectively; University of Maryland, Baltimore

Biopolymer Core facility, Baltimore, MD), pulsed with ³ H-thymidine (1 μΟ/50 μΐ/well; MP Biologicals, Santa Ana, CA) and harvested 16 hours later as described (Hanson et al., 2009, J Immunol 183(2): 937-944.10.4049/jimmunol.0804253).

[00117] Splenectomy. BALB/c mice were anesthetized with inhalational isoflurane gas (4% for induction, 1.5% maintenance). Splenectomy and sham surgeries were performed via a left subcostal laparotomy incision as described (cortez-Retamozo et al., 2012, Proc Natl AcadSci USA 109(7): 2491-2496.10.1073/pnas.1 113744109) either 10 days before or 14 days post-4Tl tumor implantation. The spleen was mobilized outside the abdomen and splenic vessels were cauterized. Buprenorphine (0.05 mg/kg) was administered i.p. for postoperative pain.

[00118] Isolation of CD8 ⁺ T cells for adoptive transfer. Mouse splenocytes were first subjected to CD1 lb ⁺ depletion using anti-CDl lb ⁺ magnetic beads (Miltenyi Biotec) unless otherwise indicated. To obtain an enriched CD8 ⁺ T cell population (routinely >90%), the CD1 lb ^neg fraction of splenocytes was then subjected to negative selection using a CD8 ⁺ magnetic bead separation kit (Miltenyi Biotec). Isolated CD8 ⁺ T cells were labeled with CFSE, CellTrace Violet, CellTracker Orange, or calcein (ThermoFisher) prior to intravenous adoptive transfer (1.5-4 x 10 ⁷ cells) into recipient mice.

[00119] In vivo depletion of MDSC Mice were injected i.p. with anti-Gr-1 antibody or isotype control antibody at 3 day intervals starting at day 3 post-tumor implantation as similarly described (Waight et al., 201 1, PLoS One 6(11):

e27690.10.1371/journal.pone.0027690). MDSC depletion was assessed by flow cytometric analysis.

[00120] In vivo and in vitro L-selectin recovery. Splenocytes from non-tumor bearing and 4Tl-bearing mice (tumor volume >1,000 mm ³ ) were depleted of CD1 lb ⁺ cells using anti- CDl lb ⁺ magnetic beads (<5 % residual CDl lb ⁺ Gr-l ⁺ ; Miltenyi Biotec). CDl lb ⁺ -depleted splenic cell populations were then labeled either with CellTrace CFSE or CellTrace Violet (Therm oFisher), respectively and co-mixed at a 1 : 1 ratio. For in vitro studies, the co-mixture was cultured at a concentration of 10 ⁶ cells ml ^-1 in complete media supplemented with 1 mM sodium pyruvate, MEM non-essential amino acids, and 25 mM HEPES. For in vivo recovery studies, 2-3 x 10 ⁷ cells of the co-mixture were adoptively transferred via the tail vein into non-tumor bearing recipients. L-selectin expression on CD4 ⁺ and CD8 ⁺ T cells was determined by flow cytometric analysis prior to transfer (input) or 4 days after culture or adoptive transfer into mice.

[00121] Adoptive transfer of human peripheral blood lymphocytes. Human peripheral blood-derived mononuclear cells from anonymous de-identified normal donors were collected from TRIMA leukoreduction filters (Trima Accel Collection System; CaridianBCT, Inc., Lakewood, CO) obtained from the Roswell Park Cancer Institute Pheresis Facility as per an approved Roswell Park Cancer Institute Review Board protocol. Leukocytes were isolated by Ficoll density gradient separation and monocyte-depleted lymphocytes were then prepared by cold aggregation as described (Tario et al., 2011, Methods Mol Biol 699: 1 19-

164.10.1007/978-l-61737-950-5_7). L-selectin expression on CD3 ⁺ CD45RA ⁺ human T cells was examined by flow cytometric analysis in the blood 24 hours post-adoptive transfer of 2-4 x 10 ⁷ cells via the tail vein into non-tumor bearing and 4Tl-bearing SCID mice. Murine CD1 lb ⁺ Gr-l ⁺ cell burden was determined by flow cytometric analysis and reported based on blood volume (i.e., number of CD1 lb ⁺ Gr-l ⁺ cells per μΐ of blood) instead of based on the %CD45 ⁺ cells since the lack of mature lymphocytes in SCID mice disproportionately skews MDSC representation within CD45 ⁺ populations.

[00122] Quantitative real-time PCR. Splenic CD4 ⁺ and CD8 ⁺ T cells from non-tumor bearing and 4T1 -bearing mice were positively selected by anti-CD4 ⁺ or anti-CD8a ⁺ - conjugated microbeads, respectively, (Miltenyi Biotec). Isolated T cell subsets were > 90% pure. Cellular RNA was extracted using the NucleoSpin R A kit (Machery-Nagel, Diiren, Germany) according to the manufacturer' s protocol. Quantification of total RNA extract was determined using the Nanodrop™ Lite Spectrophotometer (ThermoFisher Scientific, Waltham, MA). cDNA library was synthesized using i Script (Bio-Rad, Hercules, CA), which was then used for PCR amplification of murine β-actin and L-selectin. qPCR was performed using SYBR Green (ThermoFisher Scientific, Waltham, MA) on a CFX Connect™ Real- Time System (BioRad, Hercules, CA). PCR primers for β-actin: forward primer: 5'- AGAGGGAAATCGTGCGTGAC-3 ' (SEQ ID NO:3); reverse primer: 5'- CAATAGTGATGACCTGGCCGT-3 ' (SEQ ID NO:4). PCR primers for L-selectin: forward primer: 5'-CCAAGTGTGCTTTCAACTGTTC-3 ' (SEQ ID NO:5); reverse primer: 5'- AAAGGCTCACACTGGACCAC-3 ' (SEQ ID N0:6). The comparative Ct method was used to quantify L-selectin mRNA expression levels relative to endogenous β-actin.

[00123] ELISA for sL-selectin. Mouse serum was stored at -80°C for 2 months. Serum levels of soluble (s)L-selectin were measured by ELISA kit (R&D systems, Minneapolis, MN) with serum matrix equalizing diluent buffer (BIO-RAD, Kidlington, Oxford, UK).

[00124] MDSC-splenocyte co-culture assays for L-selectin modulation. Magnetic bead separation (Miltenyi Biotec) was used to isolate splenic CD1 lb ⁺ MDSC and CD 1 lb ⁺ control cells from 4T1 -bearing and NTB mice, respectively. Target splenocytes were harvested from additional age-matched NTB mice and labeled with CellTrace Violet (ThermoFisher). In studies involving L(E)-selectin or Adaml 7 ^" mice, splenocytes were shipped overnight at 4°C, and incubated at 37°C for a 5 hour-recovery period before initiation of experiments. Where indicated, MDSC and splenocytes were pretreated for 15 minutes with specific inhibitors for ADAM17 (PF-5480090, 10 μΜ; Pfizer, New York, NY) or

ADAM17/10-(INCB7839, 20 μΜ; Incyte, Wilmington, DE); inhibitors were also present throughout subsequent culture periods. MDSC (or CD1 lb ⁺ control cells) were combined with target splenocytes (at a 10: 1 ratio; i.e., 2 x 10 ⁶ myeloid cells and 2 x 10 ⁵ splenocytes) in round-bottomed 96-well plates (Corning, Corning, NY) in media (complete media supplemented with 1 mM sodium pyruvate, 1% MEM non-essential amino acids, and 25 mM HEPES) with or without 20 U/mL IFN-γ (Peprotech) and 100 ng/mL LPS (Sigma-Aldrich, St. Louis, MO). Additional cultures were set up in HTS Transwell 96-well permeable support systems with 0.4 μι polycarbonate membranes (Corning) with target splenocytes at the bottom of transwells. After 24 hours, viable T and B cells were analyzed by flow cytometry; dead cells were excluded using Zombie viability dyes (Biolegend).

[00125] PMA-induced L-selectin modulation assays. Splenocytes from wildtype NTB mice were labeled with CellTrace Violet, resuspended at a final concentration of 5 x 10 ⁶ in complete media supplemented with 1 mM sodium pyruvate, 1% MEM non-essential amino acids, and 25 mM HEPES, and cultured overnight at 37°C in round-bottomed 96-well plates (Corning). Splenocytes from L(E)-selectin mice, Adaml 7 ^~f~ mice or littermate wildtype controls were shipped overnight at 4°C, and then cultured at 37°C for 5 hour-recovery period before initiating experiments. As designated, wildtype splenocytes were pretreated for 30 minutes with specific inhibitors for ADAM17 (PF-5480090, 10 μΜ; Pfizer) or ADAM17/10, (INCB7839, 20 μΜ; Incyte) prior to addition of phorbol-12-myristate-13-acetate (PMA, 100 ng/mL; Calbiochem, San Diego, CA). After 2 hours, L-selectin expression on viable cells was assessed by flow cytometric analysis; dead cells were excluded using Zombie viability dye (Biolegend). The c/s-acting L-selectin cleavage function of AD AMI 7 was verified by PMA-stimulation (100 ng/niL, 2 hours) of co-cultures of wildtype splenocytes and fluorescently-labeled Adamll ^'1' splenocytes (both from NTB mice; cultured at 10: 1 ratio).

[00126] Intravital microscopy. Intravital microscopy of inguinal lymph nodes of non- tumor bearing mice was performed as described (Gauguet et al., 2004, Blood 104(13): 4104- 41 12; Chen et al., 2006, Nat Immunol ^' 7(12): 1299-1308.10.1038/ni l406). Briefly, mice were anesthetized (1 mg ml ^"1 xylazine and 10 mg ml ^"1 ketamine; 10 ml kg ^"1 , i.p.) and a catheter was inserted into the right femoral artery for the delivery of adoptively-transferred calcein- labeled (Therm oFisher) CD8 ⁺ T cells purified from spleens of non-tumor bearing mice and AT-3-bearing C57BL/6 mice. An abdominal skin flap was made to expose the left inguinal lymph node. CD8 ⁺ T cell interactions within postcapillary vessel walls were visualized with a customized Olympus BX51WI epi-illumination intravital microscopy system (Spectra Services, Ontario, NY). Rolling fraction, sticking fraction, and rolling velocity were determined as described using off-line measurements (Gauguet et al., 2004, Blood 104(13): 4104-41 12; Chen et al ., 2006, Nat Immunol 7(12): 1299- 1308.10.1038/ni 1406). The rolling fraction was defined as the percentage of total cells that transiently interacted with vessels during the observation period. The sticking fraction was defined as the percentage of rolling cells that adhered to vessel walls for >30 seconds. Rolling velocities in order V venules were determined using ImageJ software to measure the distances traveled by rolling cells over time.

[00127] Chemotaxis transwell assay. CD8 ⁺ T cells were negatively selected from the spleens of non-tumor bearing and AT-3-bearing mice by magnetic bead separation (Miltenyi Biotec). Purity of isolated populations was > 90%. Chemotaxis was assayed in 24 well plates with 5 μπι pore polycarbonate membranes (Corning) as described (Mikucki et al., 2015, Nat Commun 6: 7458.10.1038/ncomms8458). Media alone (complete media supplemented with 1 mM sodium pyruvate, 1% MEM non-essential amino acids, and 25 mM HEPES) or media containing 70 nM of recombinant murine CCL21 (Peprotech) was placed in the bottom chamber. CD8 ⁺ T cell migration was quantified after 3 hours using a hemocytometer.

Spontaneous migration was subtracted from all conditions, and data are reported as percentage of input cells for triplicates.

[00128] Homotypic aggregation assay. Splenic CD8 ⁺ T cells isolated by negative selection (Miltenyi Biotec) were tested for LFA-1 function by assessing PMA-induced homotypic aggregation as previously described (Isobe et al., 1991, Immunology 73(2): 159- 164). Briefly, T cells at a concentration of 5 x 10 ⁶ cells/mL in complete media supplemented with 1 mM sodium pyruvate, 1% MEM non-essential amino acids, and 25 mM HEPES (in flat-bottomed 96-well plates, Corning) were pretreated for 15 minutes with anti-CD 1 la blocking antibody specific for the OL subunit of LFA-1 (10 μg/mL; BD Biosciences). Cells were then treated with or without PMA (50 ng/mL; Calbiochem) for 18 hours. Cell aggregation was photographed using an inverted microscope. Cells were gently resuspended and counted by hemocytometer. Percentage aggregation = 100 x [1 - (number of free cells)/(number of input cells).

[00129] Whole body hyperthermia. Non-tumor bearing C57BL/6 mice were treated with fever-range whole body hyperthermia (WBH; core temperature elevated to 39.5±0.5°C for 6 hours), and allowed to return to baseline temperatures over 20 minutes before adoptive transfer of CD8 ⁺ T cells as described (Chen et al., 2006, Nat Immunol 7(12): 1299- 1308.10.1038/ni l406; Fisher et al., 201 1, J Clin Invest 121(10): 3846- 3859.10.1172/JCI44952).

[00130] Competitive short-term T cell homing assays. CD8 ⁺ T cells from non-tumor bearing mice and AT -3 tumor-bearing C57BL/6 mice were labeled with CFSE or CellTracker Orange (Therm oFisher), respectively. Labeled T cells were co-mixed at a 1 : 1 ratio and adoptively transferred via the tail vein into control and WBH-treated tumor-free mice.

Peripheral lymph nodes and spleens were harvested 1 hour after adoptive transfer and frozen in OCT compound (Sakura Finetek) for further analysis via immunofluorescence histology; quantification of cells that homed to LN was performed as described (Chen et al., 2006, Nat Immunol 7(12): 1299-1308.10.1038/ni l406; Fisher et al., 201 1, J Clin lnvest 121(10): 3846- 3859.10.1172/JCI44952). Briefly, tissue cryosections (9 μιη) were counterstained with anti- CD31 antibody to demark the position of vessels; HEV were identified based on CD31 ⁺ expression and cuboidal phenotype. Digital images were captured by observers blinded to specimen identity using an Olympus BX50 upright fluorescence microscope (Olympus Optical, Miami, FL) equipped with a SPOT RT camera (Diagnostic Instruments); all images were captured with the same settings and exposure time. The number of CFSE and

CellTracker Orange-labeled cells were quantified in >10 fields (unit area per field, 0.34 mm ² ).

[00131] In vitro and in vivo competitive T cell activation assays with DC. Bone marrow-derived dendritic cells (DC) were generated as described (Mikucki et al., 2015, Nat Commun 6: 7458.10.1038/ncomms8458) by culturing bone marrow cells (from C57BL/6 mice) for 8 days in complete media supplemented with 1 mM sodium pyruvate, MEM non- essential amino acids, 25 mM HEPES, and murine granulocyte-macrophage colony- stimulating factor (-20 ng ml ^-1 ; provided by Dr. Kelvin Lee, Roswell Park Cancer Institute). DC were matured by addition of lipopoly saccharide (0.5 pg ml ^-1 ; Sigma-Aldrich, St. Louis, MO) overnight and then pulsed with 5 μΜ OVA ₂ 57-264 peptide (SIINFEKL (SEQ ID NO: l); InvivoGen, San Diego, CA). For in vitro activation studies, SIINFEKL (SEQ ID NO: 1)- loaded DC were cultured for 4 days with a 1 : 1 co-mixture of L-selectin ¹¹¹ (from non-tumor bearing mice) and L-selectin ^l0 OT-I T cells (from AT-3-bearing mice). For in vivo studies, vaccination was performed by injecting 3 x 10 ⁶ DC in the left hind footpad of tumor-free C57BL/6 mice. Six hours post-vaccination, mice were adoptively transferred with a 1 : 1 mixture of CellTrace violet-labeled (ThermoFisher) L-selectin ¹¹¹ and CFSE-labeled

(ThermoFisher) CD8 ⁺ T cells isolated from tumor-free and AT-3-bearing OT-I mice, respectively. Organs (draining popliteal LN, confirmed by Evans blue dye; contralateral popliteal LN; and spleen) were harvested four days-post-adoptive transfer. Flow cytometry was used to analyze OT-I T cell proliferation (based on dye dilution), activation (CD44 ^M phenotype), and differentiation (intracellular IFN-γ) as described ((Fisher et al., 2011, J Clin Invest 121(10): 3846-3859.10.1 172/JCI44952; Mikucki et al., 2015, Nat Commun 6:

7458.10.1038/ncomms8458).

[00132] Statistics. All data are shown as mean±s.e.m. and group differences were calculated by 2-tailed unpaired Student's test unless otherwise indicated. Distributions of T cell rolling velocities in velocity histograms were evaluated by nonparametric Mann-Whitney U test as described (Gauguet et al., 2004, Blood 104(13): 4104-4112). For all studies, *P- values <0.05 were considered significant.

EXAMPLE 2

[00133] MDSC-induced L-selectin down-modulation occurs in a preclinical model for existing breast cancer metastasis: We originally obtained evidence that MDSC target L- selectin loss in naive T cells in preclinical murine models of primary (1°) TNBC (i.e., 4T1 tumors implanted s.c. or in the mammary fat pad; autochthonous PyMT model, and PyMT- derived implantable AT-3 line). We have extended this analysis to a robust metastatic TNBC model (Fig. 24A) to determine if MDSC execute similar systemic immunosuppressive mechanisms in a setting of metastatic disease. This question is highly relevant to current clinical trials that focus on advanced or metastatic TNBC. In this model, BALB/c mice are initially implanted with luciferase-transduced 4T1 tumors (s.c./left flank) to set up pre- metastatic niches; tumor burden is quantified by bioluminescence imaging. Once 1° tumors become established (-400 mm ³ ), they are surgically resected (Rxn) to allow subsequent outgrowth of metastases. 2° liver metastasis can be modeled by injecting 4T1 cells via the portal vein; hepatic lesions are detectable at 14 days post-Rxn (in -60% mice) while spontaneous lung metastases emerge later (4-6 weeks post-Rxn). Circulating MDSC, defined by canonical murine MDSC markers (CD1 lb, Gr-1) and T cell suppressive-function ex vivo, ²³ are strongly elevated in the 1° 4T1 model compared to non-tumor bearing (NTB) controls (Fig. 25A,B). Moreover, CD1 lb ⁺ Gr-l ⁺ MDSC from 1° 4Tl-bearing mice, but not normal CD1 lb ⁺ neutrophils or monocytes, mediate contact-dependent L-selectin

downregulation on T cells in vitro.

[00134] In setting up this model, it was critical to be able to segregate functional attributes of MDSC that arise due to 2° metastases from activities of MDSC generated by the original 1° tumor. This is achieved by selecting mice for further study at 7 days post-tumor Rxn in which there are no detectable tumors (1° or 2°) and circulating MDSC are restored to normal levels (denoted M0, Fig. 24A,B; Fig. 25A). In this scenario, subsequent metastases can be strictly linked to MDSC expansion which would not be feasible in endogenous or autochthonous metastatic models. At 14 days post-Rxn, we observe 2 mouse cohorts: (1) Ml - no detectable 1° or 2° tumor and no expansion of CD1 lb ⁺ Gr-l ⁺ MDSC (Fig. 24A,B; Fig. 25 A); some develop tumors at later time-points, not shown), (2) M2 - substantial liver lesions and MDSC expansion (Fig. 24A,B; Fig. 25A); some also have spontaneous lung metastases. MDSC expansion in the M2 metastatic group is universally associated with L-selectin loss on naive CD3 ⁺ CD44'° T cells, thus paralleling observations for 1° 4Tl-bearing mice (Fig. 25A). MDSC expansion in M2 mice is also accompanied by increases in soluble (s)L-selectin serum concentrations when compared to mice with non-detectable tumors (NTB, M0, or Ml ; Fig. 25A). SL-selectin levels are lower in M2 mice compared to 1° 4Tl-bearing mice which likely reflects the shorter time-period for sL-selectin to accumulate in the serum (7 days versus 28 days, respectively). A key feature of this model is that it does not achieve 100% penetrance for metastatic outgrowth and MDSC expansion.

[00135] MDSC form stable clusters in the circulation: Our work establishes that the blood is an active site of MDSC-induced L-selectin loss on target T cells. The importance of the blood emerged in adoptive T cell transfer studies that allowed us to track L-selectin loss on T cells that are initially retained in the blood post-transfer. Moreover, L-selectin loss is unaffected by removal of the spleen which is the only other organ site besides the blood where naive T cells co-localize with MDSC. These findings raise important questions about how MDSC execute contact-dependent L-selectin loss in fast-flowing blood. We reasoned that blood-borne MDSC might form stable interactions with target lymphocytes. Although circulating MDSC clusters have not been previously described; there is precedent for blood- borne platelet-leukocyte-tumor cell aggregates that initiate the metastatic process. We conducted studies by analyzing blood smears in the 4T1 model to determine if we could detect white blood cell (WBC) clusters containing myeloid cells (MC) or lymphocytes (L); this 'MC population includes MDSC (predominant MC in 4T1 model, neutrophils, and monocytes, since these myeloid cells are indistinguishable by morphology or phenotype. WBC clusters are not detected in MDSC ¹⁰ mice: (a) healthy NTB controls, (b) MO group, or (c) Ml group (Fig. 26A). In sharp contrast, WBC clusters are strongly elevated in MDSC ¹ " mice: (a) 1° tumor-bearing mice (no-Rxn) and (b) Ml group with 2° metastatic tumors (Fig. 26A). WBC clusters are mainly doublets (>85%) and >98% contain MDSC (MC:MC, -88% of WBC clusters; MC:L, -12% of WBC clusters). Similar results are observed in the AT-3 mammary tumor model in C57BL/6 mice {not shown). We obtained independent confirmation for circulating CD1 lb ⁺ Gr-l ⁺ CD45 ⁺ MDSC clusters in 4Tl-bearing mice using imaging flow cytometry {not shown) and a microfiltration assay (Fig 26B). We also find clusters containing PMN-MDSC (CD1 lb ⁺ CD33 ⁺ CD15 ⁺ CD14 ^l0 ) and M-MDSC

(CD1 lb ⁺ CD33 ⁺ CD15 ^' CD14 ⁺ ) in blood samples of stage IV metastatic breast cancer patients (n=4) (Fig. 26B) but not healthy donors (n=2, not shown). Collectively, these results demonstrate for the first time that blood MDSC are present in 'traveling niches' which could explain their ability to initiate contact-dependent L-selectin loss on target lymphocytes. Our published and preliminary findings support further investigation of sL-selectin and MDSC- containing clusters as metrics for functional MDSC status in the context of advanced breast cancer.

[00136] MDSC clusters represent a functional niche for L-selectin downregulation. MDSC clusters detected in the blood of 4T1 tumor-bearing mice and stage IV metastatic breast cancer patients are an active site of L-selectin loss. Support for this line of investigation is provided by our studies showing that L-selectin loss is inhibited if T cells are pretreated prior to adoptive transfer with Ab that block integrin adhesion; i.e., targeting the CD1 la integrin subunit or the integrin binding partner, intercellular adhesion molecule- 1 (ICAM-1) (Fig. 27). Thus MDSC-induced L-selectin loss may occur within integrin- stabilized clusters in the blood. This would be a highly unusual mechanism since integrins, including CD1 la (expressed by lymphocytes and myeloid cells) and myeloid-specific CD1 lb, are normally inactive on leukocytes in the free-flowing blood. EXAMPLE 3

[00137] MDSC expansion and L-selectin loss occurs in the Pan02 pancreatic model.

We expanded our analysis of immunosuppressive mechanisms to the murine Pan02 model for pancreatic ductal adenocarcinoma cancer. In this tumor system, malignant progression is associated with substantial expansion of myeloid-derived suppressor cells (MDSC) in the peripheral blood (Fig. 28A, B). Increased circulating MDSC burden in Pan02-bearing mice (i.e., at week 10) correlates with loss of L-selectin expression on naive T cells (CD3 ⁺ CD44 ^l0 ) compared to normal T cells of non-tumor bearing (NTB) C57BL/6 control mice (Fig. 28C). L-selectin was similarly reduced on CD1 lb ⁺ Gr-l ⁺ MDSC compared to normal neutrophils defined by the same phenotype in NTB control mice (Fig. 28C). Collectively, our data indicate that MDSC-induced L-selectin loss on target leukocytes occurs broadly without a gender bias in multiple tumor types in different genetic background strains including breast cancer (4T1 [BALB/c], PyMT transgenic breast cancer model and AT-3 derivative

[C56BL/6], B16 melanoma [C57BL/6], CT26 colorectal cancer [BALB/c], and Pan02 pancreatic cancer [C57BL6]. Moreover, our data indicate that multiple leukocyte subsets are targeted for L-selectin loss by MDSC including naive CD4 ⁺ and CD8 ⁺ T cells, B220 ⁺ B cells, CDl lb ⁺ Gr-l ⁺ myeloid cells (i.e., neutrophils, inflammatory monocytes, MDSC), and CD1 lb ⁺ Gr-l ^ne monocytes.

[00138] MDSC form stable conjugates with target T cells in free-flowing blood. The stringent contact requirement for MDSC-induced L-selectin loss, together with our surprising discovery of blood as the preferential site of action, predicts that stable interactions occur between MDSC effectors and target T cells within the blood compartment. We tested this hypothesis in proof-of-concept studies by analyzing cluster formation by nucleated cells in peripheral blood samples from the 4T1 murine breast tumor model by 3 independent but complementary methodologies. Our new findings, detailed below, collectively show for the first time that L-selectin loss on blood T cells correlates temporally with the appearance of circulating MDSC clusters during tumor progression.

[00139] Blood smear analysis: Smears were prepared immediately following collection of peripheral blood/EDTA specimens (10 μΐ) from non-tumor bearing (NTB) control mice or mice implanted with 4T1 breast tumors in the mammary fat pad. Blood smears were stained with Diff-Quik and evaluated for the presence of cellular clusters within an equivalent area (; unit area of high powered field [HPF] quantified = 0.34 mm ² ; > 10 HPF quantified by double-blinded analysis per specimen). Blood samples were stained in parallel to evaluate L-selectin expression on CD3 ⁺ CD44 ^l0 naive T cells by flow cytometry. This analysis reproducibly revealed that cell clusters are extremely rare in NTB control mice or early during tumor progression (e.g., 1-week post-4Tl inoculation) (<0.02 clusters per HPF) (Fig. 29). This is consistent with the lack of significant L-selectin loss (Fig. 29) at this early time-point. At later time-points (2-4 weeks post-4Tl tumor inoculation) we detected a profound increase in the density of white blood cell clusters that is inversely proportional to the extent of L-selectin loss on T cells. Maximal L-selectin loss was associated with an -500- fold increase in clusters compared to NTB controls (Fig. 29). Histological examination showed that clusters contain myeloid cells, often with segmented nuclei typical of the polymorphonuclear-MDSC subset (PMN-MDSC) (Fig. 29). Clusters additionally contained lymphocytes that were identified by their characteristic high nuclear-to-cytoplasmic ratio (Fig. 29).

[00140] We performed an additional series of experiments to formally rule out the possibility that the WBC clusters detected in blood smears were an artifact of sample preparation or were reflective of the high MDSC concentrations rather than active cell-to-cell interactions. We addressed this question by mobilizing CD1 lb ⁺ Gr-l ⁺ myeloid cells into the peripheral blood compartment by treating NTB mice with whole body hyperthermia (WBH) which raises the core body temperature of treated mice to ~39.5°C for 6. WBH treatment substantially elevated neutrophil blood counts (Fig. 30A), thus approximating the levels of blood MDSC detected in tumor-bearing mice (e.g., Fig. 28B). However, we failed to detect a significant change in WBH-treated mice compared to untreated control mice for the following parameters: blood clusters (Fig. 30B), L-selectin expression on naive T cells (Fig. 30C), or L-selectin on neutrophils (Fig. 30D). These findings are consistent with our data showing that high concentrations of mature murine neutrophils from NTB mice do not cause L-selectin loss on T cells in vitro, even if neutrophils are activated by known inducers of cell- autonomous L-selectin shedding (i.e., LPS, phorbol esters). These results support our overall conclusion that cluster formation and L-selectin loss in tumor-bearing mice represent a tightly-regulated active biological process rather than bystander events due solely to high MDSC blood concentrations.

[00141] Flow cytometry-based ImageStream analysis: Flow cytometry-based

ImageStream provided a high-throughput platform for accurate and unbiased quantification of cellular clusters in peripheral blood samples of NTB or 4T1 -bearing mice (data shown in Fig. 31 is for the 4-week time-point post-tumor implantation). Blood samples were stained for the canonical murine MDSC marker, Gr-1, and the pan-T cell marker, CD3 ⁺ . Cells were then fixed and analyzed by ImageStream. A representative profile of data obtained from a 4Tl-bearing mouse is shown in Fig. 31A; distinct populations are encircled for red blood cells (RBC), singlet T cells (CD3 ⁺ ); MDSC + T cell conjugates; singlet MDSC (Gr-1 ⁺ ), and MDSC + MDSC conjugates. Using this rigorous method, we found that Gr-1 ⁺ and/or CD3 ⁺ cell clusters are extremely rare in NTB blood (0.2 ± 0.1 clusters/μΐ blood) (Fig. 31B).

Moreover, we detected a profound ~1, 000-fold increase in stable Gr-1 ⁺ and/or CD3 ⁺ cell- containing clusters in 4Tl-bearing mice (217 ± 47 clusters/μΐ; Fig. 31B). We further determined that blood-borne clusters in 4Tl-bearing mice uniformly contain MDSC. In this regard, we found that MDSC + MDSC clusters constituted the majority of Gr-1 ⁺ or CD3 ⁺ clusters (-92%) while MDSC + T cell-containing clusters are less frequent (-8%) (Fig. 31B, C). In contrast, T cell + T cell clusters devoid of Gr-1 ⁺ MDSC are extremely infrequent (0.006%) (Fig. 31C). Application of this analytical approach to the non-Gr-l/CD3 cells revealed that clusters in this population are infrequent (0.001% of total clusters; Fig. 31C). These data provide the basis for our use of the term 'circulating myeloid cell clusters ' (CMC) to define these newly identified myeloid cell conjugates that accrue in response to tumor progression and MDSC expansion. Additional analysis showed that the CMC detected in the peripheral blood of tumor-bearing mice are predominantly MDSC + MDSC doublets (-73%; Fig. 31C), suggesting that non-MDSC cell types such as circulating tumor cells (CTC) are not a necessary constituent for stable cluster formation. Also, the restricted size of these blood-borne clusters implies that there is a threshold that cannot be exceeded for CMC to circulate through vascular beds. This notion is consistent with our observations using live- imaging microscopy that circulating MDSC labeled with Gr-1 -specific fluorescent antibody reagents form doublets in the blood of 4T1 -bearing mice whereas Gr-1+ neutrophils are uniformly singlets in NTB mice (Fig. 32).

[00142] Subset analysis of CMC further established that the majority of CMC are homotypic PMN-MDSC + PMN-MDSC clusters, defined by expression of the prototypical Ly6G ⁺ marker for the PMN-MDSC subset (i.e., representing 93.4 ± 2.2% of MDSC- containing clusters) (Fig. 33A). These data are in line with the preferential skewing of the 4T1 model toward PMN-MDSC subsets. Heterotypic clusters containing PMN-MDSC + monocytic-MDSC (M-MDSC; defined by Ly6C ⁺ Ly6G ^" phenotype) are less frequent (6.2 ± 2.2%) while M-MDSC-only containing clusters (i.e., Ly6G ^neg Ly6C ⁺ ) are extremely rare (0.4 ± 0.2%) (Fig. 33A). In this latter case, Ly6C ⁺ cells mainly form conjugates with non-MDSC (Fig. 33A) and we did not detect any homotypic M-MDSC + M-MDSC (i.e., Ly6C ⁺ + Ly6C ⁺ ) clusters (Fig. 33A). These results suggest that the PMN-MDSC subset is a central driver of cluster formation. In terms of T cells subsets, we found that both naive CD3 ⁺ T cells (i.e., defined by a CD44 ⁰ phenotype) as well as CD44 central memory and/or

effector/memory T cells (CD44 ^M ) are present within MDSC-containing clusters (Fig. 33B). Of note, MDSC within clusters express high levels of CD44. While the major nucleated cell constituents of the clusters are MDSC, we also detected CD41 ⁺ CD49 ⁺ platelets in a subfraction (-5%) of clusters (Fig. 34).

[00143] An additional series of studies determined if L-selectin loss on target leukocytes within the peripheral blood compartment is temporally linked to the formation of clusters in free-flowing blood. Our experimental design involved isolation of L-selectin ¹¹¹ splenic leukocytes from NTB mice. These cells were labeled with a fluorescent tracking dye (CFSE) prior to adoptive transfer (via the intravenous route) into MDSC ¹⁰ NTB control mice or MDSC ¹ " 4T1 -bearing mice (Fig. 35A). L-selectin expression on transferred cells and their inclusion into clusters was determined 30 min after adoptive transfer. We observed that substantial L-selectin loss occurred on CSFE ⁺ cells recovered from the blood only 30 min. after transfer into 4Tl-bearing mice whereas L-selectin was retained at a high level after transfer into NTB control mice (Fig. 35B). Restriction of our analysis to this narrow window enabled us to rigorously interrogate MDSC actions within the blood compartment since it is too short for transferred cells recovered from blood to have recirculated through solid organs (i.e., this process takes > 5 h). These data extend our prior results showing that MDSC induce L-selectin loss within a 2-hour window. L-selectin loss on transferred cells was accompanied by the formation of new clusters, as indicated by the inclusion of CFSE ⁺ cells into cellular aggregates that contained endogenous cells (i.e., CSFE ^neg cells) in 4Tl-bearing recipient mice (Fig. 35C). In sharp, leukocytes did not form clusters following transfer into NTB recipients (Fig. 35C). Evidence that L-selectin loss occurs in a high proportion of transferred cells (Fig. 35B) despite the relatively small fraction (-20%) of transferred cells within clusters at a given time-point (Fig. 35C) suggests that cluster formation is dynamic and reversible. Further analysis of this experiment established that that L-selectin was significantly reduced following adoptive transfer of L-selectin ¹¹¹ normal CD3 ⁺ CD44 ^l0 naive T cells, CD1 lb ⁺ Gr-l ⁺ neutrophils and CD 11 ⁺ Gr-1 ^' monocytes (Fig. 36).

[00144] We obtained similar results for cluster formation in co-culture experiments in which blood-derived MDSC populations (from the peripheral blood of 4Tl-bearing mice) were co-cultured with L-selectin ¹¹¹ target leukocytes labeled with CellTrace-violet (from NTB mice). In this regard, rapid formation of stable clusters was detectable by ImageStream analysis within 15 min after the addition of MDSC to target lymphocytes (Fig. 37). Cluster formation was minimal-to-nondetectable in cultures that did not contain added MDSC. [00145] Microfiltration assay: We used a dual microfiltration assay to provide independent confirmation for circulating CD1 lb ⁺ Gr-l ⁺ CD45 ⁺ MDSC clusters in 4Tl-bearing mice by (Fig. 38A). Extension of this analysis to fresh blood specimens of stage IV luminal breast cancer patients reveals the presence of clusters expressing canonical PMN-MDSC (CDl lb ⁺ CD33 ⁺ CD15 ⁺ CD14 ¹⁰ ) and M-MDSC (CDl lb ⁺ CD33 ⁺ CD15-CD14 ⁺ ) markers in 3/3 patients (Fig. 38A, B) whereas CMC are undetectable in 2/2 healthy donors. CMC in metastatic breast cancer patients were found to be highly heterogeneous with respect to the relative proportion of PM - and M-MDSC contained within clusters (Fig. 38A, B) which differs from the predominant PMN-MDSC subset representation observed within CMC in preclinical murine models (Fig. 33A). Both PMN-MDSC and M-MDSC in patient blood specimens were found to form conjugates with non-myeloid blood cells (e.g., lymphocytes). A common characteristic of human CMC and murine CMC is the absence of M-MDSC + M- MDSC conjugates (Fig. 33, Fig. 38B). The relatively high frequency of CMC, together with findings that most CMC do not contain tumor cells (i.e., <1/100 CMC have cytokeratin ⁺ tumor cells , distinguishes these structures from rare circulating myeloid cell-tumor cell clusters.

[00146] Collectively, our results provide the first evidence that MDSC exist in

'intravascular traveling niches ' which we propose explains their ability to initiate contact- dependent L-selectin loss on target leukocytes including, but not limited to T and B lymphocytes, neutrophils, and monocytes. This unprecedented mechanism of intravascular immune suppression has profound implications for cancer immunotherapy since naive or central memory T cells run the risk of encountering MDSC as they travel through blood, thereby disabling the systemic antitumor immune response during cancer immunotherapy. CMC formation could provide a platform to study the dysregulated immune-suppressive mechanisms that operate in cancer and autoimmunity. Our findings additionally suggest that CMC could serve as a blood biomarker of immune status as well as a predictor of therapeutic outcome in patients with cancer or non-malignant disorders such as autoimmune disease.

[00147] The invention has been described through some embodiments. Routine modifications to the embodiments and the disclosure will be apparent to those skilled in the art and such modifications are intended to be within the scope of the disclosure.