AND ASSOCIATED METHODS

RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application No. 62/083,730 filed on November 24 ^th , 2014, the contents of which are hereby incorporated by reference in their entirety.

FIELD

[0002] This application relates to immunotherapy and more specifically to methods and products for producing non-monocytic dendritic cells useful for immunotherapy.

INTRODUCTION

[0003] Dendritic cells (DCs) are the primary initiators of immune response, playing a key role in inducing immune reaction in autoimmune diseases, and against viral infection or cancer growth (Steinman, 2007; Steinman and Banchereau, 2007). As such, DCs have been explored both for vaccine development in chronic infections such as Hepatitis C (Zhou et al., 2012) and HIV (Garcia et al., 2013) as well as cancer immunotherapy (Steinman and Banchereau, 2007). Preclinical mouse models have demonstrated the efficacy of DC immunotherapy in reducing tumor burden (Wang and Wang, 2002; Yi et al., 201 1 ) with the success of DC immunotherapy uniquely attributable to the DCs of non-monocytic origin (Candido et al., 2001 ; Nair et al., 2000; Song et al., 1997). While these encouraging results from the mouse have formed the basis for over 100 clinical trials(Mayordomo et al., 1995; Nestle et al., 1998; Nestle et al., 2001 ) to induce DC mediated tumor-specific immunity in patients (Banchereau and Palucka, 2005; Figdor et al., 2004), the biological origins of non-monocytic DCs in humans has yet to be defined, thereby limiting these therapeutic opportunities. [0004] DCs are currently classified based on origin of cells that give rise to subsets of functional DCs in the mouse, and can be broadly divided into monocytic and non-monocytic derived DC subsets (Kushwah and Hu, 201 1 ; Shortman and Liu, 2002). Whereas most tissues are populated by non- monocytic DCs, monocytes differentiate into DCs under highly specific conditions such as lipopolysaccharide (LPS) stimulation or stimulation by gram-negative bacteria (Cheong et al., 2010). In murine models of allergy and asthma, monocytic DCs have been associated with Th2 immune responses (Plantinga et al., 2013) and have also been associated with tumor growth (Augier et al., 2010). In contrast to monocytic DCs, mouse non-monocytic DCs have been shown to have the inherent flexibility in immunomodulation and have been shown to drive antitumor immune response both in vitro and in vivo (Fields et al., 1998; Mayordomo et al., 1995). Amongst non-monocytic DCs, plasmacytoid DCs (pDCs) comprise a rare non-monocytic DC subset present in several different tissues, which are uniquely the most efficient producers of interferon (Colonna et al., 2004) and play a major role in mediating tolerance in the intestinal tract (Goubier et al., 2008). Given that the adoptive transfer of monocytes to mice under steady state conditions has failed to reconstitute a complete DC repertoire (Naik et al., 2007), and genetic tracing experiments have identified mouse DCs to be a hematopoietic lineage distinct from monocytes (Schraml et al., 2013), the use of non-monocytic DCs may be essential in expanding immunotherapeutic options in humans.

[0005] To date, DC based clinical trials have relied primarily on monocyte-derived DCs (Banchereau and Palucka, 2005; Figdor et al. , 2004; Palucka and Banchereau, 2012; Schuler et al., 2003). However, human monocytic DCs have been shown to drive Th2 responses in models of airway inflammation (Hammad et al., 2001 ; Hammad et al., 2002) and potentiate tolerance induction (Banchereau et al. , 2012; Sharma et al., 2013), which is immunosuppressive and not desirable for antitumor therapy (Dunn et al., 2006; Hung et al., 1998; Papatriantafyllou, 201 1 ), reflected in the poor efficacy of human DC cancer immunotherapy (Banchereau and Steinman, 1998; Cerundolo et al. , 2004; Palucka and Banchereau, 2012). Thus, our limited understanding of human DC development has compromised translational applications of DC based immunotherapy. Clearly, clinical applications for DC immunotherapy would benefit from a deeper understanding of human DC ontogeny.

SUMMARY

[0006] In one aspect, the inventors have identified and isolated human non-monocytic DC precursors from adult hematopoietic tissue, as well as from renewable induced pluripotent stem cells (iPSCs) reprogrammed from blood (hBiPS) that uniquely give rise to non-monocytic DCs including plasmacytoid DCs both in clonal assays along with in vivo human-mice xenografts. hBiPS cells are a potential renewable source for autologous DC vaccine development. Furthermore, using in vitro assays along with human-mice xenografts DCs derived from the human non-monocytic DC precursors were shown to be superior to clinically utilized monocytic DCs in driving a protective Th1 response along with an anti-tumor immune response, validating the efficacy of these precursors for cancer immunotherapy.

[0007] In one aspect, there is provided an isolated non-monocytic Dendritic Cell (DC) precursor cell. In one embodiment, the non-monocytic DC precursor cell is obtained from a population of CD34+CD38+CD45RA+ cells. Optionally, the non-monocytic DC precursor cell is isolated from peripheral blood or from a population of human blood iPSCs. In one embodiment, the non-monocytic DC precursor cell expresses CD1 15. In one embodiment, the non-monocytic DC precursor cell expresses CD1 15, Flt3 and HLA-DR. In one embodiment, the DC precursor cell does not express CD14 or CX3CR1 . In one embodiment, the non-monocytic DC precursor cell does not secrete IL-6 or TNF-a following stimulation with lipopolysaccharide (LPS). Also provided are cell lines and cell cultures comprising the non-monocytic DC precursor cells as described herein. [0008] In another aspect, there is provided an isolated non-monocytic dendritic cell (DC). In one embodiment, the non-monocytic DC is obtained from a non-monocytic DC precursor cell as described herein. For example, in one embodiment the non-monocytic DC is obtained by differentiating a non- monocytic DC precursor cell as described herein. In one embodiment, the monocytic DC is obtained by differentiating, or expanding and differentiating, a non-monocytic DC precursor cell in the presence of one or more of GM- CSF, IL-4, Flt3 Ligand (Flt3L) and bone marrow stromal cells (BMSCs).

[0009] In one embodiment, the non-monocytic DCs described herein exhibit an increased Th1 response relative to DCs derived from Cd14+ monocytic cells. In one embodiment, the non-monocytic DC exhibits a preferential Th1 response relative to Th2. In one embodiment, the increased Th1 response is characterized by increased IFN-γ production. In one embodiment, the Th2 response is characterized by I L-4 production.

[0010] In one embodiment, the non-monocytic DC exhibits an antitumor response. For example, in one embodiment the non-monocytic DC exhibits an increased anti-tumor response relative to CD14+ monocytic DCs.

[001 1] In another aspect, there is provided a pharmaceutical composition comprising one or more non-monocytic DC precursor cells and/or non-monocytic DCs as described herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises an anti-cancer therapeutic and/or a cell culture media.

[0012] Another aspect of the disclosure includes the use of a non- monocytic DC precursor cell or a non-monocytic DC as described herein for immunotherapy, autologous therapy, autoimmune immunotherapy, vaccination or development of vaccine adjuvants. Also provided are methods for providing immunotherapy, autologous therapy, autoimmune immunotherapy or vaccination to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a non- monocytic DC precursor cell or a non-monocytic DC as described herein. In one embodiment, the immunotherapy, autologous therapy or autoimmune immunotherapy if for the treatment of cancer. In one embodiment, the cancer is myeloma, melanoma or prostate cancer. In one embodiment, the non- monocytic DC precursor cells or a non-monocytic DCs described herein are for use in the treatment of cancer in a subject in need thereof and the cells are autologous cells from the subject.

[0013] In another aspect, there is provided a method for isolating a non-monocytic DC precursor cell from a population of cells comprising obtaining a population of CD34+CD38+CD45RA+ cells, culturing the cells and separating one or more cells that express CD1 15 from the population of cells.

[0014] Optionally, the method further comprises separating cells that express Flt3 and/or HLA-DR from the population of CD34+CD38+CD45RA+ cells wherein cells that express CD1 15, Flt3 and/or HLA-DR are non- monocytic DC precursor cells. In one embodiment, the methods described herein further comprise differentiating, or expanding and differentiating, the non-monocytic DC precursor cells into non-monocytic dendritic cells.

[0015] In one embodiment, the differentiating, or expanding and differentiating, the non-monocytic DC precursor cells comprises culturing the cells in the presence of one or more of GM-CSF, IL-4, Flt3 Ligand (Flt3L) and bone marrow stromal cells (BMSCs).

[0016] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

DRAWINGS

[0017] The disclosure will now be described in relation to the drawings in which: [0018] Figure 1 shows that human CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells represent a distinct population of non-monocytic cells isolatable from adult blood with DC generation capacity. (A) Established CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors were cultured in presence of GM-CSF and I L-4 or Flt3L alone, and screened for expression of markers expressed on murine precursors of the DC lineage. (B) Of mouse DC precursors markers examined (CD1 15, CD135, HLA-DR and CX3CR1 ), human CD1 15 showed a distinct transition expression pattern at day 2 of culture with DC differentiation inducing cytokine conditions. (C) Following 2 days of culture of CD34 ⁺ CD38 ⁺ CD45RA ⁺ cells, CD1 15 ⁺ and CD1 15 ^" sub-fractions were isolated by FACS. Representative kwik-diff stained images of isolated CD1 15 ⁺ and CD1 15 ^" subfractions from cultured CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors are shown. (D) Differential marker expression of CD1 15 ⁺ and CD1 15 ^" cells isolated from CD34 ⁺ CD38 ⁺ CD45RA ⁺ cells. (E) Gating strategy for de novo isolation of CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells derived from adult human blood. Representative analysis of CD14 and CX3CR1 expression was used to isolate CD14 X3CR1 ^" subset that was further subdivided for CD1 15, HLA-DR and Flt3 expression to allow pure isolation of CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells. Representative Kwik-Diff stained image of de novo isolated CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells is shown. (F) Non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells isolated from adult human blood lack expression of HSC, DC/Monocyte and T/B cell lineage markers, including markers expressed on monocytic CD14 ⁺ cells. (G) Non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells isolated from human blood fail to produce IL-6 and TNF-a, cytokines produced by monocytic CD14 ⁺ cells following LPS stimulation (ND-not detectable). (H) Non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells isolated from human blood fail to produce IL-12, which is produced by differentiated human DCs derived from monocytic CD14 ⁺ cells (ND-not detectable). (I) Heat map comparing gene expression profiles of monocytic CD14+ cells (n=5 samples) with non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells (n=6 samples), using all probe sets. (J) Comparison of absolute cell numbers following culture of monocytic CD14+ cells and non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells with M-CSF for 5 days. (K) Representative flow plot of CD1 a and CD14 with Kwik-Diff stained images of monocytic CD14 ⁺ cells and non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells cultured in presence of M-CSF for 5 days is shown. (L) Monocytic CD14+ cells were de novo isolated from adult human blood and failed to survive following 12 days of culture with Flt3L. (M) Non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells were de novo isolated from adult human blood and underwent expansion following 6 days of Flt3L culture. Shown is a histogram comparing absolute number of cells following Flt3L stimulation. Representative images of Kwik-Diff stained differentiated cells are shown. (N) CD4+ T cell proliferation induced by DCs derived from monocytic CD14 ⁺ cells versus non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells. (O) CD8+ T cell proliferation induced by DCs derived from monocytic CD14 ⁺ cells versus non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells. (P) Cytokine transcript analysis of DCs derived from monocytic CD14 ⁺ cells versus DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells following stimulation with LPS, normalized to immature DCs in absence of LPS stimulation. Representative of 6 human donors. *p<0.05. Error bars, s.d.

[0019] Figure 2 shows that human non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-

DR ⁺ cells represent clonal non-monocytic precursors of human DCs. (A) Monocytic CD14 ⁺ cells, and non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells were de novo isolated from human blood and then analyzed for DC differentiation. NSG recipients were transplanted with CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors and after 4 weeks, non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells were isolated from xenografts and then clonally analyzed for DC differentiation (n=440 clones). (B) Monocytic CD14 ⁺ cells isolated from adult human blood were plated on human BMSCs and cultured with GM-CSF and IL-4 for 7 days to drive DC differentiation (450 cells/well on 200 wells). Individual non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells isolated from adult human blood (n=576 clones) as well as those isolated from xenografted recipients (n=440 clones) were plated clonally on human BMSCs with Flt3L for 6 days to drive clonal expansion followed by differentiation into DCs in presence of GM-CSF and IL- 4 or Flt3L for 7 days. (C) Representative flow plots showing generation of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells from xenografts transplanted with CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors. (D) Scatterplot comparing proportions of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells generated from NSG recipients xenografted with cells treated overnight with GM-CSF and IL-4 prior to injection in comparison to untreated cells (controls) (n=10, each dot represents an individual NSG recipient). (E) Gating strategy for identification of clonal DC generation in vitro. DCs were characterized by CD1 1 c, HLA-DR, CD83 and CD1 a expression. pDCs were characterized by expression of BDCA-2 and CD123 with a lack of CD1 a expression. (F) IFN-a production by BDCA-2+CD123+ cells derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells demonstrating generation of functional pDCs. (G) Monocytic CD14 ⁺ cells isolated from human blood exclusively generated non pDCs, with a complete absence of pDC generation as shown and tabulated. Single non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells isolated from human blood or xenografted recipients clonally gave rise to either DCs alone, pDCs alone or pDCs and DCs in presence of GM-CSF and I L-4 or Flt3L as shown and tabulated. Shown are proportion of wells identifying differentiation into DCs, pDCs or both pDCs and DCs. *p<0.05. (H) de novo isolated CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells co-cultured with BMSCs in presence of 2 differentiation cytokine cocktails. (I) Combination of Flt3L, SCF, GM-CSF and IL4 (FSGMI) demonstrated better overall human cells input (CD45) and significant frequencies of differentiation into mature DCs and pDCs compared to the solely presence of GM-CSF and I L4 (GMI).

[0020] Figure 3 shows that human non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-

DR ⁺ cells generate plasmacytoid DCs in vivo. (A) NSG mice were transplanted with monocytic CD14 ⁺ cells and non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells isolated from adult human blood followed by assessment of DC differentiation in vivo. (B) Gating strategy to identify DC generation in vivo. Gating of hCD45 ⁺ cells was carried out to identify human cells and further focused on human hematopoeitic cells expressing the myeloid marker CD33. Using human CD45+CD33+ subtractions engrafting NSG mice, analysis of CD83, HLA-DR, BDCA-1 , BDCA-3 and CD14 expression were used to identify DCs (BDCA-1 ⁺ DCs, BDCA-3 ⁺ DCs, CD14 ⁺ DCs). BDCA-2 and CD123 expression on hCD45 ⁺ CD33 ^" cells were used to identify pDCs. (n=14). Representative plots are shown. (C) In vivo generation of DCs in NSG recipients transplanted with monocytic CD14 ⁺ cells versus non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells (n=14, each dot represents an individual mouse recipient). (D) In vivo generation of pDCs in NSG recipients transplanted with monocytic CD14 ⁺ cells versus non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells (n=14, each dot represents an individual mouse recipient). *p<0.05.

[0021] Figure 4 shows that DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells are superior to DCs derived from bulk CD34 ⁺ cells as well as monocytic CD14 ⁺ cells in survival and induction of Th1 response in vitro. (A) Schema for in vitro assessment of survival and function of DCs generated from non-monocytic CD1 15+Flt3+HLA-DR+ cells versus bulk CD34+ and monocytic CD14+ cells. (B) Proportions of surviving DCs, assessed by propidium iodide staining following overnight treatment with LPS. (C) Representative flow plots of BDCA-2 and CD123 expression on DCs generated from bulk CD34+ cells under different conditions, identifying lack of pDC generation. (D-F) To assess in vitro capacity of DCs to drive T cell activation in vitro, DCs were stimulated overnight with LPS and cultured with CD4 ⁺ T cells for 5 days. (D) IFN-γ and IL-4 production by CD4 ⁺ T cells. (E) Proportions of I FN-γ producing CD4 ⁺ T cells upon culture with DCs. (F) Proportions of IL-4 producing CD4 ⁺ T cells upon culture with DCs. (G-J) DCs were also treated with Th2 inducing cytokine TSLP to preferentially drive a Th2 response and were subsequently cultured with CD4 ⁺ T cells for 5 days. (G) IFN-γ and IL-4 production by CD4 ⁺ T cells. (H) Proportions of IFN-γ producing CD4 ⁺ T cells upon culture with DCs. (I) Proportions of IL-4 producing CD4 ⁺ T cells upon culture with DCs. (J) Expression of CCR7 following overnight stimulation with LPS. Representative of three independent experiments. *p<0.05. Error bars, s.d.

[0022] Figure 5 shows that human blood induced pluripotent stem cells (hBiPS) give rise to non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells, which generate non-monocytic DCs both in vivo and in vitro. (A) Experimental outline for assessment of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells generation from human iPSCs. Representative flow histograms identifying generation of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells exclusively from hBiPS but not from ES and Fib-iPS sources. Representative images of Kwik- Diff stained monocytic CD14+ cells (CD1 15-) and non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells along with fully differentiated DCs are shown. (Black arrow heads indicate presence of vacuoles in the cytoplasm). (B) Expression of pDC associated markers: BDCA-2 and CD123 is exclusively observed following culture of hB-iPS derived non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA- DR ⁺ cells with bone marrow stromal cells in presence of GM-CSF, IL-4 and Flt3L. (C) IFN-a production by DCs generated from hB-iPS derived non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells following CpG DNA stimulation demonstrating generation of functional pDCs. (D-E) DCs were derived from ES cells (hES derived DCs), hFib-iPS (hFib-iPS derived DCs), from monocytic CD14+ cells derived from hB-iPS (hB-iPS DCs from monocytic DCs) and from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells derived from hB-iPS (hB-iPS DCs from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells). (D) DCs were matured overnight with LPS and cultured with CD4+ T cells for 5 days. Representative flow plots of IFN-γ and I L-4 production by CD4+ T cells are shown. Also shown are histograms comparing proportions of I FN-γ producing CD4+ T cells and IL-4 producing CD4+ T cells upon culture with DCs. (E) DCs were also treated with Th2 inducing cytokine and cultured with CD4+ T cells for 5 days. Representative flow plots of IFN-γ and I L-4 production by CD4+ T cells are shown. Also shown are histograms comparing proportions of IFN-γ producing CD4+ T cells and IL-4 producing CD4+ T cells upon culture with DCs. (F) Gating strategy to identify DC generation in vivo following transplantation of NSG recipients with hB-iPS derived monocytic CD14+ cells or non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells. Gating of hCD45+ cells was carried out to identify human cells. Subsequently analysis of HLA-DR, CD1 1 c, CD14 and BDCA-2 expression was carried out to identify DC generation in vivo (n=8). Representative plots are shown. *p<0.05. Error bars, s.d.

[0023] Figure 6 shows that DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells are superior to DCs derived from monocytic CD14 ⁺ cells in homing and inducing Th1 response in vivo. DCs were derived from monocytic CD14 ⁺ cells, and from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells. (A) Schematic for in vivo assays in NSG mice to test the function of in vitro generated human DCs to drive Th response. Following antigen challenge with ovalbumin, T cell response(s) were assessed by human CD4 ⁺ T cells from the spleen. (B) Proportions of DCs in the thymus and lymph nodes of NSG mice. (C) IL-17, I FN-γ and I L-4 production by human CD4+ T cells. (D) Proportions of IL-17 producing (Th17), IFN-γ producing (Th1 ) and IL-4 producing (Th2) CD4 ⁺ T cells. (E) Transcript analysis of human Th17, Th1 and Th2 associated transcription factors, RORjt, T-bet and GATA-3 respectively in spleens of NSG recipients. n=14. *p<0.05. Error bars, s.d.

[0024] Figure 7 shows that DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells are superior to DCs derived from monocytic CD14 ⁺ cells in inducing anti-tumor response both in vitro and in vivo. DCs were derived from monocytic CD14 ⁺ cells, and from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells. (A-B) DCs were pulsed with Melan-A and cultured with T cells. (A) Shown are representative flow cytometry plots of Melan-A tetramer ⁺ CD8 ⁺ T cells. Non-pulsed DCs were cultured with T cells as a control. (B) Proportions of Melan-A tetramer ⁺ CD8 ⁺ T cells upon culture with DCs. (C) Schematic for in vivo assays in NSG mice to test the function of in vitro generated human DCs to drive Melan-A specific CD8 ⁺ T cell response. (D) Gating strategy for identification of human CD8 ⁺ T cells in the spleens of NSG recipients. (E) Shown are representative flow cytometry plots of Melan-A tetramer ⁺ CD8 ⁺ T cells in the spleens of NSG recipients. Non-pulsed DCs were used as control. (F) Proportions of Melan-A tetramer ⁺ CD8 ⁺ T cells in the spleens of NSG recipients. n=14. *p<0.05. Error bars, s.d.

[0025] Figure 8 shows the isolation of CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors, transcriptional profile analysis of CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells compared to mouse non-monocytic DC precursor and analysis of DC differentiation in comparison with human monocytes. (A) Gating strategy for FACS isolation of CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors from human blood. (B) Representative images of CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors following isolation by FACS. (C) Heat map comparing gene expression profiles of non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells with human common myeloid progenitor (CMP), human granulocyte and macrophage progenitor (hGMP), mouse common DC progenitor (mCDP), mouse macrophage and DC progenitor (mMDP), mouse common myeloid progenitor (mCMP) and mouse non-monocytic DC precursor (mPreDC) is shown. (D) Comparison of absolute cell numbers following culture of monocytic CD14+ cells and non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells with GM-CSF and IL-4 to drive DC generation. (E) Expression profiles of T cell stimulatory markers: CD83, CD86 and HLA-DR on DCs differentiated from monocytic CD14 ⁺ cells versus non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells. Representative of 8 independent experiments. *p<0.05. Error bars, s.d. (F) Colony forming unit (CFU) analysis of non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells in comparison to human myeloid precursors. (G) de novo isolated non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells failed to secrete IL-12 following LPS stimulation. (H) DCs derived from non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ precursors demonstrated superior CD4 ⁺ T cell proliferation potential. (I) DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ precursors demonstrated superior CD8 ⁺ T cell proliferation potential. (J) DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA- DR ⁺ precursors demonstrated significantly increased levels of Th1 inducing cytokines IL12p35, IL12p40 and I L- 1 β in comparison to DCs derived from monocytic CD14 ⁺ cell origin. [0026] Figure 9 shows the clonal expansion of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells on BMSCs with Flt3L, related to Figure 2. Non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells from adult blood or NSG recipients were sorted as single cells on a 96 well plate containing a monolayer of human bone marrow stromal cells (BMSCs). After 5-6 days of culture with/without Flt3L, expansion was observed. (A) Shown is a histogram comparing average cell count/well indicating expansion from a single non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cell. (B) Shown is a representative cytospin image of expanded non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells. Representative of 4-5 independent experiments. (C) The kinetics of clonal expansion and differentiation of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells into DCs was monitored by measuring total cell counts along with counts of pDCs generated. Shown are representative histograms for 50 clones each from two independent human adult blood samples. (D) I FN-a production by pDCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells in response to CpG stimulation. (E) qPCR analysis of Spi-B expression in pDCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells compared to DCs derived from monocytic CD14+ cells. *p<0.05. Error bars, s.d. Representative of 4-5 independent experiments.

[0027] Figure 10 shows phenotypical and functional characterization of DCs generated from human pluripotent stem cells (hPSCs), related to Figure 5. (A) Representative expression profile of DC markers (CD1 1 c, CD14, CD1 a, CD86, HLA-DR) on DCs generated from different pluripotent stem cell sources are shown. (B) CD4+ T cell proliferation induced by DCs from different pluripotent stem cell source(s). (C) CD8+ T cell proliferation induced by DCs from different pluripotent stem cell source(s). (D) Schema for in vitro assessment of survival and function of DCs generated from various pluripotent stem cell sources. (E) Proportions of DCs undergoing cell death, assessed by propidium iodide staining following overnight treatment with LPS. (F) In vivo generation of DCs in NSG recipients transplanted with hB-iPS derived monocytic CD14+ cells versus non-monocytic CD1 15+Flt3+HLA-DR+ cells *p<0.05. Error bars, s.d. Representative of 4-5 independent experiments.

[0028] Figure 1 1 shows the antigen specificity of T cell response in NSG recipients with a humanized immune system, immunized with ovalbumin pulsed DCs, related to Figure 6. Histogram comparing relative proliferation of splenocytes from NSG recipients immunized with ovalbumin (OVA)-pulsed DCs derived from monocytic CD14+ cells or DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells compared to saline-pulsed DCs in response to OVA treatment is shown (n=10). *p<0.05. Error bars, s.d.

DESCRIPTION OF VARIOUS EMBODIMENTS

[0029] Dendritic cells (DCs) are the primary initiators of immune response required for antitumor effects and DC derived from monocytes have been explored as vaccines for viral infections as well as cancer immunotherapy. While the clinical use of monocyte derived DCs that have had modest efficacy in immunotherapy, the lack of insight into the cellular origins of DC subclasses in the human has limited immunotherapeutic opportunities.

[0030] In one aspect of the description, the inventors have identified clonally derived non-monocytic DC precursors from adult humans as well as renewable induced pluripotent stem cells (iPSCs) when reprogrammed from blood (hBiPS). hBiPS are a potentially renewable source for autologous DC vaccines. Human non-monocytic DC precursor cells (hNM-DCPs) were identified to generate unique DC subtypes compared to monocytic DCs and are capable of generating a complete repertoire of DC subtypes including plasmacytoid DCs. Using infectious, as well as tumorigenic antigens, preclinical in vitro and in vivo testing demonstrated that DCs generated from hNM-DCPs are capable of superior Th1 anti-tumor response compared to sources previously utilized clinically. The use DCs derived from non- monocytic precursors, which have traditionally been absent in clinically utilized DCs derived from monocytes or bulk CD34+ cells, are therefore expected to be useful for DC-based immunotherapies including cancer immunotherapies.

[0031] In one aspect, there is provided an isolated non-monocytic Dendritic Cell (DC) precursor cell as described herein. In one embodiment, the DC precursor cell expresses CD1 15. In one embodiment, the DC precursor cell expresses CD1 15, Flt3 and HLA-DR. In one embodiment, the DC precursor cell is a human cell. Optionally, the non-monocytic DC precursor cell is obtained from blood. In one embodiment, the non-monocytic DC precursor cell is obtained from a pluripotent stem cell such as blood derived iPSCs.

[0032] In one embodiment, the non-monocytic DC precursor cell is obtained from a population of CD34+CD38+CD45RA+ cells. In one embodiments, the DC precursor cell transiently expresses CD1 15 in culture. For example, in one embodiment shown in Figure 1 B, the non-monocytic DC precursor cells express CD1 15 around day 2 of culture. In some embodiments, the non-monocytic DC precursor cells express CD1 15 from about day 0.5 to about day 4 after culturing the cells. In one embodiment, the non-monocytic DC precursor cell does not express the monocytic marker CD14. In one embodiment, the non-monocytic DC precursor cell does not express CX3CR1 .

[0033] The non-monocytic DC precursor cell described herein also exhibit certain functional characteristics that distinguish them from other cells. For example, in one embodiment the DC precursor cells do not secrete IL-6 or TNF-a following stimulation with lipopolysaccharide (LPS).

[0034] In another aspect, there is provided an isolated non-monocytic Dendritic Cell (DC) as described herein. In one embodiment, the non- monocytic DC is obtained from a non-monocytic DC precursor cell as described herein. In one embodiment, the non-monocytic DC is a plasmacytoid cell. In one embodiment, the cell is obtained from a population of pluripotent stem cells, optionally human blood pluripotent stem cells. [0035] In one embodiment, the non-monocytic DC may be obtained by differentiating and/or expanding a precursor cell as described herein in the presence of GM-CSF and/or IL-4. In one embodiment, non-monocytic DC may be obtained by differentiating and/or expanding the precursor cells in the presence of Flt3 Ligand (Flt3L). In one embodiment, non-monocytic DC may be obtained by differentiating and/or expanding the precursor cell in the presence of Flt3L and bone marrow stromal cells (BMSCs). In one embodiment, the non-monocytic DC may be obtained by differentiating and/or expanding the precursor cell in the presence of bone marrow stromal cells (BMSCs), Flt3L, Stem Cell Factor (SCF), GM-CSF and IL-4.

[0036] The non-monocytic DCs described herein also exhibit characteristics that distinguish them from other cells such as monocytic DCs. In one embodiment, the non-monocytic DCs described herein exhibit an increased Th1 response relative to DCs derived from CD14+ monocytic cells. Optionally, an increased Th1 response is characterized by increased IFN-γ production. In another embodiment, the non-monocytic DCs described herein exhibit a preferential Th1 response relative to Th2. For example in one embodiment, the non-monocytic DCs described herein exhibit a preferential Th1 response relative to Th2 compared to DCs derived from CD14+ monocytic cells. Optionally, the Th2 response may be characterized by determining IL-4 production.

[0037] In another embodiment, the non-monocytic DCs described herein exhibit superior CD4+ and CD8+ T cell proliferation relative to monocytic CD14+ cells (see for example Figures 8H and 8I). I one embodiment, the non-monocytic DCs described herein also show increased levels of Th1 inducing cytokines. For example, in one embodiment the non- monocytic DC expresses increased levels of IL12p35 and/or IL12p40 relative to DC derived from monocytic CD14+ cells (see, for example, Figure 8J).

[0038] In one embodiment, there is also provided a cell culture or cell line comprising non-monocytic DC precursor cells or non monocytic DC cells as described herein. As used herein the term "cell culture" refers to one or more cells grown under controlled conditions and optionally includes a cell line. The term "cell line" refers to a plurality of cells that are the product of a single group of parent cells. In one embodiment, the cell culture or cell line is a clonal cell culture or cell line derived from a single cell.

[0039] In one embodiment, the cells and compositions described herein are useful for immunotherapy and for eliciting and immune response in vitro or in vivo. In one embodiment, the non-monocytic DC precursor cells and/or the non-monocytic DCs of described herein are for use in immunotherapy, autologous therapy, autoimmune immunotherapy, vaccination or development of vaccine adjuvants. In one embodiment, the cells and compositions described herein are useful for the treatment of cancer in a subject in need thereof.

[0040] As used herein, the term "cancer" refers to one of a group of diseases caused by the uncontrolled, abnormal growth of cells that can spread to adjoining tissues or other parts of the body. In one embodiment, the subject has a cancer suitable for treatment by immunotherapy. In one embodiment, the subject has myeloma, melanoma or prostate cancer.

[0041] The term "treating" or "treatment" as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease (e.g. maintaining a patient in remission), preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also include prophylactic treatment. In one embodiment, treatment methods comprise administering to a subject a therapeutically effective amount of the non-monocytic DC precursor cells or non-monocytic DCs as described herein and optionally consists of a single administration, or alternatively comprises a series of administrations. In one embodiment, the cells described herein are prepared or formulated for administration to a subject in need thereof as known in the art. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0042] In one embodiment, the non-monocytic DCs described herein are also useful for inducing a preferential Th1 response and/or a preferential Th17 response in vitro or in vivo in a subject in need thereof. In one embodiment, the subject has cancer, optionally myeloma, melanoma or prostate cancer. Also provided are methods for inducing a preferential Th1 response and/or a preferential Th17 response in vivo in a subject in need thereof comprising administering to the subject the non-monocytic DC precursor cells or non-monocytic DCs as described herein.

[0043] In one embodiment, the methods and uses described herein involve cells that are autologous, such as non-monocytic DC from a subject with cancer or suspected of having cancer, or allogenic, such as non- monocytic cells from a healthy donor without cancer. Optionally, the methods described herein include obtaining a sample of peripheral blood from a subject in order to obtain autologous non-monocytic DC precursor cells or non-monocytic DCs using the methods described herein.

[0044] In another aspect, there is provided a method of identifying and/or isolating a non-monocytic Dendritic Cell (DC) precursor cell from a population of cells. In one embodiment, a non-monocytic DC precursor cell may be isolated by separating cells based on the expression of one or more biomarkers described herein to be associated with monocytic DC precursor cells. In one embodiment, the method comprises obtaining a population of cells and separating one or more cells that express CD1 15 from the population of cells. In one embodiment, the method comprises separating cells that express Flt3 and/or HLA-DR from the population of cells. In one embodiment, cells that express CD1 15, FLT3 and/or HLA-DR are non- monocytic DC precursor cells.

[0045] For example, in one embodiment there is provided a method of isolating a non-monocytic Dendritic Cell (DC) precursor cell comprising:

obtaining a population of CD34+CD38+CD45RA+ cells

culturing the cells; and

separating one or more cells that express CD1 15 from the population of cells.

[0046] In one embodiment, the method further comprises separating cells that express Flt3 and/or HLA-DR from the population of cells, wherein cells that express CD1 15, FLT3 and/or HLA-DR are non-monocytic DC precursor cells. In one embodiment, the cultured CD34+CD38+CD45RA+ cells transiently express CD1 15 between 0.5 and 5 days, between 0.5 and 4 days, or between 1 and 3 days after culturing the cells. In some embodiments, the cultured CD34+CD38+CD45RA+ cells transiently express CD1 15 about 2 days after culturing the cells.

[0047] The CD34+CD38+CD45RA+ cells may be obtained from different sources. For example, in one embodiment the CD34+CD38+CD45RA+ cells are obtained from peripheral blood cells. In one embodiment, the CD34+CD38+CD45RA+ cells are obtained from differentiating pluhpotent stem cells. In one embodiment, the pluhpotent stem cells are human blood induced pluhpotent stem cells (hB-iPSCs). In one embodiment, differentiating the hB-iPSCs comprises culturing the cells in the presence of SCF, Flt3L, II-3, II-6, GM-CSF and BMP4. [0048] In a further aspect, a person skilled in the art will appreciate that the characteristics of the non-monocytic DC precursor cells and/or the non- monocytic DC cells described herein can be used in methods to identify and/or isolate non-monocytic DC precursor cells and/or non-monocytic DC cells. For example, in some embodiments, the non-monocytic DC precursor cells and non-monocytic DC cells exhibit different patterns of gene expression relative to other cells such as monocytic DC cells. Accordingly, the present disclosure includes methods of identifying and/or isolating non-monocytic DC precursor cells and/or non-monocytic DC cells based on gene expression. Additional methods of identifying and/or isolating non-monocytic DC precursor cells and/or non-monocytic DC cells include those based on morphological and/or functional characteristics. For example, in one embodiment the non- monocytic DC precursor cells described herein do not exhibit monocytic morphological features such as cytoplasmic vacuoles or horseshoe shaped nuclei. In one embodiment, the non-monocytic DC cells exhibit a distinct cytokine response profile from CD14+ monocytic cells. In one embodiment, the non-monocytic DC cells described herein expand and differentiate in response to Flt3L. In one embodiment, the non-monocytic DC cells described herein exhibit increased levels of Th1 inducing cytokines such as I L12p35, IL12P40 and I L-1 β in comparison to DCs derived from monocytic CD14+ cells.

[0049] In one embodiment, the levels of biomarkers may be determined using fluorescent activated cell sorting (FACS). A person skilled in the art will appreciate additional methods for analyzing the expression levels of biomarkers such as PCR based methods or immunolabelling with antibodies.

[0050] Optionally, the methods described herein comprise differentiating and/or expanding a population of non-monocytic DC precursor cells to form a population of non-monocytic DCs. Also provided are methods for expanding a population of non-monocytic DCs to form an expanded population of non-monocytic DCs. The term "differentiation" as used herein refers to the process by which a less specialized cell, such as a precursor cell, becomes a more specialized cell type, such that it is committed to a specific lineage. As used herein, "precursor cell" refers to cell with a limited replicative capability that shows signs of differentiation towards a target cell, such as the non-monocytic DC precursor cells described herein.

[0051] In one embodiment, the non-monocytic DC precursor cells may be expanded by culturing the cells in the presence of Flt3 Ligand (Flt3L) and bone marrow stromal cells (BMSCs).

[0052] In another embodiment, the non-monocytic DC precursor cells may be differentiated and/or expanded by culturing the cells in the presence of one or more of GM-CSF, IL-4, Flt3 Ligand (Flt3L), Stem Cell Factor (SCF) and bone marrow stromal cells (BMSCs).

[0053] In one embodiment, the DC precursor cells may be differentiated and/or expanded by culturing the cells in the presence of GM- CSF, IL-4, Flt3 Ligand (Flt3L), Stem Cell Factor (SCF) and bone marrow stromal cells (BMSCs).

[0054] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the disclosure. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0055] The following non-limiting examples are illustrative of the present disclosure:

EXAMPLES

Example 1 : Identification and characterization of non-monocytic precursors capable of generating dendritic cells for immunotherapy

Experimental Procedures Isolation of myeloid precursors and DC differentiation

[0056] All donor samples were obtained with the approval of local human subject research ethics board at McMaster University. Human mobilized peripheral blood mononuclear cells (MNC) were isolated as described previously (Bhatia et al. , 1997). Flow cytometry was used for isolation of erythro-myeloid precursors as described previously(Manz et al., 2002). DC differentiation was performed on pre-seeded wells (6, 12, 96 plates) of confluent human BMSCs that were treated 3h with 10ug/ml of Mitomycin C (Sigma-Aldrich). Tested cells (CD14+, NMDCPs) were then added with a mixture of serum-free media (StemSpan, Stem Cell technologies) containing different cytokine combinations (Miltenyi) of SCF (20ng/ul) GM-CSF (50 ng/ul), I L-4 (10ng/ul), Flt3L (100ng/ul). All cytokines were purchased from Miltenyi. Human T cells were isolated by flow cytometry using CD4 and CD45RA for naive T cells and CD8, CD45RO, CCR7 for naive CD8 ⁺ T cells. Human CD14 ⁺ monocytic cells were isolated from human MNCs using flow cytometry.

T cell proliferation/differentiation

[0057] Purified DCs treated overnight with LPS (0.1 ug/ml) or TSLP (20 ng/ml) were added to naive CD4 ⁺ or CD8 ⁺ T cells at 1 : 10 APC:T cell ratio in flat-bottomed microtiter plates for 6 days at 37°C in a 5% C0 ₂ humidified atmosphere incubator. Cell proliferation was assessed at day 3, 5 and 7 of culture using BrdU incorporation assay following the manufacturer's instructions (Roche, QC). T cell differentiation into the Th1 or Th2 lineage was assessed on day 7 using Th1 Th2 and Th17 kit following the manufacturer's instructions (BD Biosciences, Burlington, ON).

NSG studies

[0058] OD.Cg-Prkdc ^scid ll2rg ^tm1VVil /SzJ adult mice (NSG) mice were bred and maintained in the Stem Cell and Cancer Research Institute (SCC-

Rl) animal barrier facility at McMaster University. All animal procedures received the approval of the animal ethics board at McMaster University. Mice were sub-lethally irradiated with 315 rads 24 hrs prior to injection. 50,000 non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells isolated de novo from from adult human blood were transplanted via intra femural injections. Four weeks later, engraftment was assessed by presence of hCD45+ populations and cells were phenotyped using flow cytometry. T cell response in humanized NSG mice engrafted with human MNCs followed by vaccination with OVA pulsed DCs was assessed by culturing splenocytes with OVA for 24 hours followed by assessment of T cell producing cytokines using Human Th1 Th2 and Th17 kits (BD Biosciences, Burlington, ON). Anti-Melan-A specific CD8+ T cell response was assessed in humanized mice engrafted with human MNCs followed by vaccination with Melan-A pulsed DCs. CD8+ T cell response specific to Melan-A was assessed by using Human HLA tetramers specific for Melan-A to identify Melan-A+ CD8+ T cells in the spleens of the humanized mice (BD Biosciences, Burlington, ON).

DC differentiation from pluripotent stem cells

[0059] Hematopoietic differentiation of hESCs and hiPSCs was performed as previously described(Risueno et al. , 2012). Human cord blood derived induced pluripotent stem cells were used as a source of human blood iPSCs. Day 15 embryoid bodies (EBs) were dissociated into single cells and CD34+CD45+ cells were isolated by flow cytometry on a BD FACAria, which were subsequently cultured in the presence of either GM-CSF along with TNF-a or GM-CSF along with IL-4 to drive DC generation.

Molecular and Microarray analysis

[0060] Freshly isolated cells were sorted based on surface marker expression and total RNA was extracted and amplified as reported previously(Hong et al., 201 1 ) from each purified population. For qPCR analysis, total cellular RNA was isolated using the RNeasy Plus Mini kit according to the manufacturer's instructions (Qiagen, Mississauga, ON) and cDNA was synthesized using qScript SuperMix (Quanta Biosciences, Gaithersburg, MD). For microarray analysis, total RNA was extracted using Trizol (Invitrogen) and hybridized to the Affymetrix Gene Chip Human Gene 1 .0 ST arrays (London Regional Genomics Centre, ON, Canada). Output data was normalized using Robust Multichip Averaging (RMA) algorithm and baseline transformed to the median of all samples using GeneSpring 12.5 software (Agilent Technologies). Mouse microarray data was used from publicly available GEO source (GSE15907, GSE1 1430, GSE37566, and GSE37029). Common gene entities between mouse and human data were identified and hierarchical clustering using Pearson distance metric and centroid linkage rule was done on normalized expression values using dChip software(Li and Wong, 2001 ). Multiple hypotheses testing, such as Benjamini- Hochberg false discovery rate P-value correction was performed for comparison.

Statistical analysis

[0061] Statistical analysis was performed using two-tailed f-test comparison and two-way ANOVA with GraphPad Prism (GraphPad Software Inc., San Diego, CA). Values were compared as means +/- standard error of the mean with p<0.05 considered statistically significant and indicated by asterisks in the figures.

Results

Identification of non-monocytic human DC precursors in the human

[0062] Despite the fact that bulk CD34 ⁺ populations are currently used for clinical DC based therapies (Ueno et al., 2010), primitive CD34 ⁺ CD38 ⁺ CD45RA ⁺ human progenitors have been shown to possess pDCs potential (Chicha et al., 2004). The inventors therefore hypothesized that precursors of non-monocytic DCs may emerge from this parent CD34 ⁺ CD38 ⁺ CD45RA ⁺ population upon differentiation induction. Rather than using bulk CD34 ⁺ populations used for DC based therapies (Ueno et al., 2010), CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors were isolated (Figure 8A) and cultured to screen for acquisition of markers expressed by murine non- monocytic DC precursors, including CX3CR1 , CD1 15 and CD135 (Onai et al., 2007) (Sasmono et al., 2003) (Figure 1A). Uniquely, CD1 15 showed a transient but distinct expression pattern that was maximal at day 2 of differentiation culture (Figure 1 B). Human CD1 15 ⁺ subset isolated from differentiated CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors had distinct features from monocyte DCs used clinically for immunotherapy. Unlike monocytic CD1 15 ^" cells with cytoplasmic vacuoles and distinct horseshoe shaped nuclei, CD1 15 ⁺ cells lacked monocytic morphological features (Figure 1 C). Candidate phenotypic analysis using markers associated with DCs and monocytes indicated that this unique CD1 15 ⁺ population co-expressed CD135 (Flt3) and HLA-DR, while lacking other lineage associated markers, including the hallmark monocytic marker, CD14 and CX3CR1 (Figure 1 D) (Asea et al., 2000). Based on these phenotypic screens and analyses (Figure 1 C and 1 D), we examined whether CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells could be found in the circulation of human adult peripheral blood (PB). Using the analysis and isolation strategy shown in Figure 1 E, human CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ could be detected in PB of healthy donors at a low frequency of 0.0063%. These PB derived cells lacked monocytic morphological features (Figure 1 E), as well as expression markers associated with myeloid or lymphoid progenitors (e.g. DC/monocytes or T/B cells) (Figure 1 F). The phoenotype of this subset was in sharp contrast to human monocytic CD14 ⁺ cells that express CD14, CD1 1 b and CD62L (Figure 1 F) (Geissmann et al., 2003) and are devoid of CD1 15, Flt3 or HLA-DR (Figure 1 G) and give rise to monocytic DCs. Beyond cellular morphology and phenotypic expression, de novo isolated non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells failed to secrete IL-6 and TNF-a following LPS stimulation (de Waal Malefyt et al., 1991 ) (Figure 1 G), as well as IL-12 (Ebner et al. , 2001 ) (Figure 1 H) produced by DCs derived from CD14 ⁺ monocytes. Global gene expression profiling demonstrated that human CD1 15 ⁺ Flt3 ⁺ HLA- DR ⁺ cells demonstrated similar signatures to established mouse non- monocytic DC precursors (Figure 8B) (Liu et al., 2009; Onai et al., 2007) while displaying a distinct molecular profile from human monocytic CD14 ⁺ cells (Figure 1 1). These results indicate that CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells represent a unique subset of cells of non-monocytic origin.

[0063] To functionally characterize properties of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells in the context of DC development their response to macrophage/DC differentiating inducing cytokines was compared to the response of purified monocytic CD14 ⁺ cells. Macrophage colony stimulating factor (M-CSF) drove expansion/differentiation of monocytic CD14 ⁺ cells into macrophages, whereas non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells failed to survive under these same conditions (Figure 1 J and 1 K). In contrast, Flt3 Ligand (Flt3L), shown to expand non-monocytic DC precursors in the mouse (Onai et al., 2007), inhibited survival of monocytic CD14 ⁺ cells while promoting expansion and subsequent DC differentiation of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells (Figure 1 L and 1 M). Furthermore, the combination of GM-CSF and I L-4, known to drive direct differentiation of monocytic CD14 ⁺ cells into DCs without expansion (Cavanagh et al. , 1998), induced significant expansion/differentiation of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells into DCs with increased expression of T cell stimulatory markers (CD83, CD86) (Dissanayake et al., 201 1 ) (Figure 8C and 8D). These results suggest that non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells have a unique cytokine response profile and are physiologically distinct from monocytic CD14+ cells. Biologically, DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ precursors demonstrated superior CD4 ⁺ and CD8 ⁺ T cell proliferation potential (Figure 1 N and 10) and significantly increased levels of Th1 inducing cytokines IL12p35, I L12p40 (Kano et al., 2008) and I L- 1 β in comparison to DCs derived from monocytic CD14 ⁺ cell origin (Figure 1 P). These phenotypic, molecular and in vitro analyses indicate that CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells derived from CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitor in vitro or de novo isolated from adult PB are physiologically distinct from monocytic CD14 ⁺ cells. CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells therefore appear to represent candidate non- monocytic DC precursors in the human (hNM-DCPs). In vitro clonal derivation of DC repertoire including pDCs from non- monocytic human precursors

[0064] Due to the low frequency of hNM-DCPs, the culture conditions for expansion of CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells were optimized in order to prevent the loss of their phenoptypic morphology. The combination of Flt3L in the presence of human bone marrow stromal cells (BMSCs) was determined to allow for the expansion of CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells without loss of the characteristic morphology of non-dendritic cells (Figure 9A and 9B). Consequently, de novo isolated CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells were co-cultured with BMSCs in presence of two differentiation cytokine cocktails (Figure 2H) previously shown for their potency on CD14 ⁺ monocytic DC precursors. Compared to the solely presence of GM-CSF and IL4 (GMI), the combination of Flt3L, Stem Cell Factor (SCF), GM-CSF and IL4 (FSGMI) demonstrated better overall human cells input (CD45) and significant frequencies of differentiation into mature DCs (phenotypically identified as CD1 a ^{+ "} CD1 1 c ⁺ HLA-DR ⁺ CD83 ⁺ ) and pDCs (CD1 a ^" CD123 ⁺ BDCA-2 ⁺ ) (Figure 2I), thereby delineating a robust read-out assay for hNMDCPs differentiation.

[0065] As CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells may be a heterogeneous population, these cells were clonally examined in vitro to quantitate the DC developmental capacity of this unique subset. Two sources of human non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells were used in clonal analysis as depicted in Figure 2A: 1 ) De novo isolated directly from adult human blood, and 2) Purified from human-mouse xenograft recipients transplanted with CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors. Non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells from either source were clonally deposited on human BMSCs to drive clonal expansion in presence of Flt3L followed by DC differentiation inducing conditions also in presence of Flt3L or GM-CSF and IL-4 (Figure 2B). All xenograft recipients transplanted with CD34 ⁺ CD38 ⁺ CD45RA ⁺ progenitors generated non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells in vivo (Figure 2C), where GM-CSF and IL-4 treatment significantly increased non-monocytic

CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ reconstitution compared to untreated transplanted cells (Figure 2D). In vitro monocytic CD14 ⁺ cells generated DCs that were phenotypically identified as CD1 a ^{+ "} CD1 1 c ⁺ HLA-DR ⁺ CD83 ⁺ (Figure 2E) at 100% frequency, and were completely devoid of CD1 a ^" CD123 ⁺ BDCA-2 ⁺ cells representing human pDC (Dzionek et al., 2001 ) (Figure 2E and 2G). In contrast to monocytic CD14+ cells, CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells clonally generated either DCs or pDCs exclusively, or a mixture of DCs and pDCs (Figure 2E) at a frequency of 43, 33 and 24% respectively (Figure 2G). Plasmacytoid DCs derived from non-monocytic precursors was correlated with IFN-a production, confirming functional responsiveness of pDCs generated (Dzionek et al., 2001 ) (Figure 2F). These results indicate that monocytic CD14+ populations used clinically for DCs generation are homogeneously devoid of pDC developmental capacity, whereas de novo isolated or xenograft generated human non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ subpopulations exclusively possess unipotent pDCs or bipotent DC precursors. These results reveal that the developmental origins of pDCs in the human arise from CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells.

Non-monocytic DC precursors exclusively give rise to human plasmacytoid DCs in vivo

[0066] To examine the in vivo DC development potency of non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells, transplantation studies were carried out in immune deficient recipients using de novo isolated non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells vs. clinically used monocytic CD14 ⁺ cells derived from adult donors (illustrated in Figure 3A). Human hematopoietic engrafted cells, detected by CD45 expression, were analyzed and further gated into CD33 ^" and CD33 ⁺ subfractions to separate myeloid vs. non-myeloid subsets (Figure 3B). A complete phenotypic analysis of human DC repertoire generated in vivo from transplanted CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ vs. monocytic CD14 ⁺ cells was comparatively performed using co-expression of HLA- DR ⁺ CD83 ⁺ , along with BDCA-1 (Zaba et al., 2007) and BDCA-3 (Poulin et al., 2010) recently used to characterize human DC subsets, as well as plasmacytoid DCs (Dzionek et al., 2001 ) defined by co-expression of CD123 and BDCA-2. Representative flow cytometry plots of xenografted mice transplanted with precursor subsets are shown in Figure 3B, and were quantitated for individual recipients (Figure 3C and 3D). Within the CD33+ myeloid subset, xenograft recipients transplanted with monocytic CD14 ⁺ cells showed generation of HLA-DR ⁺ CD83 ⁺ DCs, as well as BDCA-1 ⁺ and BDCA-3 ⁺ DCs, while giving rise to CD14 ⁺ cells (Figure 3B). In contrast, non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells gave rise to HLA-DR ⁺ CD83 ⁺ DCs, but were devoid of BDCA-1 ⁺ and BDCA-3 ⁺ DCs, or monocytic CD14 ⁺ cells (Figure 3B). However, unlike monocytic CD14+ precursors, only non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells were able to generate CD123 ⁺ BDCA-2 ⁺ pDCs in vivo (Figure 3B). These results were consistent in all independent experiments and recipient mice (Figure 3C and 3D). In contrast to monocytic DC precursors, CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells represent a non-monocytic precursor that gives rise to a unique repertoire of DCs that includes generation of pDCs in vivo.

Preferential Th1 vs Th2 in vitro response can be induced by human non- monocytic DCs

[0067] A Th1 response (characterized by IFN-γ production) is required to promote antitumor immune capacity, whereas Th2 response (characterized by I L-4 production) is suppressive (Cerundolo et al., 2004; Fields et al., 1998; Plantinga et al., 2013). Due to the unique in vivo capacity of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells, including pDC generation, the inventors hypothesized that these precursors may have unique properties of helper T cell activation. As illustrated in Figure 4A, their survival and ability to induce Th1 vs. Th2 response was compared to the clinical standard of conventional DCs derived from monocytic CD14+ and bulk CD34+ cells. DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells demonstrated a preferential tolerance and enhanced survival in response to bacterial infectious LPS induced cell death as compared to monocytic CD14+ and bulk CD34+ cells (Figure 4B). Analysis of DC populations derived from bulk CD34+ cells in presence of both GM-CSF and IL-4 or FLt3L identified a lack of pDC generation (Figure 4C), a property unique to non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA- DR ⁺ cells (Figure 2G) and highlighting an absence of this population in clinically utilized DC regimens for immunotherapy. DCs derived from non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells generated significantly higher proportions of I FN-γ producing Th1 T cells (Figure 4D and 4E) along with a significant reduction in generation of IL-4 producing Th2 cells when compared with DCs derived from monocytic CD14 ⁺ cells as well as bulk CD34+ cells (Figure 4D and 4F). Furthermore, upon treatment with thymic stromal lymphopoietin (TSLP), known to selectively drive Th2 response (Soumelis et al., 2002), DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ precursors preferentially induced IFN-γ production of T cells over IL-4 production with a five-fold higher frequency in Th1 cells than Th2 cells compared to DCs derived from monocytic CD14 ⁺ and bulk CD34+ cells, which preferentially drove the expected Th2 over a Th1 response (Figure 4G, 4H and 4I). Additional in vitro studies revealed preferential induction of C-C chemokine receptor type 7 (CCR7), known to mediate DC migration to lymphoid organs (Scandella et al., 2004) following LPS, in DCs from CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells (Figure 4J). These results indicate superior survival and propensity of DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells to prime a Th1 vs. Th2 response, along with a homing potential that is distinct from the clinical standard of DCs derived from monocytic CD14 ⁺ as well as bulk CD34+ cells.

Human blood derived iPSCs uniquely generate functional non- monocytic DCs

[0068] Using previously defined conditions to generate hematopoietic progenitors from human pluripotent stem cells (PSCs) (Szabo et al., 2010; Vijayaragavan et al. , 2009), primitive hematopoietic CD34+CD45+ cells were isolated from various sources of hPSCs and screened for capacity to generate non-monocytic CD1 15+Flt3+HLA-DR+ cells (Figure 5A). Human blood derived iPSCs (hB-iPS) exclusively gave rise to non-monocytic CD1 15+Flt3+HLA-DR+ cells which could be terminally differentiated into DCs (Figure 5A and S3A). hB-iPS derived non-monocytic CD1 15+Flt3+HLA-DR+ cells gave rise to pDCs, confirmed by expression of BDCA-2 and CD123 (Figure 5B) and functionally validated by production of IFN-a (Figure 5C), with a complete absence of pDC generation from other hPSC sources. In addition to the unique capacity for pDC generation, DCs generated from hB-iPS derived CD1 15+Flt3+HLA-DR+ cells also showed a significantly greater potential for both CD4+ and CD8+ T cell proliferation compared to DCs derived from other hPSC sources (Figure 10B and 10C). Due to the unique properties of hB-iPS derived CD1 15+Flt3+HLA-DR+ cells, further characterization of T cell differentiation was further carried out as shown in Figure 10D. DCs generated from hB-iPS derived non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells demonstrated a preferential tolerance and enhanced survival in response to bacterial infectious LPS induced cell death as compared to DCs from other hPSC sources (Figure 10E). DCs generated from hB-iPSCs derived non-monocytic CD1 15+Flt3+HLA-DR+ cells also generated higher proportions of IFN-γ producing Th1 T cells than DC subsets generated from other hPSC sources, along with significantly reduced generation of IL-4 producing Th2 cells (Figure 5D). Moreover, under Th2 conditions generated by TSLP treatment of DCs, DCs generated from hB- iPSCs derived non-monocytic CD1 15+Flt3+HLA-DR+ cells preferentially induced IFN-γ production in T cells with five-fold higher frequency of Th1 cells vs. Th2 cells (Figure 5E). In contrast, DCs from other hPSC sources preferentially induced Th2 differentiation over Th1 differentiation (Figure 5E). To examine the in vivo DC development potency of non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells derived from hB-iPS, transplantation studies were carried out in immune deficient recipients using non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells vs. monocytic CD14 ⁺ cells derived hB-iPS (Figure 5F). Human hematopoietic engrafted cells, detected by CD45 expression, were analyzed for DC markers: CD1 1 c, CD86, CD14 and BDCA-2. Unlike, hB-iPS derived monocytic CD14+ cells, hB-iPS derived non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells showed exclusive generation of BDCA-2+ pDCs (Figure 5F and 10F). These findings indicate that amongst the hPSC sources examined, developmental capacity and hierarchical organization of DC precursors is functionally intact in hB-iPSCs only, which have the ability to generate non-monocytic DCs that preferentially drive a Th1 response. This is likely due to epigenetic memory of hematopoietic lineage that is retained in human blood derived hB-iPSCs (Kim et al., 201 1 ; Lee et al., 2013). These data indicate that hB-iPSCs offer an abundantly available resource for non- monocytic DC-based vaccine development and related applications.

Non-monocytic DC precursors are capable of superior migration and Th1 induction in vivo

[0069] To further comparatively characterize these subsets of DC generating precursors beyond current in vitro benchmarks of T cell response (Segura et al., 2013), additional functional studies were performed in vivo. Recipient NSG mice known to support human T cells (Risueno et al., 201 1 ) were transplanted with human MNCs to reconstitute a humanized in vivo environment as shown in Figure 6A. Similar to in vitro observations of preferential CCR7 induction (Figure 4J), significantly higher proportions of DCs were observed in lymph nodes of NSG mice transplanted with DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells compared to recipients transplanted with DCs derived from monocytic CD14 ⁺ cells (Figure 6B). Furthermore, DCs were only observed in the thymus of mice transplanted with DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells compared to the recipient mice receiving DCs derived from monocytic CD14 ⁺ cells (Figure 6B). These in vivo observations indicate a superior homing capacity of DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells to migrate to lymphoid tissues. These same xenografted mice were next challenged with ovalbumin in complete Freund's adjuvant (Vidard et al., 1995) to compare T cell response in the spleen of recipients (Figure 6C). The specificity of the in vivo immune response was confirmed by lack of ovalbumin induced splenocytic proliferation from control mice transplanted with DCs in the absence of antigen pulse stimulation (Figure 1 1 ). DCs derived from monocytic CD14 ⁺ cells selectively gave rise to a preferential Th2 response over Thl with 6-7 fold higher IL-4 ⁺ CD4 ⁺ T cells compared to IFN-y ⁺ CD4 ⁺ T cells (Figure 6C and 6D). In contrast, DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells led to a preferential induction of Th1 over Th2 response with 2-3 fold higher proportions of IFN-γ secreting T cells than I L-4 producing T cells (Figure 6C and 6D). Concomitantly, expression of Th1 associated transcription factor T-bet (Zhu et al., 2010) was several fold higher than that of the Th2 associated transcription factor GATA-3 (Zhu et al., 2010) observed in spleens of recipients transplanted with DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells compared to monocytic CD14 ⁺ cells (Figure 6E). In addition to the Th1 and Th2 cells, another subset of T cells characterized by IL-17 production, termed Th17, has recently been identified (Yang et al., 2008) to have potent tumor eradication capacity (Martin-Orozco et al. , 2009). As such, Th17 induction was examined using novel non-monocytic DC precursors. Although DCs derived from monocytic CD14 ⁺ cells showed poor induction of Th17 response in vivo, DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells generated a significantly higher splenic Th17 response along with increased expression of Th17 associated transcription factor, RORyT (Yang et al., 2008) (Figures 6C, 6D and 6E).

Non-monocytic human DCs drive superior anti-tumor immune response in vivo

[0070] Based on the biological potential of DCs derived from non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells possessing a superior Th1 vs. Th2 and Th17 induction capacity that is accompanied by superior homing capacity to lymphoid organs in vivo, their ability to potentiate a CD8 ⁺ T cell response directed against tumor antigens was examined. Melan-A is an antigen widely expressed in melanoma, where CD8 ⁺ T cell response against Melan-A is indicative of an anti-myeloma immune response (Boon and van der Bruggen, 1996) and is used as a surrogate for measuring the levels of protective immune response in melanoma patients (Romero et al., 2002). DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells possessed nearly 10-fold higher capacity to drive induction of Melan-A specific CD8 ⁺ T cells compared to DCs derived from monocytic CD14 ⁺ cells in vitro (Figure 7A and 7B). To test the in vivo efficacy of DCs to drive an anti-tumor immune response from non-monocytic DC precursors, a human-xenograft models was used as illustrated in Figure 7C. To identify CD8 ⁺ T cells, engrafted human CD45 ⁺ cells were gated and further sub-fractionated into hCD3 ⁺ hCD8 ⁺ subsets (Figure 7D). Consistent with our in vitro observations, DCs derived from non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells increased in vivo induction of Melan-A specific CD8 ⁺ anti-tumor T cell response relative to monocytic CD14 ⁺ cells (Figure 7E and 7F). Collectively, these results demonstrate that DCs derived from non-monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells have a superior capacity in inducing anti-tumor T cell response compared to the standard clinical regimen of using DCs derived from monocytic CD14 ⁺ cells (Cerundolo et al., 2004; Palucka and Banchereau, 2012).

Discussion

[0071] Although murine studies have formed the basis for DC-mediated cancer immunotherapy, their clinical applicability has been hindered due to inadequate delineation of analogous DC precursors in the human. In one aspect of the description, the inventors have identified human non-monocytic DC precursors capable of giving rise DC with unique properties, including generation of pDCs. As demonstrated both in vitro and in vivo, human DCs generated from CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ non-monocytic precursors possess preferential Th1 inducibility critical for cancer immunotherapy, even under conditions that selectively drive Th2 response. Specific to anti-tumor immunotherapy, human DCs derived from novel non-monocytic precursors demonstrated a > 10 fold enhanced ability to drive Melan-A specific CD8+ T cell response in vivo compared to clinically utilized monocytic DCs, further supporting unique applications and suitability for cancer therapy. Mouse DC generation proceeds from macrophage/dendritic cell precursor (MDP) (Fogg et al., 2006), which differentiates into common dendritic cell precursor (CDP) (Onai et al., 2007), giving rise to non-monocytic DCs(Liu et al., 2009). MDP and CDP are phenotypically overlapping populations within the mouse BM with both populations identified by CX3CR1 expression (Auffray et al., 2009) (Fogg et al., 2006; Onai et al., 2007). Surprisingly, the data indicates that non- monocytic CD1 15 ⁺ Flt3 ⁺ HLA-DR ⁺ cells lack CX3CR1 expression, highlighting unique differences between mouse and human hematopoiesis in the context of DC biology. Given that the current understanding of human DC biology largely comes from studies in the mouse (Ardavin, 2003; Geissmann et al., 2010), these differences are critical in informing and revisiting clinical applications.

[0072] In addition, novel sources of these non-monocytic DCs were identified, where only hBiPS have the ability to generate CD1 15 ⁺ Flt3 ⁺ HLA- DR ⁺ non-monocytic precursors with a capacity to generate non-monocytic DCs both in vitro and in vivo that can preferentially drive a Th1 response. This not only offers a novel source of non-monocytic human DCs, but also provides a system to model and study DC deficiencies recently described in humans (Bigley et al., 201 1 ; Collin et al., 201 1 ) that cannot be studied using human ESCs or skin-derived iPSC sources due to their preferential ability to generate monocytic DCs alone. Moreover, DCs have also been explored as vaccines for viral infections such as HCV (Zhou et al., 2012) and HIV (Garcia et al., 2013) and hB-iPS derived non-monocytic CD1 15+Flt3+HLA-DR+ cells offers a renewable source for autologous vaccine development. Although preclinical work with hB-iPS derived non-monocytic CD1 15+Flt3+HLA-DR+ cells provides validation for their utility in vaccine development due to their propensity for enhanced survival, T cell proliferation and Th1 differentiation, further optimization has been limited by the complexities of generating functional T cells from pluripotent stem cell sources.

[0073] The methodology described herein can be applied with ease in a translational setting for the development of clinical grade DC vaccines for immunotherapy as well as use in anti-tumor DC therapies. Additionally, identification of human non-monocytic DC precursors allows for these cells to be used in screening platforms for identification of human vaccine adjuvants, which has been limited with murine DC populations. Moreover, identification of human non-monocytic DC precursors also allows for investigation of DC biology in human autoimmune disorders, which have previously relied on DCs derived from monocytes. Although non-monocytic precursors comprise a rare population in blood (<1 %), identification of human precursors capable of clonal expansion to several hundred-fold in the presence of Flt3L provides a method to apply to patient immunotherapy, in addition to generation from patient specific iPSCs generated from blood cells. Importantly, it has been demonstrated that standard clinical conditions to expand DCs using GM-CSF do not support human non-monocytic DC precursors, suggesting that these same non-monocytic DCs have not participated in clinical therapies used to date. The present Example provides a proof of principle for generation and utility of unique human DC precursors for immunotherapy as compared to current clinically used sources of DCs (Banchereau and Steinman, 1998; Cerundolo et al., 2004; Palucka and Banchereau, 2012).

[0074] While the present disclosure has been described with reference to what are presently considered to be the examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0075] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. References:

Ardavin, C. (2003). Origin, precursors and differentiation of mouse dendritic cells. Nature reviews Immunology 3, 582-590.

Asea, A., Kraeft, S.K. , Kurt-Jones, E.A., Stevenson, M.A., Chen, L.B., Finberg, R.W., Koo, G.C., and Calderwood, S.K. (2000). HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nature medicine 6, 435-442.

Auffray, C, Fogg, D.K. , Narni-Mancinelli, E., Senechal, B. , Trouillet, C, Saederup, N., Leemput, J. , Bigot, K., Campisi, L, Abitbol, M., et al. (2009). CX3CR1 + CD1 15+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation. The Journal of experimental medicine 206, 595-606.

Augier, S., Ciucci, T., Luci, C, Carle, G.F. , Blin-Wakkach, C, and Wakkach, A. (2010). Inflammatory blood monocytes contribute to tumor development and represent a privileged target to improve host immunosurveillance. Journal of immunology 185, 7165-7173.

Banchereau, J., and Palucka, A.K. (2005). Dendritic cells as therapeutic vaccines against cancer. Nature reviews Immunology 5, 296-306.

Banchereau, J., and Steinman, R.M. (1998). Dendritic cells and the control of immunity. Nature 392, 245-252.

Banchereau, J., Zurawski, S., Thompson-Snipes, L, Blanck, J. P., Clayton, S., Munk, A., Cao, Y., Wang, Z., Khandelwal, S. , Hu, J., et al. (2012). Immunoglobulin-like transcript receptors on human dermal CD14+ dendritic cells act as a CD8-antagonist to control cytotoxic T cell priming. Proceedings of the National Academy of Sciences of the United States of America 109, 18885-18890.

Bhatia, M., Wang, J.C., Kapp, U., Bonnet, D., and Dick, J.E. (1997). Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proceedings of the National Academy of Sciences of the United States of America 94, 5320-5325. Bigley, V. , Haniffa, M., Doulatov, S., Wang, X.N. , Dickinson, R., McGovern, N., Jardine, L, Pagan, S., Dimmick, I., Chua, I., et al. (201 1 ). The human syndrome of dendritic cell, monocyte, B and NK lymphoid deficiency. The Journal of experimental medicine 208, 227-234.

Boon, T., and van der Bruggen, P. (1996). Human tumor antigens recognized by T lymphocytes. The Journal of experimental medicine 183, 725-729.

Candido, K.A., Shimizu, K., McLaughlin, J.C., Kunkel, R., Fuller, J.A., Redman, B.G., Thomas, E.K., Nickoloff, B.J., and Mule, J.J. (2001 ). Local administration of dendritic cells inhibits established breast tumor growth: implications for apoptosis-inducing agents. Cancer research 61 , 228-236.

Cavanagh, L. L., Saal, R.J. , Grimmett, K.L., and Thomas, R. (1998). Proliferation in monocyte-derived dendritic cell cultures is caused by progenitor cells capable of myeloid differentiation. Blood 92, 1598-1607.

Celluzzi, CM., Mayordomo, J. I ., Storkus, W.J. , Lotze, M.T., and Falo,

L.D., Jr. (1996). Peptide-pulsed dendritic cells induce antigen-specific CTL- mediated protective tumor immunity. The Journal of experimental medicine 183, 283-287.

Cerundolo, V., Hermans, I.F., and Salio, M. (2004). Dendritic cells: a journey from laboratory to clinic. Nat Immunol 5, 7-10.

Cheong, C, Matos, I ., Choi, J.H., Dandamudi, D.B., Shrestha, E., Longhi, M.P., Jeffrey, K.L., Anthony, R.M., Kluger, C, Nchinda, G. , et al. (2010). Microbial stimulation fully differentiates monocytes to DC- SIGN/CD209(+) dendritic cells for immune T cell areas. Cell 143, 416-429.

Chicha, L, Jarrossay, D., and Manz, M.G. (2004). Clonal type I interferon-producing and dendritic cell precursors are contained in both human lymphoid and myeloid progenitor populations. The Journal of experimental medicine 200, 1519-1524.

Collin, M., Bigley, V., Haniffa, M., and Hambleton, S. (201 1 ). Human dendritic cell deficiency: the missing ID? Nature reviews Immunology 1 1 , 575- 583. Colonna, M., Trinchieri, G., and Liu, Y.J. (2004). Plasmacytoid dendritic cells in immunity. Nat Immunol 5, 1219-1226.

de Waal Malefyt, R., Abrams, J., Bennett, B., Figdor, C.G. , and de Vries, J.E. (1991 ). Interleukin 10(l L-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of I L-10 produced by monocytes. The Journal of experimental medicine 174, 1209-1220.

Dissanayake, D., Hall, H. , Berg-Brown, N., Elford, A.R. , Hamilton, S.R., Murakami, K., Deluca, L.S., Gommerman, J.L., and Ohashi, P.S. (201 1 ). Nuclear factor-kappaBI controls the functional maturation of dendritic cells and prevents the activation of autoreactive T cells. Nature medicine 17, 1663- 1667.

Dunn, G.P., Koebel, CM., and Schreiber, R.D. (2006). Interferons, immunity and cancer immunoediting. Nature reviews Immunology 6, 836-848.

Dzionek, A., Sohma, Y., Nagafune, J., Cella, M., Colonna, M., Facchetti, F., Gunther, G., Johnston, I. , Lanzavecchia, A., Nagasaka, T., et al. (2001 ). BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction. The Journal of experimental medicine 194, 1823-1834.

Ebner, S., Ratzinger, G., Krosbacher, B., Schmuth, M., Weiss, A., Reider, D., Kroczek, R.A. , Herold, M., Heufler, C, Fritsch, P., et al. (2001 ). Production of I L-12 by human monocyte-derived dendritic cells is optimal when the stimulus is given at the onset of maturation, and is further enhanced by I L-4. Journal of immunology 166, 633-641 .

Fields, R.C., Shimizu, K., and Mule, J.J. (1998). Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America 95, 9482-9487.

Figdor, C.G., de Vries, I. J., Lesterhuis, W.J., and Melief, C.J. (2004). Dendritic cell immunotherapy: mapping the way. Nature medicine 10, 475- 480. Fogg, D.K., Sibon, C, Miled, C, Jung, S., Aucoutuher, P., Littman, D.R., Cumano, A. , and Geissmann, F. (2006). A clonogenic bone marrow progenitor specific for macrophages and dendritic cells. Science 31 1 , 83-87.

Garcia, F., Climent, N., Guardo, A.C., Gil, C, Leon, A., Autran, B., Lifson, J.D., Martinez-Picado, J., Dalmau, J., Clotet, B., et al. (2013). A dendritic cell-based vaccine elicits T cell responses associated with control of HIV-1 replication. Sci Transl Med 5, 166ra162.

Geissmann, F., Jung, S., and Littman, D.R. (2003). Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19, 71 -82.

Geissmann, F., Manz, M.G., Jung, S., Sieweke, M.H., Merad, M., and Ley, K. (2010). Development of monocytes, macrophages, and dendritic cells. Science 327, 656-661 .

Goubier, A., Dubois, B. , Gheit, H., Joubert, G., Villard-Truc, F., Asselin- Paturel, C, Trinchieri, G., and Kaiserlian, D. (2008). Plasmacytoid dendritic cells mediate oral tolerance. Immunity 29, 464-475.

Hammad, H., Charbonnier, A.S., Duez, C, Jacquet, A., Stewart, G.A., Tonnel, A.B., and Pestel, J. (2001 ). Th2 polarization by Der p 1 -pulsed monocyte-derived dendritic cells is due to the allergic status of the donors. Blood 98, 1 135-1 141 .

Hammad, H., Lambrecht, B.N., Pochard, P., Gosset, P., Marquillies, P., Tonnel, A.B., and Pestel, J. (2002). Monocyte-derived dendritic cells induce a house dust mite-specific Th2 allergic inflammation in the lung of humanized SCID mice: involvement of CCR7. Journal of immunology 169, 1524-1534.

Hong, S.H., Rampalli, S., Lee, J.B., McNicol, J., Collins, T., Draper,

J.S., and Bhatia, M. (201 1 ). Cell fate potential of human pluripotent stem cells is encoded by histone modifications. Cell stem cell 9, 24-36.

Hung, K., Hayashi, R., Lafond-Walker, A., Lowenstein, C, Pardoll, D., and Levitsky, H. (1998). The central role of CD4(+) T cells in the antitumor immune response. The Journal of experimental medicine 188, 2357-2368. Kano, S., Sato, K., Morishita, Y., Vollstedt, S., Kim, S., Bishop, K., Honda, K., Kubo, M., and Taniguchi, T. (2008). The contribution of transcription factor IRF1 to the interferon-gamma-interleukin 12 signaling axis and TH 1 versus TH-17 differentiation of CD4+ T cells. Nat Immunol 9, 34-41 .

Kim, K., Zhao, R., Doi, A., Ng, K., Unternaehrer, J., Cahan, P., Huo, H.,

Loh, Y.H., Aryee, M.J., Lensch, M.W., et al. (201 1 ). Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells. Nat Biotechnol 29, 1 1 17-1 1 19.

Kushwah, R., and Hu, J. (201 1 ). Complexity of dendritic cell subsets and their function in the host immune system. Immunology 133, 409-419.

Lake, R.A., and Robinson, B.W. (2005). Immunotherapy and chemotherapy-a practical partnership. Nature reviews Cancer 5, 397-405.

Lee, J.H., Lee, J.B. , Hong, S.H., Shapovalova, Z., Fiebig-Comyn, A., Risueno, R. M., Laronde, S., Szabo, E., and Bhatia, M. (2013). Retention of the somatic transcriptome governs differentiation potential of induced pluripotent stem cells. J Cell Biol, Under review.

Li, C, and Wong, W.H. (2001 ). Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proceedings of the National Academy of Sciences of the United States of America 98, 31 -36.

Liu, K., Victora, G.D., Schwickert, T.A., Guermonprez, P., Meredith, M. M., Yao, K., Chu, F.F., Randolph, G.J., Rudensky, A.Y., and Nussenzweig, M. (2009). In vivo analysis of dendritic cell development and homeostasis. Science 324, 392-397.

Manz, M.G., Miyamoto, T., Akashi, K. , and Weissman, I.L. (2002).

Prospective isolation of human clonogenic common myeloid progenitors. Proceedings of the National Academy of Sciences of the United States of America 99, 1 1872-1 1877.

Martin-Orozco, N., Muranski, P., Chung, Y., Yang, X.O., Yamazaki, T., Lu, S., Hwu, P., Restifo, N.P. , Overwijk, W.W., and Dong, C. (2009). T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity 31 , 787-798.

Mayordomo, J. I., Zorina, T., Storkus, W.J. , Zitvogel, L, Celluzzi, C, Falo, L.D., Melief, C.J., lldstad, ST., Kast, W.M., Deleo, A. B., et al. (1995). Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nature medicine 1 , 1297-1302.

Naik, S.H., Sathe, P. , Park, H.Y., Metcalf, D., Proietto, A.I. , Dakic, A., Carotta, S., O'Keeffe, M., Bahlo, M., Papenfuss, A., et al. (2007). Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. Nat Immunol 8, 1217-1226.

Nair, S.K., Heiser, A. , Boczkowski, D., Majumdar, A., Naoe, M., Lebkowski, J.S., Vieweg, J., and Gilboa, E. (2000). Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells. Nature medicine 6, 101 1 -1017.

Nestle, F.O., Alijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dummer, R., Burg, G., and Schadendorf, D. (1998). Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nature medicine 4, 328-332.

Nestle, F.O. , Banchereau, J. , and Hart, D. (2001 ). Dendritic cells: On the move from bench to bedside. Nature medicine 7, 761 -765.

Onai, N., Obata-Onai, A., Schmid, M.A., Ohteki, T., Jarrossay, D., and Manz, M.G. (2007). Identification of clonogenic common Flt3+M-CSFR+ plasmacytoid and conventional dendritic cell progenitors in mouse bone marrow. Nat Immunol 8, 1207-1216.

Palucka, K., and Banchereau, J. (2012). Cancer immunotherapy via dendritic cells. Nature reviews Cancer 12, 265-277.

Papatriantafyllou, M. (201 1 ). Tumour immunology: TSLP drives human tumour progression. Nature reviews Immunology 1 1 , 235.

Plantinga, M., Guilliams, M. , Vanheerswynghels, M. , Deswarte, K.,

Branco-Madeira, F., Toussaint, W., Vanhoutte, L, Neyt, K., Killeen, N., Malissen, B., et al. (2013). Conventional and monocyte-derived CD1 1 b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen. Immunity 38, 322-335.

Poulin, L.F., Salio, M. , Griessinger, E., Anjos-Afonso, F., Craciun, L, Chen, J.L., Keller, A.M., Joffre, O., Zelenay, S., Nye, E., et al. (2010). Characterization of human DNGR-1 + BDCA3+ leukocytes as putative equivalents of mouse CD8alpha+ dendritic cells. The Journal of experimental medicine 207, 1261 -1271 .

Risueno, R.M., Campbell, C.J., Dingwall, S., Levadoux- Martin, M., Leber, B., Xenocostas, A., and Bhatia, M. (201 1 ). Identification of T- lymphocytic leukemia-initiating stem cells residing in a small subset of patients with acute myeloid leukemic disease. Blood 1 17, 71 12-7120.

Risueno, R.M., Sachlos, E., Lee, J.H., Lee, J. B., Hong, S.H. , Szabo,

E. , and Bhatia, M. (2012). Inability of human induced pluripotent stem cell- hematopoietic derivatives to downregulate microRNAs in vivo reveals a block in xenograft hematopoietic regeneration. Stem Cells 30, 131 -139.

Romero, P., Valmori, D., Pittet, M.J., Zippelius, A., Rimoldi, D., Levy,

F. , Dutoit, V., Ayyoub, M., Rubio-Godoy, V., Michielin, O., et al. (2002). Antigenicity and immunogenicity of Melan-A/MART-1 derived peptides as targets for tumor reactive CTL in human melanoma. Immunol Rev 188, 81 -96.

Sasmono, R.T., Oceandy, D., Pollard, J.W., Tong, W., Pavli, P., Wainwright, B.J. , Ostrowski, M.C., Himes, S.R., and Hume, D.A. (2003). A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood 101 , 1 155-1 163.

Scandella, E., Men, Y., Legler, D.F., Gillessen, S., Prikler, L., Ludewig, B., and Groettrup, M. (2004). CCL19/CCL21 -triggered signal transduction and migration of dendritic cells requires prostaglandin E2. Blood 103, 1595-1601 .

Schraml, B.U., van Blijswijk, J., Zelenay, S., Whitney, P.G., Filby, A., Acton, S.E., Rogers, N.C., Moncaut, N., Carvajal, J.J. , and Reis, E.S.C. (2013). Genetic Tracing via DNGR-1 Expression History Defines Dendritic Cells as a Hematopoietic Lineage. Cell 154, 843-858.

Schuler, G., Schuler-Thurner, B., and Steinman, R. M. (2003). The use of dendritic cells in cancer immunotherapy. Curr Opin Immunol 15, 138-147.

Segura, E., Touzot, M., Bohineust, A., Cappuccio, A., Chiocchia, G. ,

Hosmalin, A., Dalod, M., Soumelis, V., and Amigorena, S. (2013). Human inflammatory dendritic cells induce Th17 cell differentiation. Immunity 38, 336- 348.

Sharma, M., Hegde, P., Aimanianda, V., Beau, R., Senechal, H., Poncet, P., Latge, J. P., Kaveri, S.V., and Bayry, J. (2013). Circulating human basophils lack the features of professional antigen presenting cells. Sci Rep 3, 1 188.

Shortman, K., and Liu, Y.J. (2002). Mouse and human dendritic cell subtypes. Nature reviews Immunology 2, 151 -161 .

Song, W., Kong, H.L., Carpenter, H., Torii, H., Granstein, R. , Rafii, S.,

Moore, M.A., and Crystal, R.G. (1997). Dendritic cells genetically modified with an adenovirus vector encoding the cDNA for a model antigen induce protective and therapeutic antitumor immunity. The Journal of experimental medicine 186, 1247-1256.

Soumelis, V., Reche, P.A., Kanzler, H., Yuan, W., Edward, G. , Homey,

B., Gilliet, M., Ho, S., Antonenko, S., Lauerma, A., et al. (2002). Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat Immunol 3, 673-680.

Steinman, R. M. (2007). Lasker Basic Medical Research Award. Dendritic cells: versatile controllers of the immune system. Nature medicine 13, 1 155-1 159.

Steinman, R.M., and Banchereau, J. (2007). Taking dendritic cells into medicine. Nature 449, 419-426.

Szabo, E. , Rampalli, S., Risueno, R. M., Schnerch, A., Mitchell, R., Fiebig-Comyn, A., Levadoux-Martin, M., and Bhatia, M. (2010). Direct conversion of human fibroblasts to multilineage blood progenitors. Nature 468, 521 -526.

Ueno, H., Schmitt, N., Klechevsky, E., Pedroza-Gonzalez, A., Matsui, T., Zurawski, G., Oh, S., Fay, J., Pascual, V., Banchereau, J., et al. (2010). Harnessing human dendritic cell subsets for medicine. Immunol Rev 234, 199-212.

Vidard, L, Colarusso, L.J., and Benacerraf, B. (1995). Specific T-cell tolerance may reflect selective activation of lymphokine synthesis.

Proceedings of the National Academy of Sciences of the United States of America 92, 2259-2262.

Vijayaragavan, K., Szabo, E., Bosse, M., Ramos-Mejia, V., Moon, R.T., and Bhatia, M. (2009). Noncanonical Wnt signaling orchestrates early developmental events toward hematopoietic cell fate from human embryonic stem cells. Cell stem cell 4, 248-262.

Wang, R.F., and Wang, H.Y. (2002). Enhancement of antitumor immunity by prolonging antigen presentation on dendritic cells. Nat Biotechnol

20, 149-154.

Yang, X.O., Pappu, B.P., Nurieva, R., Akimzhanov, A., Kang, H.S., Chung, Y., Ma, L, Shah, B., Panopoulos, A.D., Schluns, K.S., et al. (2008). T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity 28, 29-39.

Yi, H., Guo, C , Yu, X., Gao, P., Qian, J., Zuo, D., Manjili, M.H., Fisher, P.B., Subjeck, J.R., and Wang, X.Y. (201 1 ). Targeting the immunoregulator SRA/CD204 potentiates specific dendritic cell vaccine-induced T-cell response and antitumor immunity. Cancer research 71 , 661 1 -6620.

Zaba, L.C., Fuentes-Duculan, J., Steinman, R.M., Krueger, J.G., and Lowes, M.A. (2007). Normal human dermis contains distinct populations of CD1 1 c+BDCA-1 + dendritic cells and CD163+FXIIIA+ macrophages. J Clin Invest 1 17, 2517-2525. Zhou, Y., Zhang, Y. , Yao, Z., Moorman, J. P., and Jia, Z. (2012). Dendritic cell-based immunity and vaccination against hepatitis C virus infection. Immunology 136, 385-396.

Zhu, J., Yamane, H., and Paul, W.E. (2010). Differentiation of effector CD4 T cell populations. Annual review of immunology 28, 445-489.