INTERLEUKIN-27 PRODUCING B CELLS AND USES THEREOF

Abstract

The present invention relates to an isolated population of mammalian cells comprising about 75% or more B-1a regulatory cells that express the inhibitory cell surface receptors lymphocyte-activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and CXC chemokine receptor type 4 (CXCR4) and secrete interleukin-27 (IL-27). The present invention also relates to methods of preparing and using the population of cells to suppress the immune system and / or treat or prevent disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of co-pending U.S. Provisional Patent Application No. 62 / 863,054, filed June 18, 2019, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with federal support by the National Eye Institute of the National Institutes of Health under Project No. Z01EY000350-18. The federal government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Filed Materials The computer readable nucleotide / amino acid sequence listing, which was submitted contemporaneously herewith and is identified as follows, is hereby incorporated by reference in its entirety: One 2,692 byte ASCII (text) file entitled "749447_ST25.TXT", dated June 12, 2020. [Background technology]

[0004] Uveitis, age-related macular degeneration (AMD), graft-versus-host disease (GVHD), and multiple sclerosis (MS) are diseases that initiate or progress as a result of adverse immunological activity. These diseases can cause blindness, paralysis, and significant morbidity affecting quality of life. Uveitis consists of a diverse group of potentially sight-threatening intraocular inflammatory diseases of infectious or autoimmune etiology, in which autoreactive lymphocytes contribute to ocular pathology by attacking and damaging uveal tissue. Similarly, autoimmune processes contribute significantly to the progression of retinal degeneration associated with AMD, although the process that initiates AMD has not been clearly identified. MS is caused in part by lymphocytes that attack and / or destroy myelinated neurons, thereby disrupting synaptic transmission and interneuronal communication. In GVHD, allogeneic transplants are viewed as foreign by the recipient's body, and the transplanted tissue attacks the host individual. Steroids are an effective treatment for uveitis and multiple sclerosis, but cannot be used long-term due to serious side effects. Similar to uveitis and multiple sclerosis, there may be side effects associated with the use of steroids and immunosuppressants to treat GVHD. Furthermore, there is currently no effective cure for AMD, and current treatments aim to slow the progressive retinal degeneration. Thus, there remains an unmet need for safe and effective long-term treatments for the aforementioned diseases. Summary of the Invention

[0005] The present invention provides an isolated population of mammalian cells comprising about 75% or more B-1a regulatory cells that express the inhibitory cell surface receptors lymphocyte-activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and CXC chemokine receptor type 4 (CXCR4), and secrete interleukin-27 (IL-27).

[0006] The invention also provides a method of preparing a population of mammalian cells of an embodiment of the invention, comprising: (a) isolating clusters of differentiation 5 positive (CD5+) expressing cells from a sample of mammalian peripheral lymphoid tissue, mammalian umbilical cord blood, mammalian ascites, or mammalian bone marrow using fluorescence activated cell sorting (FACS) to provide isolated CD5+ expressing cells; (b) culturing the isolated CD5+ expressing cells in a cell culture medium to provide cultured cells; (c) activating the cultured cells with a BCR (B cell receptor) or TLR (Toll-like receptor) agonist to provide activated cells; and (d) exposing the activated cells to IL-27.

[0007] The present invention further provides a method of suppressing the immune system of a mammal, comprising administering to the mammal a population of mammalian cells of the embodiments of the present invention.

[0008] The invention further provides a method of treating a mammal suffering from graft-versus-host disease, comprising administering to the mammal suffering from graft-versus-host disease a population of mammalian cells of the embodiments of the invention.

[0009] The invention provides a method of preventing or reducing the severity of graft-versus-host disease in a mammal, comprising administering to the mammal a population of mammalian cells of an embodiment of the invention prior to the mammal receiving an allogeneic transplant.

[0010] The present invention provides a method for preventing or reducing the severity of graft-versus-host disease in a mammal, the method comprising: (a) mixing a population of mammalian cells of an embodiment of the present invention with a transplant material to form a transplant mixture; and (b) administering the transplant mixture to the mammal. [Brief description of the drawings]

[0011] [Figure 1]Figure 1 is a set of confocal microscopy images showing CD19+ B cells sorted from C57BL / 6 mice. The cells were activated in vitro for 48 hours by stimulation with lipopolysaccharide (LPS) or anti-CD40 / anti-IgM (BR) antibodies. The cells were incubated with fluorescently labeled anti-p28 or anti-Ebi3 antibodies. IL-27 (co-expressing p28 and Ebi3) expressing cells were detected by confocal microscopy (white arrows). [Figure 2A] Figure 2A is a series of flow cytometry plots showing sorted CD19+ B cells isolated from the peritoneum or spleen of C57BL / 6J mice activated in vitro for 48 hours by stimulation with LPS or BCR. The plots show the percentage of B-1a and B2 cells expressing IL-27. [Figure 2B] FIG. 2B is a bar graph showing the percentage of B-1a and B2 cells from the peritoneal cavity of FIG. 2A that express IL-27. [Figure 2C] FIG. 2C is a bar graph showing the percentage of B-1a and B2 cells from FIG. 2A from the spleen that express IL-27. [Figure 2D] FIG. 2D is a graph showing the results of analysis of the culture supernatant of FIG. 2A by enzyme-linked immunosorbent assay (ELISA). [Diagram 3] 3 is a set of flow cytometry plots showing CD19+ B cells sorted from C57BL / 6J mice activated in vitro for 48 hours by stimulation with anti-CD40 / anti-IgM (BCR) in the presence or absence of IL-27. The plots show the frequency of various cells in the culture. The numbers in the quadrants indicate the percentage of B cells expressing p28, Ebi3 or p28 and Ebi3 (IL-27). [Figure 4] FIG. 4 is a bar graph showing the quantified frequency of the various cells in the cultures shown in the plot of FIG. [Diagram 5]FIG. 5 is a graph depicting the results of NanoString RNA analysis (NanoString Technologies, Inc., Seattle, WA) of various cells in the cultures depicted in the plots in FIG. 3, demonstrating that BCR / IL-27 synergistically upregulated expression of the IL-27 subunits p28 and IL-27Rα and altered the pattern of chemokine receptor expression. [Figure 6] Figure 6 is a set of images showing the results of immunofluorescence / confocal microscopy analysis of various cells in the cultures shown in the plots in Figure 3, demonstrating that BCR / IL-27 synergistically upregulated expression of the IL-27 subunits p28 and IL-27Rα and altered the pattern of chemokine receptor expression. Cells expressing IL-27 (co-expressing p28 and Ebi3) were detected by confocal microscopy (white arrows). [Figure 7] Figure 7 is a graph showing sorted CD19+ B cells from wild-type or IL-27RαKO mice activated in vitro for 48 hours by stimulation with anti-CD40 / anti-IgM (BCR) in the presence or absence of IL-27. B cells expressing p28, Ebi3, or p28 and Ebi3 (IL-27) were detected by intracellular cytokine assays, and the bar graph shows the percentage of IL-27-producing B cells in the various cultures. [Figure 8] FIG. 8 is a graph showing qPCR results for expression of IL-27Rα in cells isolated from the peritoneal cavity and spleen of wild-type mice and sorted into B-1a or B2 cells. [Figure 9] FIG. 9 shows CD19+ B cells isolated from human peripheral blood mononuclear cells (PBMCs) of human volunteers activated with phorbol myristate acetate (PMA) in the presence of IL-27. [Figure 10] FIG. 10 is a graph showing the CD19+ B cells of FIG. 9 in the presence of IL-27. [Figure 11]FIG. 11 is a graph showing the frequency of human B cells expressing p28, Ebi3, or both p28 and Ebi3 (IL-27) after CD19+ B cells were isolated from PBMCs of human volunteers and activated with PMA in the absence of IL-27. [Figure 12] FIG. 12 is a flow cytometry plot showing the frequency of the cells of FIG. 11 that express either p28, Ebi3, or both p28 and Ebi3 (IL-27). [Figure 13A] Figure 13A is a bar graph showing the frequency of IL-27-producing B-1a cells in the peritoneal cavity. C57BL / 6J mice were injected (iv) with LPS (50 μg / mouse) and the frequency of IL-27-producing B-1a cells in the peritoneal cavity was assessed daily up to day 4 post-injection. B-1a cells were isolated from the peritoneal cavity at various time points and analyzed by intracellular cytokine staining assay. [Figure 13B] Figure 13B is a bar graph showing the frequency of IL-27-producing B2 cells in the peritoneal cavity. C57BL / 6J mice were injected (iv) with LPS (50 μg / mouse) and the frequency of IL-27-producing B2 cells in the peritoneal cavity was assessed daily up to day 4 post-injection. B2 cells were isolated from the peritoneal cavity at various time points and analyzed by intracellular cytokine staining assay. [Figure 14A] Figure 14A is a bar graph showing the frequency of IL-27-producing B-1a cells in the spleen. C57BL / 6J mice were injected (iv) with LPS (50 μg / mouse) and the frequency of IL-27-producing B-1a cells in the spleen was assessed daily up to day 4 post-injection. B-1a cells were isolated from the spleen at various time points and analyzed by intracellular cytokine staining assay. [Figure 14B] Figure 14B is a bar graph showing the frequency of IL-27-producing B2 cells in the spleen. C57BL / 6J mice were injected (iv) with LPS (50 μg / mouse) and the frequency of IL-27-producing B2 cells in the spleen was assessed daily up to day 4 post-injection. B2 cells were isolated from the spleen at various time points and analyzed by intracellular cytokine staining assay. [Figure 15]Figure 15 is a flow cytometry bar graph showing the percentage of CXCR3+ chemokine receptor expressing CD19+ CD5+ CD23- B-1a B cells. Data are representative of at least three independent experiments (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). [Figure 16] Figure 16 is a flow cytometry bar graph showing the percentage of CXCR4+ chemokine receptor expressing CD19+ CD5+ CD23- B-1a B cells. Data are representative of at least three independent experiments (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). [Figure 17] Figure 17 is a flow cytometry bar graph showing the percentage of CXCR5+ chemokine receptor expressing CD19+ CD5+ CD23- B-1a B cells. Data are representative of at least three independent experiments (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). [Figure 18] Figure 18 is a set of fundus images of the retina showing the improvement of clinical scores after injection of IL-27. Experimental autoimmune uveitis (EAU) was induced by immunization of C57BL / 6J mice with IRBP651-670-peptide in Freund's adjuvant (CFA) (n=12). Mice were treated by intraperitoneal injection of IL-27 (100 ng / mouse) or PBS on day (-1) of immunization and every other day until day 12 after immunization. Eyes were analyzed 14 or 21 days after immunization by fundus examination, histology, optical coherence tomography (OCT), or electroretinogram (ERG). [Figure 19] Figure 19 is a graph showing EAU scores for the retinas shown in Figure 18. The clinical scores and disease severity of EAU were assessed based on changes in the optic disc or retinal vessels, and retinal and choroidal infiltration. [Figure 20]Figure 20 is a set of images of hematoxylin and eosin histological sections of the retina of Figure 18. Scale bar = 200 mm; V = vitreous; GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer; RPE / CH = retinal pigment epithelium and choroid. [Figure 21] Figure 21 is a set of images showing OCT analysis of the retina of Figure 18. The layered structure of the retina is shown. The white arrows indicate inflammatory cells (white arrow) in the vitreous or optic nerve. [Figure 22] Figure 22 is a graph showing ERG analysis of the retina of Figure 18 on day 20 after induction of EAU. Mean dark-adapted ERG wave amplitude is plotted as a function of flash luminance, and values ​​are the mean ± SEM from four animals in each group. [Diagram 23] Figure 23 is a graph showing ERG analysis of the retina of Figure 18 20 days after induction of EAU. Mean dark-adapted ERG b-wave amplitude is plotted as a function of flash luminance and values ​​are the mean ± SEM from 4 animals in each group. [Figure 24] Figure 24 is a graph showing ERG analysis of the retina of Figure 18 20 days after induction of EAU. Mean light-adapted ERG wave amplitude is plotted as a function of flash luminance, and values ​​are the mean ± SEM from 4 animals in each group. [Diagram 25] Figure 25 is a graph showing ERG analysis of the retinas of Figure 18 on day 20 after induction of EAU. Mean light-adapted ERG b-wave amplitude is plotted as a function of flash luminance and values ​​are the mean ± SEM from four animals in each group. [Figure 26] FIG. 26 is a graph showing analysis of the cytokine IL-27 in the serum of the mice from FIG. [Figure 27] FIG. 19 is a graph showing analysis of the cytokine IL-17 in the serum of the mice from FIG. 18. [Figure 28] FIG. 28 is a graph showing analysis of the cytokine IL-10 in the serum of the mice from FIG. [Figure 29] FIG. 29 is a graph showing analysis of the cytokine IL-35 in the serum of the mice from FIG. [Diagram 30] Figure 30 is a flow cytometry plot showing the percentage of B cells expressing IL-27. The numbers in the quadrants indicate the percentage of CD19+ cells, CD19+ CD5+ CD1low cells, or CD19+ CD5+ CD1low cells in the spleens of control (PBS-treated) or IL-27-treated EAU mice. The gating strategy is as shown. [Diagram 31] FIG. 31 is a bar graph showing the percentage of IL27-expressing B cells from FIG. [Diagram 32] Figure 32 is a flow cytometry plot showing the percentage of IL-27 expressing B cells in the spleens of control (PBS treated) or IL-27 treated EAU mice. Gating strategy is as shown. Numbers in the quadrants indicate the percentage of CD19+ B cells, CD19+ CD5+ CD1low B cells, or CD19+ CD5+ CD1low B cells expressing p28, Ebi3, or p28 and Ebi3 (IL-27). [Diagram 33] FIG. 33 is a bar graph showing the percentage of IL27-expressing B10 cells in the spleens of control (PBS-treated) or IL27-treated EAU mice from FIG. [Diagram 34] FIG. 34 is a bar graph showing the percentage of IL27-expressing B-1a cells in the spleens of control (PBS-treated) or IL27-treated EAU mice from FIG. [Diagram 35] Figure 35 is a set of fundus images of retinas from mice 17 days after adoptive transfer by fundus examination. Purified peritoneal B-1a cells (5x105 cells / mouse; >80% i27-Bregs) from wild-type donor CD45.2+ EAU mice were transferred into naive syngeneic wild-type or IL-27RαKO CD45.1+ mice. 24 hours after adoptive transfer, EAU was induced in recipient mice by immunization with IRBP651-670 (n=12). Clinical disease was monitored by fundus examination up to 17 days after adoptive transfer. [Diagram 36] FIG. 36 is a graph showing EAU scores for the retinas shown in FIG. [Figure 37]Figure 37 is a set of flow cytometry plots from CD4+ T cells subjected to FACS and intracellular cytokine assays. The numbers in the quadrants indicate the percentage of CD4+ T cells expressing IL-17. Data are representative of at least three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 38A] Figure 38A is a set of flow cytometry plots from CD4+ T cells subjected to FACS and intracellular cytokine assays. The numbers in the quadrants indicate the percentage of CD4+ T cells expressing IL-10. [Figure 38B] FIG. 38B is a bar graph showing the percentage of CD4+ T cells expressing IFN-γ. [Figure 38C] FIG. 38C is a bar graph showing the percentage of CD4+ T cells expressing IL-17. [Figure 38D] FIG. 38D is a bar graph showing the percentage of CD4+ T cells expressing IFN-γ and IL-17. [Figure 38E] FIG. 38E is a bar graph showing the percentage of CD4+ T cells expressing IL-10. [Figure 39] Figure 39 is a set of flow cytometry plots from CD19+ T cells (eye) subjected to FACS. The numbers in the quadrants indicate the percentage of CD19+ CD5+ CD23- B-1a cells expressing p28, p35, Ebi3, p28, and Ebi3 (IL-27). Data are representative of at least three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Diagram 40] Figure 40 is a set of flow cytometry plots from CD19+ T cells (eye) subjected to FACS. The numbers in the quadrants indicate the percentage of CD19+ CD5- CD23+ B2 expressing either p28 and Ebi3 (IL-27) or p35 and Ebi3 (IL-35). Data are representative of at least three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Diagram 41]FIG. 41 is a bar graph showing the percentage of B-1a cells in the eyes of FIG. 39 that express p28 and Ebi3. [Diagram 42] FIG. 42 is a bar graph showing the percentage of B2 cells in the eye of FIG. 40 that express p28 and Ebi3. [Diagram 43] FIG. 43 is a bar graph showing the percentage of B2 cells in the eye of FIG. 40 that express p35 and Ebi3. [Diagram 44] FIG. 44 is a set of photomicrographs of hematoxylin and eosin stained sections of mouse brain (top row) and spinal cord (middle row) on day 17 after immunization (original magnification ×200). Arrows indicate inflammatory cells in the brain or spinal cord. The extent of EAE-induced demyelination was assessed by Luxol fast blue staining (bottom row; Luxol fast blue is an alcohol-soluble copper phthalocyanine dye that is attracted to bases found in lipoproteins of the myelin sheath). Arrows indicate areas of demyelination in the spinal cord. EAE was induced by immunization of C57BL / 6J mice with MOG35-55-peptide in CFA (n=12). Mice were treated by intraperitoneal injection of IL-27 (100 ng / mouse) or PBS on day 0 after immunization and every other day until day 12 after immunization. [Diagram 45] Figure 45 is a graph showing the EAU scores of the spinal cord described in Figure 44. Clinical scores and disease assessments of EAE were confirmed by two masked investigators according to a well-established scoring system. [Figure 46] Figure 46 is a set of flow cytometry plots from inflammatory cells in the brain and spinal cord after intracellular cytokine analysis of untreated or IL-27 treated mice isolated and collagenase digested on day 17 after immunization. The numbers in the quadrants indicate the percentage of CD4 T cells expressing IL-17 or IFN-γ in the spinal cord and brain. [Figure 47]Figure 47 is a set of flow cytometry plots from inflammatory cells in the brain and spinal cord after collagenase digestion of untreated or IL-27 treated mice isolated on day 17 post-immunization and intracellular cytokine analysis. The numbers in the quadrants indicate the percentage of IL-10 expressing cells and CD4 T cells in the spinal cord and brain. [Figure 48] FIG. 48 is a bar graph showing the percentage of CD4 T cells from FIG. 46 and FIG. 47 that express IFN-γ. [Figure 49] FIG. 49 is a bar graph showing the percentage of CD4 T cells from FIG. 46 and FIG. 47 that express IL-17. [Figure 50] FIG. 50 is a bar graph showing the percentage of CD4 T cells from FIG. 46 and FIG. 47 that express IL-17 and IFN-γ. [Figure 51] FIG. 51 is a bar graph showing the percentage of CD4 T cells from FIG. 46 and FIG. 47 that express IL-10. [Figure 52] Figure 52 is a set of flow cytometry plots showing the percentage of IL-27 expressing B cells from the spinal cord and brain of unimmunized, PBS-treated or IL-27-treated EAE mice analyzed by intracellular cytokine staining assay for IL-27 (p28 and Ebi3) expression. The numbers in the quadrants indicate the percentage of CD19+ CD5+ CD1dhi B cells or CD19+ CD5+ CD1dlow B cells in the spinal cord or brain that express p28, Ebi3, or p28 and Ebi3 (IL-27). [Diagram 53] FIG. 53 is a bar graph showing the percentage of CD19 T cells from FIG. 52 that express CD19+ CD5+ CD1dlow. [Figure 54]Figure 54 is a set of flow cytometry plots showing the percentage of IL-27 expressing B cells from spleens of unimmunized, PBS-treated or IL-27-treated EAE mice analyzed for expression of IL-27 (p28 and Ebi3) by intracellular cytokine staining assay. The numbers in the quadrants indicate the percentage of total CD19+ CD5+ CD1dhi B cells or total CD19+ CD5+ CD1dlow B cells in the spleen expressing p28, Ebi3, or p28 and Ebi3 (IL-27). [Figure 55] FIG. 55 is a bar graph showing the percentage of CD19 T cells from FIG. 54 that express p28 and Ebi3. [Figure 56] Figure 56 is a set of flow cytometry plots showing analysis of spleen cells from PBS-treated or IL-27-treated EAE mice for IL-27 proliferation. Numbers in quadrants indicate the percentage of CD19+ CD5+ CD1dlow B-1a cells. [Figure 57] FIG. 57 is a bar graph showing the percentage of CD19 T cells from FIG. 56 that express CD19+ CD5+ CD1dhi. [Figure 58] FIG. 58 is a bar graph showing the percentage of CD19 T cells from FIG. 56 that express CD19+ CD5+ CD1dlow. [Figure 59] Figure 59 is a graph showing EAU scores of splenocytes from MOG35-55 immunized (PBS-treated EAE or IL-27-treated) CD45.2+ mice restimulated ex vivo and transferred into naive CD45.1+ WT mice (1x107 / mouse). EAE clinical scores and disease assessments were confirmed by two masked investigators. [Figure 60]Figure 60 is a set of flow cytometry plots showing the percentage of CD4+ T cells. Spinal cord, brain, lymph nodes (LN) or spleen of PBS-treated or IL-27-treated mice were isolated 20 days after adoptive transfer, collagenase digested, and CD4+ T cells and IL-27-producing B-1a were analyzed by intracellular cytokine staining assay. Numbers in the quadrants indicate the percentage of CD4+ T cells expressing IL-17 or IFN-γ. Data are representative of more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 61] FIG. 61 is a bar graph showing the percentage of cells in FIG. 60 that express IL-17. [Figure 62] FIG. 62 is a bar graph showing the percentage of cells in FIG. 60 that express IL-17 and IFN-γ. [Figure 63] Figure 63 is a set of flow cytometry plots showing the percentage of IL-27 producing B-1a cells. Spinal cord, brain, lymph nodes (LN) or spleen of PBS-treated or IL-27-treated mice were isolated 20 days after adoptive transfer, collagenase digested, and CD4+ T cells and IL-27 producing B-1a were analyzed by intracellular cytokine staining assay. Numbers in quadrants indicate the percentage of CD19+ CD5+ CD11b+ B-1a cells expressing p28, Ebi3, or p28 and Ebi3 (IL-27). Data are representative of more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 64] FIG. 64 is a bar graph showing the percentage of B-1a cells from the spinal cord of FIG. 63 that express p28 and Ebi3 (IL-27). [Figure 65] FIG. 65 is a bar graph showing the percentage of B-1a cells from FIG. 63 from the brain that express p28 and Ebi3 (IL-27). [Figure 66] FIG. 66 is a bar graph showing the percentage of B-1a cells from FIG. 63 that express p28 and Ebi3 (IL-27) from the spleen. [Figure 67]Figure 67 is a graph showing a set of flow cytometry plots (top) and EAE clinical scores (bottom) from purified peritoneal B-1a cells (5x105 cells / mouse; >80% i27-Bregs) from WT donor CD45.2+ engrafted into naive syngeneic wild-type mice and 24 hours after adaptive engraftment, EAE was induced in recipient mice by immunization with MOG35-55 (n=12). EAE clinical scores and disease assessments were performed by two masked investigators. [Figure 68] Figure 68 is a set of flow cytometry plots showing the percentage of CD4+ T cells expressing IL-10, IL-17, or IFN-γ. Spinal cords and brains of PBS-treated or B-1a-treated mice were isolated 15 days after immunization, digested with collagenase, and analyzed by intracellular cytokine staining assay. Data are representative of more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 69] FIG. 69 is a bar graph showing the percentage of spinal cord and brain cells from FIG. 68 that express IFN-γ. [Figure 70] FIG. 70 is a bar graph showing the percentage of spinal cord and brain cells from FIG. 68 that express IL-17. [Figure 71] FIG. 71 is a bar graph showing the percentage of spinal cord and brain cells from FIG. 68 that express IL-10. [Figure 72] Figure 72 is a set of flow cytometry plots showing the percentage of CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells expressing p28, Ebi3, or p28 and Ebi3 (IL-27) in the spinal cord. Spinal cords of PBS-treated or B-1a-treated mice were isolated 15 days after immunization, digested with collagenase, and analyzed by intracellular cytokine staining assay. Data are representative of more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 73] FIG. 73 is a bar graph showing the percentage of spinal cord cells from FIG. 72 that express p28 and Ebi3 (IL-27). [Figure 74] Figure 74 is a set of flow cytometry plots showing the percentage of CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells expressing p28, Ebi3, or p28 and Ebi3 (IL-27) in the brain. Brains from PBS- or B-1a-treated mice were isolated 15 days after immunization, digested with collagenase, and analyzed by intracellular cytokine staining assay. Data are representative of more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 75] FIG. 75 is a bar graph showing the percentage of brain cells from FIG. 74 that express p28 and Ebi3 (IL-27). [Figure 76] Figure 76 is a set of flow cytometry plots showing the percentage of CD19+ CD5+ CD23B-1a cells or CD19+ CD5 CD23+ B2 cells expressing p28, Ebi3, or p28 and Ebi3 (IL-27) in the peritoneal cavity. Fluids from the peritoneal cavities of PBS-treated or B-1a-treated mice were isolated 15 days after immunization, digested with collagenase, and analyzed by intracellular cytokine staining assay. Data are representative of more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 77] FIG. 77 is a bar graph showing the percentage of cells in FIG. 74 from the peritoneal cavity that express p28 and Ebi3 (IL-27). [Figure 78] Figure 78 is a schematic diagram showing macrophages from EAU mice cultured in a transwell system with B-1a cells from wild-type EAU mice in the bottom well. The effect of macrophages on the proliferation of B-1a cells was assessed by [3H]-thymidine incorporation assay. [Figure 79] FIG. 79 is a set of flow cytometry plots showing the percentage of B-1a cells from FIG. 78 that express either p28, Ebi3, or p28 and Ebi3 (IL-27). [Figure 80]Figure 80 is a bar graph showing the mean CPM values ​​of B-1a cells and macrophages from Figure 78. Proliferative responses were analyzed in five replicate cultures. Data are representative of at least three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 81] FIG. 81 is a bar graph showing the percentage of B-1a cells and macrophages from FIG. 78 that express p28 and Ebi3 (IL-27). [Figure 82] Figure 82 is a graph showing ELISA analysis of IL-27 secretion from primary mouse peritoneal macrophages activated with LPS in the presence or absence of lentiviral guide RNA targeting p28 (sgp28-1 or spg28-2). [Figure 83] Figure 83 is a graph showing ELISA analysis of IL-27 secretion from mouse peritoneal primary B-1a cells activated with LPS in the presence or absence of lentiviral guide RNA targeting p28 (sgp28-1 or spg28-2). [Figure 84] Figure 84 is a schematic diagram showing EAU mouse-derived pathogenic (uveitogenic) T cells cultured in a transwell system containing B-1a cells infected with lentiviral guide RNA (sgp28 / Ebi3) targeting the suppression of IL-27. The effect of B-1a cells on the proliferation of uveitogenic T cells was evaluated by [3H]-thymidine incorporation assay. [Figure 85] FIG. 85 is a bar graph showing the average CPM values ​​of the cells in FIG. [Figure 86] FIG. 86 is a set of flow cytometry plots showing the percentage of uveocyte-derived CD4+ T cells expressing IL-10, IL-17 and / or IFN-γ as determined by intracellular cytokine staining assays. [Figure 87] FIG. 87 is a bar graph showing the percentage of cells in FIG. 86 that express IFN-γ. [Figure 88] FIG. 88 is a bar graph showing the percentage of cells in FIG. 86 that express IL-17. [Figure 89]FIG. 89 is a bar graph showing the percentage of cells in FIG. 86 that express IFN-γ and IL-17. [Figure 90] FIG. 90 is a bar graph showing the percentage of cells from FIG. 86 that express IL-10. [Figure 91] Figure 91 is a schematic diagram showing pathogenic (uveitogenic) T cells from EAU mice cultured in a transwell system containing wild-type EAU mouse B-1a cells or B-1a cells infected with lentiviral guide RNA (sgp28 / Ebi3) targeting suppression of IL-27. The effect of B-1a cells on the proliferation of uveitogenic T cells was evaluated by [3H]-thymidine incorporation assay. [Figure 92] FIG. 92 is a set of flow cytometry plots showing the percentage of uveocyte-derived CD4+ T cells expressing LAG-3, as determined by an intracellular cytokine staining assay. [Figure 93] FIG. 93 is a bar graph showing the percentage of the cells in FIG. 92 that express LAG-3. [Figure 94] Figure 94 is a schematic diagram showing pathogenic (uveitogenic) T cells from EAU mice cultured in a transwell system containing wild-type EAU mouse B-1a cells or B-1a cells infected with lentiviral guide RNA (sgp28 / Ebi3) targeting suppression of IL-27. The effect of B-1a cells on the proliferation of uveitogenic T cells was evaluated by [3H]-thymidine incorporation assay. [Figure 95] Figure 95 is a set of flow cytometry plots of CD4+ CD25+ Foxp3+ and CD4+ CD25+ Foxp3- expressing p35, Ebi3, or IL-35 (p35 / Ebi3). [Figure 96] FIG. 96 is a bar graph showing the percentage of cells in FIG. 95 that express p35 and Ebi3. [Figure 97]FIG. 97 is a set of flow cytometry plots of CD4+ CD25+ Foxp3+ and CD4+ CD25+ Foxp3− expressing p35, Ebi3, or IL-35 (p35 / Ebi3). [Figure 98] FIG. 98 is a bar graph showing the percentage of cells in FIG. 97 that are CD4+ CD25+ Foxp3+. [Figure 99] FIG. 99 is a bar graph showing the percentage of cells in FIG. 97 that are CD4+ CD25+ Foxp3-. [Figure 100] Figure 100 is a set of flow cytometry plots and graphs showing the results of CD19+ CD5+ CD23- B-1a cells sorted from the peritoneal cavity of C57BL / 6J mice activated in vitro for 48 hours by LPS stimulation, analyzed by ELISA (left flow cytometry plots). Supernatants from intraperitoneal cultures were analyzed by qPCR (right). [Figure 101] FIG. 101 is a bar graph showing the results of qPCR analysis of B-1a cells purified from the peritoneal cavity of C57BL / 6J mice injected (iv) with LPS 48 hours prior. [Figure 102] FIG. 102 is a flow cytometry plot showing B-1a or plasma cells (B2) from C57BL / 6J mouse peritoneal cavity or spleen, respectively, after sorting using magnetic beads and activation with anti-CD40 / anti-IgM (BCR). [Figure 103] FIG. 103 is a graph of qPCR analysis of RNA from the cells of FIG. 102 quantified for expression of Pd1 mRNA transcripts. [Figure 104] Figure 104 is a graph of qPCR analysis of RNA from the cells of Figure 102 quantified for expression of Lag3 mRNA transcripts. [Figure 105] FIG. 105 is a set of graphs showing RNA isolated at various time points analyzed by qRT-PCR from CD19+ B cells from C57BL / 6J mouse spleens activated with anti-CD40 / anti-IgM in the presence or absence of IL-27. [Fig. 106]Figure 106 is a Volcano plot analysis 24 hours after detecting genes differentially induced by IL-27 using the NanoString transcription factor panel. [Figure 107] Figure 107 is an image of a Western blot. CD19+ B cells were isolated from the spleens of C57BL / 6J mice 24 hours after immunization with LPS (in vivo) in the presence or absence of IL-27, or from mouse CD19+ B cells after activation with LPS (in vitro) in the presence or absence of IL-27. Nuclear extracts were prepared from the cells and analyzed by electrophoretic mobility shift assay (EMSA) to detect AICE complexes induced by IL-27. Transcription factors recruited to the CTLA4-AICE or p28-AICE locus were identified by supershift assay. Total cell extracts prepared from CD19+ B cells of C57BL / 6J mice immunized with LPS in the presence or absence of IL-27 were analyzed by Western blotting. [Figure 108] FIG. 108 is a bar graph showing relative gene expression in B-1a cells from the peritoneal cavity. [Fig. 109] Figure 109 is a set of flow cytometry plots of FACS analysis of CD19+ B cells showing the percentage of B-1a or plasmablasts expressing p28, Ebi3, or p28 and Ebi3 (IL-27). The CD19+ B cells were derived from the spleen of C57BL / 6J (wild type) or mice with targeted deletion of irfH in B cells (CD19-IRF8KO) after activation with anti-CD40 / anti-IgM for 3 days. Gating strategy is as shown. Data are representative of more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 110] FIG. 110 is a bar graph showing the amount of CD19+ CD27+ CD383+ cells from FIG. [Figure 111] FIG. 111 is a bar graph showing the amount of CD19+ CD5+ CD11b+ cells in FIG. [Figure 112]Figure 112 is a bar graph showing chromatin immunoprecipitation (CHIP) analysis performed on B-1a cells stimulated with LPS or LPS+IL-27 for 24 hours and analyzed for STAT1 binding to the p28 or ehi3 promoter regions. Cell lysates were immunoprecipitated with anti-STAT1 antibody or control IgG. Immunoprecipitates and input DNA were analyzed by qPCR using primers corresponding to the p28 or ehi3 promoter sites. [Figure 113] Figure 113 is a bar graph showing CHIP analysis performed on B-1a cells stimulated with LPS or LPS+IL-27 for 24 hours and analyzed for STAT3 binding to the p28 or ehi3 promoter region. Cell lysates were immunoprecipitated with anti-STAT3 antibody or control IgG. Immunoprecipitates and input DNA were analyzed by qPCR using primers corresponding to the p28 or ehi3 promoter sites. [Fig. 114] Figure 114 is a set of flow cytometry plots of FACS analysis of healthy human PBMCs cultured with TLR9 agonist CpG and BCR (anti-CD40 or anti-IgM) for 3 days. Gating on human B-1 cells (CD19+ CD20+ CD27+ CD43+ ) revealed that 19.9% ​​of BCR-activated B cells in human PBMCs produced IL-27. [Figure 115] FIG. 115 is a bar graph showing the amount of CD19+ CD20+ CD27+ CD43+ p28+ Ebi3+ cells in FIG. [Fig. 116] Figure 116 is a set of flow cytometry plots of FACS analysis of healthy human PBMCs cultured with TLR9 agonist CpG and BCR (anti-CD40 or anti-IgM) for 3 days. Gating on CD19+ CD20+ CD27+ CD43+ CD11b+ B-1 cells revealed to contain as much as 35% of BCR-activated human B-1 cells. [Figure 117] FIG. 117 is a bar graph showing the amount of CD19+ CD20+ CD27+ CD43+ CD11 b+ p28+ Ebi3+ cells in FIG. 116. [Fig. 118]FIG. 118 is a bar graph showing the amount of CD19+ CD20+ CD27+ CD43+ CD11b- p28+ Ebi3+ cells in FIG. [Figure 119] Figure 119 is a set of flow cytometry plots of FACS analysis of human cord blood from healthy human donors. As many as 18.1% of resting B-1a cells constitutively produce IL-27, and stimulation of BCR-activated cord blood B cells with IL-27 increased the percentage of cord blood i27-Bregs to 73.9%. [Figure 120] FIG. 120 is a bar graph showing the amount of cells in FIG. 119 (top row). [Figure 121] FIG. 121 is a bar graph showing the amount of cells in FIG. 119 (lower panel). [Figure 122] Figure 122 is a set of graphs showing the relative abundance of i27-Breg compared to other Breg subtypes (IL-10 producing Bregs and i35-Bregs). Activated human umbilical cord blood cells were expanded for 6 days. The majority of Breg cells were i27-Bregs (largest slice of each pie chart, ranging from 61.2 + / - 5.3 to 87.1 + / - 3.1%), with much lower levels of IL-10 producing Bregs (ranging from 2.6 + / - 0.3 to 6.7 + / - 1.1%) and i35-Bregs (ranging from 10.2 + / - 2.7 to 32 + / - 6.8 5%) detected. [Figure 123] Figure 123 is a set of graphs showing the relative abundance of B-2 cells. These plots revealed that most i27-Bregs were in either the naive or memory B cell pool. [Figure 124] FIG. 124 is a graph showing that similar to mouse species, human i27-Breg cells constitutively express the inhibitory receptors PD-1 and LAG3. [Fig. 125] FIG. 125 is a bar graph showing the amount of PD1+ cells in FIG. [Fig. 126] FIG. 126 is a bar graph showing the amount of LAG3+ cells in FIG. [Figure 127]Figure 127 is a set of flow cytometry plots of FACS analysis of human i27-Breg cells. i27-Breg cells suppressed the proliferative response of inflammatory CD4+ T cells producing TNF-α, IL-17, and IFN-γ. [Figure 128] FIG. 128 is a bar graph showing the average CPM values ​​of the cells in FIG. [Figure 129] FIG. 129 is a bar graph showing the amount of TNFα+ CD4+ T cells from FIG. [Fig. 130] FIG. 130 is a bar graph showing the amount of IFNγ+ CD4+ T cells from FIG. [Fig. 131] FIG. 131 is a bar graph showing the amount of IL-17A+ CD4+ T cells from FIG. [Fig. 132] FIG. 132 shows the results of a proximity ligation assay (PLA) demonstrating the physical interaction between p28 and Ebi3. [Fig. 133] Figure 133 is a gel showing that B cells produce the heterodimeric (p28 / Ebi3) IL-27 cytokine. C57BL / 6J mice were injected (iv) with LPS or LPS+IL-27, and 24 hours later, lysates or supernatants from cultured B-1a cells were subjected to reciprocal immunoprecipitation / western blot analysis. Antibodies used for IP or western blotting are indicated. [Fig. 134] Figure 134 is a graph showing the results of NanoString RNA analysis demonstrating that IL-27 altered the pattern of chemokine receptor expression in activated B cells. [Fig. 135A] FIG. 135A shows flow cytometry plots demonstrating the difference in secretion of natural IgM antibodies by unchallenged B-1a and i27-Breg cells in the peritoneal cavity. [Fig. 135B] FIG. 135B shows a set of graphs depicting the difference in secretion of natural IgM antibodies by unchallenged B-1a and i27-Breg cells in the peritoneal cavity. [Fig. 136] FIG. 136 shows a principal component analysis (PCA) plot showing the separation of cells into four different populations. [Fig. 137] FIG. 137 shows gene ontology (GO) analysis showing functional pathway enrichment in i27-Breg cells. [Figure 138] Figure 138 is a heat map of the gene signature of i27-Breg cells compared to the signature program of unchallenged B-1a cells. [Figure 139] Figure 139 is a set of heatmaps showing genes that encode transcription factors, signaling proteins, cytokines, chemokines, or encode cell surface proteins that are differentially expressed in i27-Bregs and B-1a cells. [Fig. 140] FIG. 140 is a set of graphs from qPCR expression of genes encoding inhibitory receptors. [Fig. 141A] FIG. 141A is a set of representative flow cytometry plots showing a significant expansion of CD19+ CD20+ CD27+ CD43+ B-1 cells secreting IL-27 in response to anti-CD40 or BCR. [Fig. 141B] FIG. 141B is a scatter plot showing a significant increase in CD19+ CD20+ CD27+ CD43+ B-1 cells secreting IL-27 in response to anti-CD40 or BCR. [Fig. 142A] FIG. 142A is a set of representative flow cytometry plots showing a significant expansion of CD27+ CD43+ CD11+ or CD27+ CD43+ CD11- B-1 cells secreting IL-27 in response to anti-CD40 or BCR. [Fig. 142B] FIG. 142B is a set of scatter plots showing a significant increase in CD27+ CD43+ CD11+ or CD27+ CD43+ CD11- B-1 cells secreting IL-27 in response to anti-CD40 or BCR. [Fig. 143A]Figure 143A is a set of representative flow cytometry plots of human cord blood CD19+ B cells (top) or blood sorted B-1a cells (bottom) activated with either BCR or BCR and IL-27, showing a significant expansion of IL-27-producing CD27+ CD43+ B-1a cells. [Fig. 143B] FIG. 143B is a set of scatter plots of human cord blood CD19+ B cells (top) or blood sorted B-1a cells (bottom) activated with either BCR or BCR and IL-27, showing significant expansion of IL-27-producing CD27+ CD43+ B-1a cells. [Fig. 144] FIG. 144 shows representative t-SNE clustering plots and flow cytometry pie charts showing the distribution and relative abundance of IL-27 (i27-Breg), IL-35 (i35-Breg) and IL-10 secreting Bregs in the B-1 compartment of activated human umbilical cord blood. [Fig. 145] FIG. 145 is a graph and pie chart showing the amount of different Breg subsets (e.g., i27-Bregs, i35-Bregs, and B10 cells) in cultures of human CD19+ B cells in human blood after 6 days of activation and analyzed by intracellular cytokine assay. [Fig. 146] FIG. 146 shows the results of RNA-Seq analysis using RNA from conventional CD19+ B-2, i27-Breg, i35-Breg, or B10 cells. [Fig. 147] FIG. 147 is a graph showing a heatmap analysis showing differentially expressed genes between i27-Breg and i35-Breg cells. [Fig. 148] FIG. 148 is a graph showing a heatmap analysis showing genes differentially expressed between conventional CD19+ B-2 cells and i27-Breg cells. [Fig. 149A] FIG. 149A is a set of representative flow cytometry plots showing the percentage of IL-27-secreting CD11b+ B-1a cells following co-culture (1:1) of activated IL-27-producing B-1a with plasmacytoid dendritic cells. [Fig. 149B] FIG. 149B is a representative bar graph showing the percentage of IL-27-secreting CD11b+ B-1a cells following co-culture (1:1) of activated IL-27-producing B-1a cells with plasmacytoid dendritic cells. [Fig. 150] Figure 150 is a scatter plot showing significant suppression of EAE after transfer of purified IL-27-secreting peritoneal B-1a cells (>80% i27-Bregs) from WT donor CD45.2+ mice into naive syngeneic CD45.1+ mice and 24 hours later, EAE was induced in recipient mice by immunization with MOG35-55 (n=12). [Fig. 151A] FIG. 151A is a set of representative flow cytometry plots showing attenuation of EAE symptoms in mice treated with i27-Breg, as indicated by the percentage of CD4+ T cells expressing IL-10, IL-17, or IFN-γ. [Fig. 151B] FIG. 151B is a set of scatter plots showing attenuation of EAE symptoms in mice treated with i27-Breg, as shown by the percentage of CD4+ T cells expressing IL-10, IL-17, or IFN-γ. [Fig. 152A] FIG. 152A is a set of representative flow cytometry plots showing CD19+ CD5+ CD23- B-1a cells or CD19+ CD5- CD23+ B2 cells secreting IL-27 in the spinal cord. [Fig. 152B] Figure 152B is a set of scatter plots showing CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells secreting IL-27 in the brain. [Fig. 153A] FIG. 153A is a set of representative flow cytometry plots showing CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells secreting IL-27 in the brain. [Fig. 153B] FIG. 153B is a set of scatter plots showing CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells secreting IL-27 in the brain. [Fig. 154A]FIG. 154A is a set of representative flow cytometry plots showing CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells secreting IL-27 in the peritoneal cavity. [Fig. 154B] FIG. 154B is a set of scatter plots showing CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells secreting IL-27 in the peritoneal cavity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention Regulatory B cells (Bregs) suppress autoimmune diseases by producing IL-10 or IL-35 alone or in combination with inhibitory cell surface receptors. However, Bregs described so far (e.g., U.S. Pat. No. 9,629,897) are antigen-specific and derived from the B2 lymphocyte lineage. The present invention provides an isolated population of human cells that includes a non-naturally occurring enriched population of regulatory B cells of the B-1a lineage that produce and secrete interleukin-27 (i27-Bregs).

[0013] Interleukin-27 (IL-27) is a member of the IL-12 cytokine family. IL-27 is a heterodimeric cytokine composed of two distinct protein subunits encoded by ebi3 (Epstein-Barr virus-induced gene 3) and IL-27p28. IL-27 is expressed by cells and interacts with the IL-27 receptor (IL-27R), which is composed of two proteins, IL-27α (IL-27 alpha) and gpl30. IL-27 induces the differentiation of diverse populations of T cells of the immune system. Natural activation of B-1a regulatory cells by inflammatory stimuli leads to IL-27 production and the concomitant egress of i27-Bregs to the spleen, where they reprogram conventional lymphocytes to acquire immunoregulatory functions.

[0014] The population of cells of the present invention can comprise about 25% or more B-1a regulatory cells (e.g., about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, about 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, or about 90% or more B-1a regulatory cells). Such a relatively high percentage of B-1a cells compared to other cell types in the cell population does not exist in the human body or in nature. B-1a cells in the human body are detected in small numbers (<2%) in peripheral lymphoid tissues. Within this minority population of less than 2%, i27-Bregs represent less than 2%, approximately 4 / 10,000 of the naturally occurring human cell population (i.e., 0.02 × 0.02 = 0.0004).

[0015] The population of cells of the present invention expresses the inhibitory cell surface receptors lymphocyte activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and CXC chemokine receptor type 4 (CXCR4). The population of cells may have the receptors on their surface or may be capable of having the receptors on their surface.

[0016] LAG-3 (or cluster of differentiation 223 (CD223)) is a protein encoded by the human LAG3 gene. LAG3 is an immune checkpoint receptor.

[0017] PD-1 (or cluster of differentiation 279 (CD279)) is a protein encoded by the PDCD1 gene in humans. PD-1 is also an immune checkpoint receptor. PD-1 promotes apoptosis of antigen-specific T cells in lymph nodes and reduces apoptosis of regulatory T cells (anti-inflammatory, suppressor T cells).

[0018] CXCR4 (or fusin or cluster of differentiation 184 (CD184)) is a protein encoded by the human CXCR4 gene. CXCR4 is an alpha chemokine receptor specific for stromal-derived factor 1 (SDF-1 or CXCL12), a molecule with chemotactic activity for lymphocytes.

[0019] The population of cells optionally also expresses the inhibitory cell surface receptor glucocorticoid-inducible TNFR-related protein (GITR or tumor necrosis factor receptor superfamily member 18 (TNFRSF18) or activation-inducible TNFR family receptor (AITR)). GITR is a protein encoded by the TNFRSF18 gene in humans. GITR has been shown to be upregulated upon T cell activation.

[0020] The population of cells of the present invention optionally also expresses the inhibitory cell surface receptor OX40 (or tumor necrosis factor receptor superfamily member 4 (TNFRSF4) or cluster of differentiation 134 (CD134)). OX40 is a protein encoded by the human TNFRSF4 gene. OX40 is not constitutively expressed in resting naive T cells.

[0021] The population of cells of the present invention also optionally expresses the inhibitory cell surface receptor cytotoxic T-lymphocyte-associated protein 4 (CTLA4 or cluster of differentiation 152 (CD152)). CTLA4 is a protein encoded by the human CTLA4 gene. CTLA4 is an immune checkpoint that downregulates immune responses. CTLA4 is constitutively expressed in regulatory T cells, but is only upregulated in conventional T cells after activation.

[0022] The population of cells of the present invention may be derived from a mammal. The term "mammal" includes, but is not limited to, Rodentia such as mice, Logomorpha such as rabbits, Carnivora including felines (cats) and dogs (canines), Artiodactyla including cats (bovines) and pigs (porcines), Perssodactyla including equidae, Primates, Ceboids or Simioids (monkeys) and Anthropoids (humans and apes). More preferably, the population of cells is derived from a human.

[0023] The present invention provides a method for preparing a population of cells (e.g., human cells), comprising: (a) isolating differentiation cluster 5 positive (CD5+) expressing cells from a mammalian tissue or liquid sample to provide isolated CD5+ expressing cells; (b) culturing the isolated CD5+ expressing cells in a cell culture medium to provide cultured cells; (c) activating the cultured cells with a BCR (B cell receptor) or TLR (Toll-like receptor) agonist to provide activated cells; and (d) exposing the activated cells to IL-27. In this regard, isolation of CD5+ (CD5 is expressed on the surface of T cells and B-1a cells) expressing cells can be performed by any suitable method, for example, by using fluorescence-activated cell sorting (FACS), microfluidic cell sorting, or magnetic cell sorting.

[0024] The mammalian tissue or fluid sample may be from any suitable source, such as mammalian peripheral lymphoid tissue, mammalian umbilical cord blood, mammalian ascites, mammalian bone marrow, induced pluripotent cells (iPSCs) or other samples that contain B-1a cells. In at least some embodiments, it may be desirable to use ascites or umbilical cord blood as the sample, since these sources usually have a higher percentage of B-1a cells than other samples (e.g., peripheral lymphoid tissue). In some embodiments, the preferred source of tissue or fluid may be from a donor subject to be treated with the population of cells of the present invention.

[0025] Any suitable cell culture medium capable of supporting the growth of B-1a cells can be used, for example, Roswell Park Memorial Institute Medium (RPMI 1640) medium can be used.

[0026] The cultured cells are exposed to a BCR or TLR agonist. Any suitable BCR or TLR agonist that can activate the cells can be used. Examples of BCR agonists include anti-CD40 and anti-IgM antibodies. Examples of TLR agonists include TLR9 and TLR4 agonists. As with all lymphocytes, B-1a cells need to be activated to elicit biological activity, and thus CD5+ B-1a cells are activated with a BCR or TLR agonist. CD40 is a costimulatory protein found on antigen-presenting cells and is required for B cell activation following the interaction of the B cell receptor with antibodies against IgM. However, maximal secretion of IL-27 by activated B-1a cells requires an IL-27 signal provided by binding of IL-27 to its cognate receptor on B-1a cells and further upregulation of the IL-27 receptor.

[0027] As used herein, the terms "Toll-like receptor" and "TLR" refer to any member of a family of highly conserved mammalian proteins that recognize pathogen-associated molecular patterns and act as key signaling elements in innate immunity. TLR polypeptides share a characteristic structure that includes an extracellular domain with leucine-rich repeats, a transmembrane domain, and an intracellular domain involved in TLR signaling.

[0028] The terms "Toll-like receptor 4" and "TLR4" refer to a nucleic acid or polypeptide having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a publicly available TLR4 sequence. A suitable TLR4 agonist is LPS.

[0029] The terms "Toll-like receptor 9" and "TLR9" refer to a nucleic acid or polypeptide having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a publicly available TLR9 sequence. Suitable TLR9 agonists are oligonucleotides containing CpG motifs (CpG ODN).

[0030] Activated cells are exposed to IL-27, which promotes the expansion of i27-Bregs and efficiently and continuously increases the proportion and abundance of i27-Bregs.

[0031] The methods of the present invention are useful for treating disease in a mammal. Treatment may result in desirable suppression of the immune system.

[0032] The method of the present invention is useful for treating, suppressing, or preventing GVHD. The patient can receive a solid organ or allogeneic bone marrow or hematopoietic stem cell transplant. To prevent or reduce the severity of GVHD, a population of mammalian cells of the present invention is administered to the mammal before the mammal receives an allogeneic transplant. Alternatively, GVHD can be prevented or suppressed by mixing an i27-Breg population of cells of the present invention with transplantation material to form a transplantation mixture, and then administering the transplantation mixture to the mammal. In this regard, the transplantation material can include allogeneic lymphocytes. In one embodiment, the transplanted cells are cells derived from iPS cells (e.g., cardiac cells, pancreatic cells, retinal cells).

[0033] The population of mammalian cells of the present invention can be mixed with the transplant material ex vivo. "Ex vivo" refers to methods carried out in or on cells or tissues in an artificial environment outside of an organism with minimal alteration of the natural conditions. In contrast, the term "in vivo" refers to methods carried out within an organism under normal, intact conditions, whereas "in vitro" methods are carried out using components of an organism that have been isolated from their normal biological context.

[0034] The population of mammalian cells can be administered in the form of a pharma- ceutically acceptable (e.g., physiologically acceptable) composition. The composition can include a carrier, preferably a pharma- ceutically (e.g., physiologically acceptable) carrier, and a population of mammalian cells. Any suitable carrier can be used within the context of the present invention, and many such carriers are known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition can be administered and the particular method used to administer the composition. The composition can be optionally sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The composition can be made according to conventional techniques, for example, as described in Remington: Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0035] The population of mammalian cells may be administered to a mammal (as defined herein above). Preferably, the mammal is a mouse or a human.

[0036] The present invention provides a method for suppressing the immune system of a mammal, comprising administering a population of mammalian cells of the present invention to a mammal in need thereof to suppress the immune system of the mammal. Thus, the present invention provides a method for suppressing autoimmunity in a mammal, comprising administering an isolated population of IL-27-producing B-1a cells to a mammal, whereby in vivo IL-27 production in said mammal is artificially increased to a high level, thereby suppressing autoimmunity in said mammal. Although IL-27 is rapidly cleared in vivo, administration of the isolated population of IL-27-producing B-1a cells allows for the expansion of i27-Bregs and sustained IL-27 secretion in vivo. This provides a distinct advantage over therapeutic approaches that may rely on direct administration of IL-27.

[0037] IL-27 and IL-35 are two immunosuppressive members of the IL-12 family of cytokines. Although IL-35 or IL-27 have shown great promise in suppressing autoimmune diseases, the main drawbacks of using cytokines, especially heterodimeric cytokines, as biologics are their relatively short half-life, transient biological activity, and unpredictable pharmacokinetic characteristics. Another important obstacle relates to dosing issues. Because the binding of IL-35 or IL-27 subunit proteins is not strong (non-covalent), IL-35 and IL-27 subunit proteins dissociate easily, making it difficult to ascertain the effective dose administered or required to ameliorate disease of bioactive p35:Ebi3 or p28:Ebi3 heterodimers. The therapeutic use of i27-Breg offers several therapeutic advantages over the use of biologics such as IL-10, IL-27, or IL-35, which are the most effective cytokines produced by Breg or Treg cells. i27-Bregs expanded in vivo, thereby sustaining the production of IL-27 in recipient host tissues; (ii) ex vivo generated i27-Bregs could expand in vivo and reprogram recipient lymphocytes into IL-10-, IL-27-, and IL-35-producing Bregs and Tregs, thereby sustaining the production of these immunosuppressive cytokines in recipient host tissues; (iii) disease suppression by innate i27-Bregs does not require prior activation by disease-inducing self-antigens, offering potential therapeutic advantages over disease-specific Breg / Treg therapies used for autoimmune diseases.

[0038] The term "autoimmunity" as used herein refers to the failure of an organism (e.g., a mammal such as a human or mouse) to recognize its own constituent parts as self, which results in an immune response against the organism's own cells and tissues. In other words, autoimmunity is an adaptive immune response against "self" antigens, and is characterized by the production of inflammatory cytokines that mediate pathology by damaging host tissues, or the production of "autoantibodies" that can cause complement-mediated diseases.

[0039] "Autoimmune disease" refers to any one of a group of diseases or disorders in which tissue damage is associated with humoral and / or cellular immune responses against body components, or in a broader sense, immune responses against self. The pathological immune response may be systemic or organ-specific. For example, immune responses against self may affect the joints, skin, brain, the myelin sheath that protects neurons, kidneys, liver, pancreas, thyroid, adrenal glands, eyes (e.g., uveitis), and ovaries. The formation of immune complexes is involved in the pathogenesis and progression of autoimmune diseases. Increased immune complex formation correlates with the presence of antibodies directed against self (autoantibodies). The presence of autoantibodies, either as part of immune complexes or unbound to antigens (free antibodies), may contribute to tissue inflammation. In some autoimmune diseases, the presence of free autoantibodies significantly influences the pathology of the disease. Another aspect of the pathogenesis and progression of autoimmune diseases is the role of inflammatory cytokines. Under normal circumstances, inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) play a protective role in response to infection and cellular stress. However, pathological consequences resulting from chronic and / or excessive production of TNF-α and IL-1 are thought to underlie the progression of many autoimmune diseases, such as rheumatoid arthritis, Crohn's disease, inflammatory bowel disease, uveitis, and psoriasis. Other inflammatory cytokines involved in autoimmune diseases include interleukin-6, interleukin-8, and granulocyte-macrophage colony-stimulating factor (see, e.g., U.S. Pat. No. 8,080,555).

[0040] The cell population and method of the present invention can be used to suppress the autoimmunity associated with any autoimmune disease.There are more than 80 autoimmune diseases known in the art, including multiple sclerosis (MS), insulin-dependent diabetes mellitus, systemic lupus erythematosus (SLE), psoriasis, autoimmune hepatitis, thyroiditis, insulitis, uveitis, orchitis, gravitational myasthenia, idiopathic thrombocytopenic purpura, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), encephalomyelitis, systemic autoimmune disease (e.g., rheumatoid arthritis (RA), scleroderma, and juvenile arthritis).

[0041] Autoimmunity is "suppressed" when one or more symptoms of an autoimmune disease are reduced or alleviated in a mammal (e.g., a human) affected by the autoimmune disease. The improvement, worsening, regression, or progression of symptoms can be determined by any objective or subjective measure, many of which are known in the art. Those skilled in the art will understand that symptoms of autoimmune disease vary based on the disease and the location of the abnormal immune response. Symptoms common to some autoimmune diseases include, for example, fatigue, muscle and / or joint pain, muscle weakness, fever, swollen glands, inflammation, susceptibility to infection, weight gain or loss, allergies, digestive problems, changes in blood pressure, and dizziness.

[0042] The cell populations and methods of the invention can be used to reduce or inhibit pancreatic inflammation.

[0043] The cell populations and methods of the invention can be used to reduce or inhibit the symptoms of AMD.

[0044] As used herein, the terms "therapy," "treatment," and the like refer to obtaining a desired pharmacological and / or physiological effect.

[0045] Preferably, the pharmacological and / or physiological effect is therapeutic, i.e., the effect partially or completely cures the disease and / or the adverse symptoms resulting from the disease. To this end, the method of the present invention includes administering a "therapeutically effective amount" of the isolated IL-27-producing B-1a cell population. A "therapeutically effective amount" refers to an amount effective at the dosage and for the period necessary to achieve the desired therapeutic result. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the IL-27-producing B-1a cell population to induce the desired response in the individual.

[0046] Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents an autoimmune disease or its symptoms. In this regard, the method of the present invention comprises administering a "prophylactically effective amount" of an isolated IL-27-producing B-1a cell population to a mammal predisposed to or otherwise at risk of developing an autoimmune disease. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., preventing the onset of disease or preventing recurrence of the disease).

[0047] The isolated IL-27-producing B-1a cell population of the present invention or a composition comprising an isolated IL-27-producing B-1a cell population can be administered to a mammal using any suitable administration technique, many of which are known in the art, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The composition is preferably suitable for parenteral administration. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to a mammal using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0048] When the method of the present invention includes administering an isolated IL-27-producing B-1a cell population to a mammal, the isolated IL-27-producing B-1a cell population is administered to the mammal in a dose sufficient to induce the generation of B cells that produce IL-27 and suppress autoimmunity in the mammal. The therapeutic or prophylactic effect can be monitored by periodic evaluation of the treated patient. In the case of repeated administration over several days or more, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other administration schedules may be useful and are within the scope of the present invention. The desired dosage can be delivered by a single bolus administration of the composition, multiple boluses of the composition, or continuous infusion administration of the composition.

[0049] A typical amount of cells to be administered to a mammal (e.g., a human) can range, for example, from 500,000 to 100 million cells, although amounts below or above this exemplary range can be appropriate in the context of the present invention. For example, the daily amount of cells can be about 500,000 to about 50 million cells (e.g., about 5 million cells, about 15 million cells, about 25 million cells, about 35 million cells, about 45 million cells, or a range defined by any two of the foregoing values), preferably about 10 million to about 100 million cells (e.g., about 20 million cells, about 30 million cells, about 40 million, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, or a range defined by any two of the foregoing values), more preferably about 10 million cells to about 50 million cells (e.g., about 12 million cells, about 25 million cells, about 35 million cells, about 45 million cells, or a range defined by any two of the foregoing values).

[0050] The present invention can be utilized in combination with other existing treatments for autoimmune diseases. For example, the cell population of the present invention can be administered in combination with immunosuppressants or immunomodulators or other anti-inflammatory agents for the treatment or prevention of autoimmune diseases, such as the autoimmune diseases disclosed herein. In this regard, the method of the present invention can be used in combination with disease-modifying antirheumatic drugs (DMARDs) (e.g., gold salts, sulfasalazine, antimalarials, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, cyclosporine A, tacrolimus, sirolimus, minocycline, leflunomide, and glucocorticoids).), calcineurin inhibitors (e.g., cyclosporine A or FK 506), modulators of lymphocyte recirculation (e.g., FTY720 and FTY720 analogs), mTOR inhibitors (e.g., rapamycin, 40-0-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573, or TAFA-93), ascomycins with immunosuppressive properties (e.g., ABT-281, ASM981, etc.), corticosteroids, cyclophosphamide, azathioprine, methotrexate, leflunomide, mizoribine, mycophenolic acid, mycophenolate mofetil, 15-deoxyspergualin, or immunosuppressive homologs, analogs, or derivatives thereof, immunosuppressive monoclonal antibodies (e.g., monoclonal antibodies against leukocyte receptors, such as MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD58, CD80, CD86, or their ligands), other immunomodulatory compounds, adhesion molecule inhibitors (e.g., LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists, or VLA-4 antagonists), chemotherapeutic agents (e.g., paclitaxel, gemcitabine, cisplatinum, doxorubicin, cyclosporine ... xorubicin, or 5-fluorouracil), anti-TNF agents (e.g., infliximab, adalimumab, monoclonal antibodies against TNF such as CDP870, or receptor constructs against TNF-RI or TNF-RII such as ENBREL™ (Etanercept) or PEG-TNF-RI, blockers of pro-inflammatory cytokines, IL-1 blockers (e.g., KINERET™ (anakinra) or IL-1 trap, AAL160, ACZ 885, and IL-6 blockers), chemokine blockers (e.g., inhibitors or activators of proteases), anti-IL-15 antibodies, anti-IL-6 antibodies, anti-CD20 antibodies, NSAIDs, and / or anti-infective agents.

[0051] The present invention can be utilized in combination with the administration of B cells that produce interleukin-35 (IL-35). The IL-35 producing B cells (i35-Breg) can be administered to a mammal sequentially (before or after) or simultaneously with the cell population of the present invention.

[0052] The embodiments of the present invention may be useful alone or in combination with one or more other embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the present invention are provided below as numbered embodiments 1 to 26. As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or subsequent individually numbered embodiments. Thus, the present invention provides all combinations of these embodiments and is not limited to the combinations of embodiments explicitly provided below.

[0053] (1) (a) expresses the inhibitory cell surface receptors lymphocyte activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and CXC chemokine receptor type 4 (CXCR4); and (b) secrete interleukin-27 (IL-27); An isolated population of mammalian cells comprising greater than about 75% B-1a regulatory cells.

[0054] (2) The population of mammalian cells of embodiment (1), wherein the regulatory cells further express the inhibitory cell surface receptor glucocorticoid-inducible TNFR-related protein (GITR).

[0055] (3) The population of mammalian cells of embodiment (1) or (2), wherein the regulatory cells further express the inhibitory cell surface receptor OX40.

[0056] (4) The population of mammalian cells described in any one of embodiments (1) to (3), wherein the regulatory cells further express the inhibitory cell surface receptor cytotoxic T-lymphocyte-associated protein 4 (CTLA4).

[0057] (5) A method for preparing a population of mammalian cells according to any one of embodiments (1) to (4), comprising: (a) isolating cluster of differentiation 5 positive (CD5+) expressing cells from a sample of mammalian peripheral lymphoid tissue, mammalian umbilical cord blood, mammalian ascites, induced pluripotent cells (iPSCs), or mammalian bone marrow using fluorescence activated cell sorting (FACS) to provide isolated CD5+ expressing cells; (b) culturing the isolated CD5+ expressing cells in a cell culture medium to provide a cultured cell; (c) activating the cultured cells with a BCR (B cell receptor) or TLR (Toll-like receptor) agonist to provide activated cells; and (d) exposing the activated cells to IL-27.

[0058] (6) A method for suppressing the immune system of a mammal, the method comprising administering to the mammal a population of mammalian cells described in any one of embodiments (1) to (4).

[0059] (7) The method of embodiment (6), further comprising sequentially or simultaneously administering to the mammal B cells that produce interleukin-35 (IL-35).

[0060] (8) The method of embodiment (6) or (7), wherein the administering treats a disease in the mammal.

[0061] (9) The method according to any one of embodiments (6) to (8), wherein the mammal has an autoimmune disease.

[0062] (10) The method of embodiment (9), wherein the autoimmune disease is an eye disease.

[0063] (11) The method according to embodiment (9), wherein the autoimmune disease is a disease of the central nervous system.

[0064] (12) The method according to embodiment (9), wherein the autoimmune disease is a brain disease.

[0065] (13) The method of embodiment (9), wherein the autoimmune disease is uveitis.

[0066] (14) The method of embodiment (9), wherein the autoimmune disease is encephalomyelitis.

[0067] (15) The method according to any one of embodiments (6) to (8), wherein the mammal has multiple sclerosis.

[0068] (16) The method according to any one of embodiments (6) to (8), wherein the administration suppresses inflammation of the pancreas.

[0069] (17) The method of embodiment (6) or (7), wherein the mammal has received an allogeneic bone marrow or hematopoietic stem cell transplant.

[0070] (18) The method of embodiment (6) or (7), wherein the mammal is a recipient of an allogeneic solid organ transplant.

[0071] (19) The method of embodiment (17) or (18), wherein the mammal has graft-versus-host disease (GVHD).

[0072] (20) The method according to any one of embodiments (6) to (8), wherein the mammal has age-related macular degeneration (AMD).

[0073] (21) A method for treating a mammal having graft-versus-host disease, comprising administering to the mammal having graft-versus-host disease a population of mammalian cells described in any one of embodiments (1) to (4).

[0074] (22) The method of embodiment (21), wherein the mammal has received an allogeneic bone marrow or hematopoietic stem cell transplant prior to administration of the population of mammalian cells.

[0075] (23) The method of embodiment (21), wherein the mammal has received an allogeneic solid organ transplant prior to administration of the population of mammalian cells.

[0076] (24) A method for preventing or reducing the severity of graft-versus-host disease in a mammal, comprising administering to the mammal a population of mammalian cells described in any one of embodiments (1) to (4) before the mammal receives an allogeneic transplant.

[0077] (25) The method according to embodiment (24), wherein the allogeneic transplant is an allogeneic bone marrow or hematopoietic stem cell transplant.

[0078] (26) The method according to embodiment (24), wherein the allogeneic transplant is an allogeneic solid organ transplant.

[0079] (27) A method for preventing or reducing the severity of graft-versus-host disease in a mammal, comprising: (a) mixing a population of mammalian cells according to any one of embodiments (1) to (4) with a transplant material to form a transplant mixture; and (b) administering said transplant mixture to a mammal.

[0080] (28) The method of embodiment (27), wherein the transplant material comprises allogeneic lymphocytes.

[0081] (29) The population of mammalian cells according to any one of embodiments (1) to (4) or the method according to any one of embodiments (5) to (28), wherein the mammal is a human. EXAMPLES

[0082] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0083] The following materials and procedures were used in Examples 1-5.

[0084] CD19 in mouse and human PBMCs and human umbilical cord blood + B cells. Six- to eight-week-old C57BL / 6J and IL-27RαKO mice were purchased from Jackson Laboratory (Bar Harbor, ME). Female mice were used, and mice were randomized for all studies described. Human peripheral blood mononuclear cells (PBMCs) were obtained from the National Institutes of Health (NIH) Blood Bank managed by the NIH Office of Blood Transfusion. Primary human umbilical cord blood CD19 + B cells were purchased from STEMCELL™ Technologies (Vancouver, Canada).

[0085] Isolation of mouse and human B cells. PBMCs from normal human subjects were isolated from buffy coats by density gradient centrifugation using commercially available lymphocyte separation medium (Mediatech Inc., Manassas, VA). + B cells were sorted using anti-CD19 antibody-conjugated magnetic beads (Miltenyl Biotec, Bergisch Gladbach, Germany). Mouse B2 cells were isolated from the spleen using a B cell isolation kit (130-090-862), CD19 microbeads (130-052-201), and a plasma cell isolation kit (130-092-530) (all available from Miltenyl Biotec). B1 cells were isolated from the peritoneal cavity of C57BL / 6J mice. Some mice were immunized with LPS in the presence or absence of IL-27. For B-1a cells, isolation was performed in a two-step procedure using the B-1a Cell Isolation Kit; Cat. No. 130-097-413) as recommended by the manufacturer. Briefly, B-1a cells from the peritoneal cavity were negatively selected with a MACS™ magnetic cell column consisting of B-1a cells and magnetic beads labeled with a cocktail of biotin-conjugated non-B-1a antibodies, then B-1a cells were positively selected with magnetic beads conjugated with a B-1a-specific antibody.

[0086] Immunofluorescence staining and confocal imaging analysis CD19 +B cells were activated in vitro for 48 h by stimulation with LPS or anti-CD40 / anti-IgM antibodies in the presence or absence of IL-27. Cells were fixed and blocked with 5% goat serum, then incubated with fluorescently labeled anti-p28 (Invitrogen, Waltham, MA) or anti-Ebi3 antibodies (Santa Cruz Biotechnology, Dallas, TX). Cells were washed and incubated with ALEXAFLUOR™ 568-, ALEXA FLUOR™ 488-, or ALEXAFLUOR™ 647-labeled secondary antibodies (Invitrogen) containing 4',6-diamidino-2-phenylindole (DAPI) and imaged under a laser scanning confocal microscope (FV1000, Olympus Corporation, Tokyo, JP, or LSM700, Carl's AG) (Oh et al., J.Biol. See Chem., 287:30436-30443 (2012).

[0087] Experimental autoimmune uveitis (EAU) EAU was performed by injecting the interphotoreceptor retinoid-binding protein (IRBP) in a 0.2 ml emulsion containing Mycobacterium tuberculosis H37RA strain (2.5 mg / ml) in complete Freund's adjuvant (CFA) in a 1:1 volume ratio. 651-670The disease was induced by active immunization of C57BL / 6J and IL-27RαKO mice with the peptide. Mice were also administered Bordetella pertussis toxin (1 μg / mouse) at the same time as immunization. Mice were treated by intraperitoneal injection of IL-27 (100 ng / mouse) or phosphate-buffered saline (PBS) on day -1 of immunization and every other day until day 12 after immunization. Eight mice per group were used in each study, and mice were age- and sex-matched. Clinical disease was established and scored by fundus examination and histology (see Wang et al., Nat. Med., 20:633-641 (2014), and Oh et al., J. Immunol., 187:3338-3346 (2011)). The severity of ocular disease was examined using a binocular microscope equipped with coaxial illumination. Eyes for histology were enucleated 21 days after immunization, fixed in 10% buffered formalin, and serially sectioned in the perpendicular interpupillary plane. All sections were stained with hematoxylin and eosin.

[0088] Fundus examination Fundus examinations were performed on days 10–21 after EAU induction. Briefly, general anesthesia was administered systemically (intraperitoneal injection of ketamine (1.4 mg / mouse) and xylazine (0.12 mg / mouse)) followed by dilatation of the pupils with topical administration of 1% tropicamide eye drops (Alcon Inc., Fort Worth, TX). Fundus images were captured using a Micron III retinal imaging microscope for small rodents (Phoenix Research Labs, Pleasanton, CA) or a modified KarlStorz veterinary ear endoscope (see Oh et al., (2012), supra, and Paques et al., Invest Ophthalmol. Vis. Sci., 48:2769-2774 (2007)) in combination with a Nikon D90 digital camera. Evaluation of fundus photographs was performed by a masked observer without knowledge of the identity of the mice to avoid subjective bias. At least six images (two posterior central and four peripheral retinal images) were taken from each eye by positioning the endoscope and observing from superior, inferior, lateral, and medial views to identify, map, and record individual lesions. A clinical scoring system for retinal inflammation was used (see Xu et al., Exp. Eye Res., 87:319-326 (2008), and Chan et al., J. Autoimmun., 3:247-255 (1990)).

[0089] Imaging the mouse retina with spectral domain optical coherence tomography (SD-OCT) Optical coherence tomography (OCT) is a non-invasive technique that allows visualization of the internal microstructure of various ocular structures in living animals. An SD-OCT system (Bioptigen Inc., Morrisville, NC) with a central wavelength broadband light source of 820 nm was used for in vivo non-contact imaging of control or EAU mouse eyes. Mice were anesthetized and pupils were dilated as described above. Mice were then immobilized using an easily rotatable adjustable holder that allowed for horizontal or vertical scan scanning. Each scan was performed at least twice, realigning each time. Scan dimensions (depth and lateral extent) were adjusted until optimal signal intensity and contrast were achieved. Retinal thickness was measured from the central retinal region of all images obtained from both horizontal and vertical scans from the same eye using the system software and averaged. Retinal thickness was determined in the system software using known methods (see Gabriele et al., Invest. Ophthalmol. Vis. Sci., 52: 2250-2254 (2011)).

[0090] Electroretinogram (ERG) Prior to ERG recording, mice were dark-adapted overnight and experiments were performed under dim red lighting. Mice were anesthetized with a single intraperitoneal injection of ketamine (1.4 mg / mouse) and xylazine (0.12 mg / mouse), and pupils were dilated with MIDRIN™ P (Santen Pharmaceuticals, Osaka, Japan) containing 0.5% tropicamide and 0.5% phenylephrine hydrochloride. ERGs were recorded using an electroretinogram console (Espion E2; Diagnosys LLC, Lowell, MA), which generates and controls the light stimuli. Dark-adapted ERGs were recorded using a single flash in a Ganzfeld dome, with intensities ranging from -4 to 1 log cd s / m 2 , delivered in 6 steps. The light-adapted ERG was 20 cd / m 2 The background was obtained, and the light stimulation was 0.3–30 cd s / m in 5 steps. 2ERG recordings were started with a goniotomy. Goniotomy prism solution (Alcon Labs, Fort Worth, TX) was used to provide good electrical contact and maintain corneal moisture. A reference electrode (gold wire) was placed in the mouth and a ground electrode (subdermal stainless steel needle) was placed at the base of the tail. Signals were differentially amplified and digitized at a rate of 1 kHz. Amplitudes of the major ERG components (a-wave and b-wave) were measured using automated and manual methods (Espion software; Diagnosys LLC, Lowell, MA). Immediately after ERG recording, fundus imaging was performed as previously described.

[0091] Isolation of retinal cells To characterize inflammatory cells crossing the blood-retinal barrier during EAU, mice were anesthetized and perfused with 1x PBS. Enucleated eyes were placed in a Petri dish containing culture medium (Roswell Park Memorial Institute medium (RPMI 1640)) for immediate isolation of the retina under a dissecting microscope. Eyes were cut along the limbus, and the lens and cornea were carefully removed. The retina was then detached and the attached optic nerve was removed, before the freshly isolated retina was digested with collagenase (1 mg / ml) in RPMI 1640 medium containing 10 μg / ml DNase (Sigma-Aldrich, St. Louis, Missouri) at 37 °C for 2 h. During incubation, cells were intermittently pipetted every 30 min, and the digestion reaction was stopped with 5–10 volumes of 10% fetal bovine serum (FBS) in RPMI 1640 medium. Cells were washed twice with complete RPMI 1640 medium, and cells were counted using a VI-CELL™ XR cell viability analyzer (Beckman Coulter, Brea, CA).

[0092] Cell co-culture Uveocytes isolated from lymph nodes and spleens of mice with EAU, B-1a, macrophages, and dendritic cells were isolated from EAU-immunized mice on day 17. B-1a, macrophages, and dendritic cells were separated by magnetic column beads (MiltenyiBiotech). Coculture experiments were performed in a transwell system (Corning Incorporated, Corning, New York) in RPMI1640 medium containing 10% FBS. Uveocytes or B-1a cells (5 × 10 5 cells), followed by macrophages or dendritic cells (5 × 10 5 1 piece) was seeded in the upper chamber (pore size: 0.4 pm) and IRBP 651-670 After 72 hours of co-culture, cells were harvested for analysis by flow cytometry and thymidine incorporation assay. For functional analysis of human B-1a cells, CD19 + CD20 + CD27 + CD43 + B1 cells were purified by cell sorting and stimulated with anti-CD40 (10 μg / ml) and anti-IgM (5 μg / ml) for 72 hours in the presence or absence of rhIL-27 (100 ng / ml).

[0093] Experimental autoimmune encephalomyelitis (EAE) EAE was induced by injecting 200 μg myelin oligodendrocyte glycoprotein peptide 35-55 (MOG) in a CFA emulsion containing 2.5 mg / ml of heat-killed, ground Mycobacterium tuberculosis strain H37RA. 35-55 Mice were also injected intraperitoneally (ip) twice with 0.3 μg of Bordetella pertussis toxin (Sigma-Aldrich) in 100 μl of RPMI 1640 medium containing 0.1% normal mouse serum on day 0 and day 2 after immunization. 35-55IL-27 (100 ng / mouse) was administered simultaneously with immunization with IL-27 and every other day until day 12 after immunization. Control or IL-27-treated groups (n=12) were euthanized on day 17 of immunization. Mice were monitored and assessed daily for disease severity by a masked observer. Clinical signs of EAE were graded according to the following scale: 0, no clinical symptoms; 1, clumsy, incontinent or weak bladder, floppy tail; 2, mild paraplegia (trouble initiating movement); 3, moderate paraplegia (weakness of hind limbs); 4, complete forelimb and hind limb paralysis; 5, moribund (see Liu et al., J.Immunol.,180:6070-6076(2008)). Spinal cords and brains were harvested 17 days after immunization and stained with hematoxylin and eosin (H&E). For adoptive transfer studies, mice with EAE treated with or without IL-27 were sacrificed 10 days after immunization and used as donors for passive induction of EAE by adoptive transfer of encephalitogenic cells. Spleen cells were isolated and cultured to obtain MOG 35-55 The mice were stimulated with peptide (20 μg / ml) and anti-CD40 antibody (10 μg / ml) in the presence or absence of IL-27 for 3 days and then transplanted intravenously (iv) into naive syngeneic recipient mice. 6 100% / mouse; n=12). Twenty days after adoptive cell transfer, disease was assessed and brain or spinal cord tissue was harvested from recipient mice, fixed in 10% buffered formalin, and sectioned for histopathological examination. Central nervous system (CNS) infiltrates were harvested from brain and spinal cord, and lymphocytes / mononuclear cells were separated by collagenase digestion followed by Percoll gradient for analysis.

[0094] Adoptive transfer of B-1a cells B-1a cells were isolated from the peritoneal cavity of donor mice and sorted using magnetic beads. B-1a cells were cultured for 48 h in complete RPMI 1640 containing LPS (1 μg / ml), washed (2×) to remove residual LPS, and adoptively transferred (5× l0) into C57BL / 6J and IL-27RαKO mice. 5 pcs).

[0095] In vivo model of LPS-induced inflammation LPS (50 μg / mouse) was injected into C57BL / 6J mice, and some mice received IL-27 (100 ng / mouse) 1 h prior to intravenous LPS injection. Mice in the control and IL-27-treated groups (n=5) were euthanized 24 h after injection, and spleen cells were subjected to fluorescence-activated cell sorting (FACS) analysis.

[0096] Proliferation assay Uveocytes or B-1a cells were harvested from IRBP-immunized C57BL / 6J or IL-27RαKO mice on day 17 after immunization. The cells were restimulated in vitro for 72 hours with IRBP peptides in the presence or absence of B-1a, dendritic cells, and macrophages. For in vitro studies, CD19 + B cells were stimulated with anti-CD40 (10 μg / ml) and anti-IgM (5 μg / ml) antibodies in the presence or absence of IL-27. 3 Cells were pulsed with H-thymidine (0.5 μCi / 10 μl / well). Data presented are the mean CPM±SEM of responses of five replicate cultures.

[0097] Detection of cytokine-expressing lymphocytes by FACS CD19 +B cells (>98%) were stimulated with LPS (2 μg / ml) or activated with anti-CD40 (10 μg / ml) and anti-IgM (5 μg / ml) as described above. For intracellular cytokine detection, cells were restimulated with phorbol myristate acetate (PMA) (50 ng / ml) / ionomycin (500 ng / ml) for 5 h. GOLGIPLUG™ (BD Pharmingen, San Diego, CA) was added for the last 3 h, and intracellular cytokine staining was performed using the recommended BD CYTOFIX / CYTOPERM™ kit (BD Pharmingen). FACS analysis was performed on a MACSQUANT™ analyzer (Miltenyi Biotec) using protein-specific monoclonal antibodies and corresponding isotype control antibodies (BD Pharmingen) (see Amadi-Obi et al., Nat.Med.,13:711-718(2007), and Wang et al., Nat.Med.,20:633-641(2014)). FACS analysis was performed on samples stained with monoclonal antibodies conjugated to fluorochromes, including CD19, CD20, CD24, CD27, CD38, CD43, CD138, and CD11b. Cells were color compensated and quadrant gates were set using isotype controls with background less than 0.3%. Live cells were subjected to side scatter (SSC) and forward scatter (FSC) analysis.

[0098] Characterization of regulatory B cells (Breg) and regulatory T cells (Treg) Primary B cells isolated from the brain, spinal cord, retina, peritoneal cavity, blood, spleen, or draining lymph nodes (LNs) of unimmunized EAE or EAU mice express CD19 + The cells were sorted and used for surface and intracellular FACS analysis. Some cells reacted with LPS, IRBP, 651-670 -Peptides and anti-CD40 antibodies, MOG 35-55-peptide and anti-CD40 (see Wang et al., Nat. Med., 20:633-641 (2014), and Choi et al., Front Immunol., 8:1258 (2017)). For intracellular cytokine detection, cells were restimulated with PMA (50 ng / ml) and ionomycin (500 ng / ml) for 5 h. GOLGIPLUG™ (BD Pharmingen) was added for the last hour, and intracellular cytokine staining was performed using the recommended BD CYTOFIX / CYTOPERM™ kit (BD Pharmingen). FACS analysis was performed on a MACSQUANT™ analyzer (Miltenyi Biotec) using protein-specific monoclonal antibodies and corresponding isotype control antibodies (BD Pharmingen) as previously described. Dead cells were stained with a dead cell exclusion dye (Fixable Viability DyeEFLUOR™ 450, Thermo Fisher Scientific) and live cells were subjected to side scatter (SSC) and forward scatter (FSC) analysis. Breg and Treg cells were characterized by analysis of CD4, CD19, CD5, CD27, CD38, CD138, B220, CD1d, IL-10, p28, p35 or Ebi3 expression. FACS analysis was performed on cells stained with monoclonal antibodies conjugated to fluorescent dyes, dead cells were excluded and each tube of cells was color compensated. Quadrant gates were set using isotype controls with background less than 0.5%.

[0099] CRISPRJCas9-mediated gene deletion sgRNAs were generated using known techniques (see Sanjana et al., Nat. Med.,11:783-784(2014)) and cloned into lentiCRISPR v2, pMD2.G. sgRNAs were selected by CRISPRSCAN, an online tool that racks sgRNA sites by on-target binding efficiency and off-target hit probability. For IL-27, three sgRNAs were selected and cloned into a lentiviral vector carrying a SpCas9 sgRNA scaffold driven by a U6 promoter. The sgRNA sequences were as follows: sgp28 target site 1, 5'-GCTTCCTCGCTACC AC ACT-3' (SEQ ID NO:1), site 2; 5'-GGGCCATGAGGCTGGAT CTC-3' (SEQ ID NO:2); site 3 5'-GATGGTATCCCAGGGGCAGG-3' (SEQ ID NO:3). For Ebi3 targeting, the same lentiviral vector was used for cloning of three sgRNAs: site 1 5'-GTCGGGGATGGTGC ATCGGG-3' (SEQ ID NO: 4); site 2 5'-TCTCTGATGGGTCACTAACT-3' (SEQ ID NO: 5); site 3 5'-CAGGAGCAGTCCACGGCCAC-3' (SEQ ID NO: 6). To delete IL-27, purified B-1a cells or macrophages were transduced with lentiviral clones expressing sgRNAs. Two days after infection, cells were activated with LPS for 48 h and analyzed by FACS or ELISA.

[0100] Detection of cytokine secretion by ELISA CD19 + B or B-1a cells were activated in vitro with or without LPS, anti-CD40 and anti-IgM, and / or IL-27. Supernatants were collected after 48 h of culture. IL-27 and IL-35 were quantified using heterodimer ELISA kits specific for mouse IL-27 or IL-35 (BioLegend, San Diego, CA). IL-17 or IL-10 were quantified using R&D Systems kits as recommended by the manufacturer.

[0101] RNA extraction, NanoString analysis, and PCR Total RNA was isolated from the abdominal cavity or spleen using RNEASY™ plus mini kit (Qiagen, Hilden, Germany). cDNA synthesis, RT-PCR and qPCR analysis were performed according to known techniques (see Amadi-Obi et al., Nat.Med.,13:711-718(2007)). Each gene-specific primer pair used for RT-PCR analysis spans at least one intron. The following primers and probes used for qPCR were purchased from Applied Biosystems (Foster City, California): IRF8 (Mm_00492567), IRF4 (Mm_00516431), BCL6 (Mm_00477633), Blimpl (Mm_00476128), Pax5 (Mm_00435501), Lag-3 (Mm_01185091), PD-1 (Mm_00435532), IL-1 (Mm_00435533), and IL-2 (Mm_00435534). 27 (Mm_004461162), IL-12a (Mm_00434169), IL-10 (Mm_00439614), IL-27Rα (Mm_00497259), p21 (Mm_00817699), p27 (Mm_00438168), Cdk1 (Mm_00772472), Cdk2 (Mm_00443947), and Cdk4 (Mm_00726334). mRNA expression was normalized to the level of the GADPH (Mm_99999915) gene. A total of 100 ng of RNA was used per sample for NanoString nCounter analysis. A custom nCounter Gene Expression CodeSet Immunology Panel was used. Data were normalized using housekeeping genes and analyzed with nSolverAnalysis software version 3.

[0102] Immunoprecipitation and immunoblotting Total cell lysates were prepared according to known techniques (see Li et al., Invest. Ophthalmol. Vis. Sci.,40:976-982(1999)). Clarified lysates or cell supernatants were immunoprecipitated with antibodies prebound to protein G-Sepharose beads according to known techniques (see Oh et al., J.Biol.Chem.,286:30888-30897(2014)). Immunoprecipitates were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and blots were probed with specific antibodies. The following antibodies were used for immunoprecipitation and / or Western blotting: p28 (Invitrogen), Ebi3, and β-actin (Santa CruzBiotechnology). Preimmune serum was used in parallel as a control, and signals were detected with HRP-conjugated secondary F(ab')2 (Zymed Labs, San Francisco, California) using the ECL system (Amersham, Arlington Heights, Illinois).

[0103] Western blotting analysis Preparation of whole cell lysates and performance of Western blot analysis were performed according to known techniques (see Wang et al., Nat. Med., 20:633-641 (2014) and Egwuagu et al., J. Immunol., 168:3181-3187 (2002)). Cell extracts (20-40 μg / lane) were fractionated by 10% gradient SDS-PAGE under reducing conditions and Western blot analysis was performed using antibodies specific for pSTAT1, pSTAT3, STAT1, STAT3, p28, p35, Ebi3, IL-27Rα, GP130, IRF8 or β-actin (Santa Cruz Biotechnology and Cell Signaling Technology, Danvers, MA). Preimmune serum was used in parallel as a control and the signal was detected with HRP-conjugated secondary F(ab')2 Ab (Zymed Laboratories) using the ECL-PLUS system (Amersham). Each Western blotting analysis was repeated at least three times.

[0104] Chromatin immunoprecipitation (ChIP) analysis ChIP assays were performed using the EZ-CHIP™ chromatin immunoprecipitation kit (Millipore Sigma, Darmstadt, Germany). B cells were activated with LPS in the presence or absence of IL-27, and DNA-protein complexes were crosslinked for 10 min by adding fresh formaldehyde (Sigma-Aldrich) to the medium at a final concentration of 1% and then quenched with 135 mM glycine. Cells were then washed with cold PBS (2×) and lysed (EZ-CHIP™ Lysis Buffer) by sonication (5×) in 15-s bursts (Sonic Dismembrator Model 1000, power 5, Thermo Fisher Scientific). Lysates were then cleared with protein G-agarose for 1 h, pelleted, and incubated overnight with control IgG or anti-STAT1 or STAT3 antibodies (Cell Signaling Technology). Prior to antibody incubation, input samples were removed from the lysates and stored at -80°C until extraction. Immunoprecipitation was performed according to the manufacturer's instructions (EZ-CHIP™). Immunoprecipitated and input DNA were subjected to PCR and qPCR using primers to detect the binding activity of STAT1 and STAT3. Primers for IL-27p28 gene promoter (5'-CTGAAACCCCAGCTTCCTGCCA-3' (SEQ ID NO: 7) and 5'-CATCTCCTGGGTAGGGGGGTCTTATACT-3' (SEQ ID NO: 8)) are from -134 to -303, and the STAT binding motif is GGAAGGGAAA 77 ACGTT (SEQ ID NO: 9). Primers for the promoter region of EBI3 gene (5'-CTGATTCTGTCTCTGTTTCTCTCAGTT-3' (SEQ ID NO: 10) and 5'-GTGGGGAAAGGCCTTGAGGTAGA-3' (SEQ ID NO: 11)) are from -1 to -150, and the STAT binding motif is CCTCAAGGCCTTTCC (SEQ ID NO: 12).

[0105] Electrophoretic mobility shift assay (EMSA) EMSA was performed according to well-known procedures (see Yu et al., J. Immunol., 157:126-137 (1996)). A double-stranded oligonucleotide containing a motif from the AP1-IRF-1 composite element (AICE), 5'TGAnTCA / GAAA-3' (SEQ ID NO: 13), was synthesized by fill-in reaction using Klenow polymerase (New England BioLabs, Beverly, Massachusetts) to [α-P 32 ] were labeled with dATP or (alpha-32P)dGTP (3000 Ci / mmol) (PerkinE labeled; Immel Inc., Waltham, MA). + B cells were stimulated with LPS (1 μg / ml) in the presence or absence of IL-27 (20 μg / ml) for 3 days, and nuclear extracts were prepared in buffer containing the following protease inhibitors: 2 μM leupeptin, 2 μM pepstatin, 0.1 μM aprotinin, 1 mM [4-(2-aminoethyl)benzenesulfonyl fluoride, hydrochloride], 0.5 mM phenylmethylsulfonyl fluoride, and 1 μM E-64 [N-(Nl-trans-carboxyoxirane-2-carbonyl)-1-leupeptin]agmatine, according to a published procedure (Yu et al., J. Immunol., 157:126-137 (1996)). Protein levels were measured by the recommended BCA method, and extracts were stored at −70°C until use. DNA-protein binding reactions were performed in a 20 μl mixture containing 5 μg of nuclear protein and 1 μg of double-stranded poly(dLC) (Boehringer Mannheim, Barcelona, ​​Spain), 12 mM HEPES (pH 7.9), 60 mM KCI, 0.5 mM DTT, 12% glycerol, and 2.5 mM MgCl. After incubation on ice for 15 min, the samples were diluted with 1 μl P 32 The samples were further incubated with the labeled probe (15,000 cpm) for 20 min at room temperature and then fractionated on a 5% native polyacrylamide gel in 0.25x Tris-borate-EDTA buffer. 32Before adding the P-labeled probe, extracts were preincubated with 1 μl of antibodies specific for basic leucine zipper transcription factor (BATF) (Cell Signaling Technology), Jun B, Jun D, ​​IRF-4, IRF-8, or IRF-1 (Santa CruzBiotechnology).

[0106] Proximity ligation assay Proximity ligation assays (PLA) were performed using the Duolink PLA kit (Sigma Aldrich, St. Louis, MO). Activated B cells were attached to slides and blocked with blocking solution for 1 h, followed by overnight incubation with mouse anti-p28 (rabbit) and anti-Ebi3 (mouse) primary antibodies. A pair of oligonucleotide-labeled secondary antibodies (PLA probes) that bind to the primary antibodies was then added, incubated for 1 h, and then ligation solution containing a hybridizing connector oligo was added. Proximal (within 40 nm) PLA probes then interacted and were ligated to the connector oligo. The resulting closed circular DNA template was amplified by DNA polymerase. Complementary detection oligos conjugated to fluorescent dyes hybridized to the repeat sequences of the amplicon and p28:Ebi3 heterodimers were then detected as individual fluorescent spots by confocal microscopy (LSM 700, Carl Zeiss AG, Oberkochen, Germany).

[0107] RNA-Seq and analysis For RNA-Seq, mRNA was isolated by oligo-dT beads and libraries were sequenced using standard Illumina, Inc. library protocols (Kit RS-122-2101 TruSeq Stranded mRNA LT Sample prep kit, Illumina, Inc., San Diego, CA). Libraries were sequenced on a NovaSeq 6000 system (Illumina, Inc.). Relative abundance of genes was measured by Read Count using StringTie. Statistical analysis was performed to find differentially expressed genes using abundance estimates of each gene in the samples. Genes with one more than the zeroed Read Count value in the sample were excluded. To facilitate log2 transformation, one was added to each Read Count value of filtered genes. Filtered data was log2 transformed and subjected to the trimmed mean M value (TMM) normalization method. Statistical significance of differential expression data was determined using exact t-tests using edgeR with fold change being the null hypothesis that no differences existed between groups. P-values ​​were adjusted for multiple testing using the Benjamini and Hochberg false discovery rate (FDR) correction. For heatmaps, counts were normalized using the R package heatmap and the BroadInstitute tool Morpheus. Hierarchical clustering analysis was performed using complete linkage and Euclidean distance as measures of similarity to display expression patterns of differentially expressed transcripts that met |fold change| > 2 and raw p-values ​​< 0.05 of an independent t-test.

[0108] statistical analysis Graphs were plotted and analyzed using GraphPad Prism 7.0, two-tailed unpaired Student's t-test, nonparametric Mann-Whitney U test, or one-way analysis of variance, depending on the experiment. Probability values ​​of <0.05 were considered statistically significant. Some data are presented as mean + SEM. Asterisks denote p-values ​​as follows: * P < 0.05,** P < 0.01, *** P < 0.001, and **** P<0.0001.

[0109] Sample sizes are indicated in the figures or figure legends and refer to the number of animals. In vitro assays using human umbilical cord blood or PBMCs were repeated independently using cells from at least three unrelated donors. Results shown represent at least three independent experiments, as described in the legend. Optical coherence tomography, ERG and confocal image analysis were performed blindly. EAE and EAU scoring was performed by a masked investigator. The intrinsic immunotherapeutic effect of i27-Breg in EAU was validated and reproduced in the EAE model. Mice were age / sex matched, randomized and consisted of equal numbers of males and females.

[0110] Example 1 This example demonstrated that peritoneal B1 cells secrete IL-27 (i27-Bregs) and activation of i27-Bregs during inflammation triggers egress into secondary lymphoid tissues.

[0111] Immunohistochemistry / confocal microscopy of activated murine CD19 +We colocalized the expression of p28 and Ebi3 on B cells (Figure 1, white arrows), indicating that B lymphocytes produce IL-27. B1 lymphocytes (B-1a and B-lb) are the B cells of innate immunity that are mainly localized in the peritoneal cavity, while B2 are conventional antigen-specific B cells in the spleen. Intracellular cytokine staining by flow cytometry of activated B cells in mouse peritoneal cavity or spleen revealed that both of these developmentally and functionally distinct B cell lineages can produce IL-27 (Figure 2). However, regardless of the activation stimulus or source of B-cells, B-1a cells were the major producers of IL-27 (Figure 2A), and the production of IL-27 by B-1a cells was confirmed by ELISA (Figure 2D). Reciprocal IP / Western analysis detected co-expression of p28 and Ebi3 in lysates and supernatants of activated B-1a cells, providing further evidence that B-1a cells indeed secrete heterodimeric IL-27 (p28 / Ebi3). PLA further demonstrated a physical interaction between p28 and Ebi3 (Figure 132), providing direct evidence that B cells secrete heterodimeric IL-27 cytokines. Reciprocal immunoprecipitation and Western blot (IP / Western) analysis of whole cell extracts or supernatants of activated B-1a cells detected co-expression of p28 and Ebi3 (Figure 133), further confirming that B cells secrete heterodimeric IL-27.

[0112] FACS analysis of activated B cells revealed a distinct population of IL-27-producing B cells (approximately 7.73%) that increased in response to IL-27 (2.85-fold) (Figure 3), suggesting that exposure to IL-27 may induce the expansion of i27-Bregs. NanoString RNA analysis (Figure 5) and Western blotting also showed that BCR / IL-27 synergistically upregulated the expression of IL-27 subunits p28, IL-27Rα, and altered the pattern of chemokine receptor expression (Figure 5). Immunohistochemical / confocal microscopy analysis also detected upregulated expression of IL-27 (white arrows) by B cells in response to IL-27 / BCR signaling (Figure 6), suggesting that BCR and IL-27 signals may be required for optimal expansion of i27-Bregs. Furthermore, chromatin immunoprecipitation assays demonstrated that IL-27 mediated its effects by inducing the binding of activated STAT1 and STAT3 to the U27a proximal promoter (Figures 112 and 113). Although BCR / IL-27-induced signals promoted the expansion of IL-27-producing cells, BCR / IL-27-induced signals failed to expand these cells in cultures of B cells lacking the IL-27 receptor (IL-27RαKO) (Figure 7), clarifying the necessity of IL-27 signals for the generation of IL-27-producing B cells. Consistent with the requirement of IL-27 for the autocrine proliferation of IL-27-producing B-1a cells is the observation that IL-27 upregulates IL-27Rα expression in B1 cells (Figure 8). Importantly, in the context of the applicability of IL-27-producing B cells to immunotherapy, innate-like human B1 cells were also found to produce IL-27 (Figures 9-11).

[0113] To examine whether B cells can produce IL-27 in vivo, we injected C57BL / 6J mice with LPS (i.v.) and measured the percentage of IL-27-producing B-1a or B2 cells in the peritoneal cavity or spleen. Approximately 19.4% of B-1a cells in the peritoneal cavity of PBS-treated mice were producing IL-27 at 24 h, whereas the percentage of these cells increased to approximately 55.6% in mice injected with LPS (Figures 13A-14B). The rapid kinetics of this response indicates recruitment rather than proliferation. Interestingly, the percentage of IL-27-secreting B-1a cells rapidly declined over time in the peritoneal cavity, eventually returning to basal levels by day 4 of inflammation (Figures 13A-14B). Similar analyses revealed distinct patterns of recruitment of IL-27-producing B-1a cells to the spleen. Starting from day 1 after LPS injection, the percentage of B-1a cells recruited to the spleen gradually increased from 2.01%, reaching a peak of 8.8% by day 3, and then returning to basal levels on day 4 of inflammation (Figures 13A-14B). Note that IL-27-producing B2 cells never exceeded 2% in the spleen or peritoneal cavity (Figures 13B and 14B). These results indicate that injection of LPS induced a rapid increase in IL-27-producing B-1a cells, followed by their egress from the peritoneal cavity, and that these events were temporally correlated with the subsequent recruitment of B-1a cells to the spleen.

[0114] The present data show a time-dependent increase in CXCR3- and CXCR5-expressing B-1a cells in the spleen, which coincided temporally with a significant decrease in CXCR4-expressing B-1a cells in the peritoneal cavity (Figures 15-17). These results are consistent with the NanoString data (Figure 5) which showed upregulation of Cxcr5 and downregulation of Cxcr4 transcription by B-1a cells in response to IL-27 (Figures 5 and 134).

[0115] Taken together, these observations suggest that differential regulation of chemokine receptor expression by B-1a cells in response to IL-27 promotes the egress of B-1a cells from the peritoneal cavity and their subsequent trafficking to the spleen.

[0116] Example 2 This example demonstrated that IL-27-producing B-1a cells (i27-Bregs) conferred protection from severe uveitis.

[0117] EAU is an animal model of human uveitis and is a predominantly T cell-mediated intraocular inflammatory disease induced by immunization with retinal proteins / peptides in CFA. We used the EAU model to investigate whether i27-Bregs contribute to immune regulation during uveitis. EAU expresses interphotoreceptor retinoid-binding protein (IRBP)-dependent inflammatory pathways that mediate ocular inflammation. 651-670 EAU was induced in C57BL / 6J mice by immunization with peptides derived from uveitis-associated retinal vasculitis (IL-27) and mice were treated with PBS (control) or IL-27 simultaneously with immunization. Fundus images of PBS-treated mice revealed features of uveitis including haze at the optic nerve head margin, enlargement of the parapapillary area, moderate to severe retinal vasculitis, and cellular infiltration (Figure 18). In contrast, IL-27-treated mice were protected from EAU and showed mild EAU with fewer cells and lower disease scores (Figure 19). Histological analysis of PBS-treated eyes shows inflammatory cells in the vitreous, choroiditis, photoreceptor cell damage and retinal folds, whereas these characteristic features of uveitis were not observed in the eyes of IL-27-treated mice (Figure 20). Optical coherence tomography (OCT) showed substantial accumulation of inflammatory cells in the vitreous and optic nerve head of PBS-treated mice, but not in IL-27-treated mice (Figure 21), and electroretinograms (ERGs) of IL-27-treated mice did not detect visual impairment in control mice (Figures 22-25). Consistent with the improvement of EAU, an increase in IL27 and a decrease in IL17 were detected in the serum of IL27-treated mice (Figures 26-29). Other immunosuppressive cytokines, including IL-10 and IL-35, were also elevated in the serum of IL-27-treated mice (Figures 26-29). Intracellular cytokine analysis showed that approximately 8.2% of B cells in the spleens of PBS-treated mice secreted IL-27, while the percentage of i27-Breg increased to more than 15% in IL-27-treated mice (Figures 30 and 31), indicating a correlation between the increase in i27-Breg and the improvement of EAU. B10(CD19 + CD5+ CD1d hi ) and B-1a (CD19 + CD5 + CD1d low Since i27-Breg cells are CD5+ and exhibit innate immune-like Breg functions, we investigated whether i27-Breg induced during EAU were derived from the B-1a or B10 pool. Although spleens of PBS-treated mice contained moderate levels of IL-27-producing B10 cells (approximately 2.97%), the proportion of these i27-Breg did not increase during EAU in IL-27-treated mice (Figures 32-34). In contrast, approximately 6% or more of B-1a cells in the spleens of PBS-treated mice were i27-Breg, which increased to approximately 11% or more in IL-27-treated mice (Figures 32-34), and in vivo exposure to IL-27 further induced an expansion of i27-producing B-1a cells during EAU.

[0118] To investigate the potential therapeutic importance of i27-Bregs, we cloned peritoneal B-1a cells (>80% i27-Bregs) from WT donor CD45.2 + Purified from mice with EAU, 5 x 10 5 1 mouse / day was cultured as naïve syngeneic WT or IL-27RaKOCD45.1 + Mice were transplanted with IL-27Rα-deficient uveitis and EAU was induced 24 hours after prophylactic administration of B-1a cells. Fundus images 17 days after immunization showed severe uveitis in IL-27Rα-deficient mice (Figures 35 and 36), which correlated with an increase in Thl and Thl7 cells in the eye (Figures 37-38E). The PBS-injected group showed features of uveitis, albeit less severe, compared to IL-27RαKO mice. In contrast, mice given prophylactic B-1a cells developed only mild EAU (Figures 35 and 36), which correlated with a decrease in Thl / Thl7 cells (Figure 37) and a concomitant increase in IL-27-producing B-1a cells (about 10.7%) in the eye (Figure 39). This improvement was not observed in IL-27Rα recipients, proving that the improvement was mediated by IL-27. Interestingly, B-1a therapy induced approximately 2.2-fold expansion of IL-35-producing Breg cells (i35-Breg) (FIG. 40).

[0119] Furthermore, plasmacytoid dendritic cells were found to induce the proliferation of i27-Breg cells, as confirmed by flow cytometry after co-culture (1:1) of activated IL-27-producing B-1a and plasmacytoid dendritic cells (CD11b cells secreting IL-27). + See Figures 149A-149B, showing the percentage of B-1a cells.

[0120] Example 3 This example demonstrated that i27-Breg in the brain and spinal cord suppresses neuroinflammation and encephalomyelitis.

[0121] These studies used the EAE model, which represents a progressive and relapsing-remitting human disease that shares essential immunopathogenic features with multiple sclerosis (MS). 35-55 IL-27-treated mice were induced by immunization of C57BL / 6J mice with IL-27-peptide / CFA. Control PBS-treated mice developed EAE characterized by infiltration of inflammatory cells into the brain and spinal cord, flaccid tail, paraplegia, fore / hind limb paralysis, and moribundity (Figure 44). However, these hallmarks of EAE were much reduced in IL-27-treated mice, as shown by histology and lower EAE clinical scores (Figure 45). Attenuation of disease was demonstrated by a significant reduction in the frequency of Thl7 or IFN-γ / IL-17-expressing Thl7 cells in the brain and spinal cord of IL-27-treated mice, as well as a significant reduction in the frequency of IL-10-expressing CD4+ cells in the brain and spinal cord of IL-27-treated mice. + This correlated with an increase in T cells (Figures 46-51). More importantly, we detected i27-Breg cells in the spinal cord and brain of EAE mice (Figures 52 and 53), as well as significant levels of IL-27-producing B-1a cells in the spinal cord (Figures 52 and 53) and in the spleens of mice treated with IL-27 (Figures 54-58).

[0122] The role of i27-Breg cells in suppressing EAE is related to CD45.1 + and CD45.2 + This was further substantiated in adoptive transfer studies using congenic mouse lineages. CD45.2 + Mice, MOG 35-55 -peptide / CFA and treated with PBS or IL-27. Encephalitogenic cells were harvested from spleens and LNs 21 days after immunization and expressed either PBS-treated or IL-27-treated CD45.2 + 10x10 from mouse 6 10 cells were cultured in a 30-well platelet-free control, with non-immunized CD45.1 + The mice were then adoptively transferred with CD45.2 IL-27 into the PBS-treated mice and the onset and severity of EAE was assessed. Transfer of cells from PBS-treated mice induced disease with characteristics of EAE, whereas transfer of CD45.2 IL-27-treated mice induced disease with characteristics of EAE. + CD45.1 cells were administered + Mice developed mild, delayed EAE (Figure 59). The reduced EAE in recipient mice was due in part to suppression of Thl7 responses (Figure 60) and concomitant expansion of IL-27-producing B-1a cells (Figures 63-66). + It is noteworthy that the levels of IL-27-producing B-1a cells were slightly increased in the spinal cord, brain, and spleen of recipient IL-27-treated mice (Figures 63-66), which may be due to the increased expression of transplanted CD452 + We showed that i27-Bregs cells could expand in vivo. Most notably, they transduced CD45.2 into CNS tissues. + Mobilization of Bregs is mediated by endogenous CD45.1 + Thus, the increase in the number of transplanted i27-Bregs and endogenous CD45.1 in the spinal cord and brain was significantly increased by 1.0%. + Expansion of Bregs would have sustained long-term production of IL-27 in host tissues, and thus i27-Breg therapy may offer a therapeutic advantage over administration of IL-27, which is rapidly cleared in vivo.

[0123] Example 4 This example demonstrated that innate immune IL-27-producing B-1a cells suppress EAE and EAU in an antigen-independent manner.

[0124] Bregs are largely antigen-specific and are effective in suppressing disease mediated by lymphocytes that recognize the same cognate self-antigen. Therefore, we investigated whether IL-27-producing B-1a cells induced by an irrelevant stimulus such as LPS could suppress encephalitogenic lymphocytes that mediate EAE. We also investigated whether IL-27-producing B-1a cells induced by an irrelevant stimulus such as LPS could suppress encephalitogenic lymphocytes that mediate EAE. + C57BL / 6J mice were injected with LPS, and purified B-1a cells were obtained from the peritoneal cavity 2 days later (>80% B-1a i27-Bregs). + EAE was induced 24 hours after adoptive transfer into MOG congenic mice. 35-55 (n=7) + Mice were induced with ex vivo generated B-1a i27-Bregs (5 × 10 5 Transplantation of IL-17 / IFN-γ double-positive T cells in the brain and spinal cord and IL-10-producing regulating CD4 + The suppression of EAE correlated with an increase in i27-Breg cells in the spinal cord (Figures 72 and 73), brain (Figures 74 and 75) and peritoneal cavity (Figures 76 and 77), with the majority of i27-Breg cells observed to be B-1a cells. Similar results were obtained in the EAU model. Thus, consistent with their developmental origin, suppression of CNS autoimmune disease by innate immune i27-Breg cells does not require prior activation by the self-antigen that induced EAE or EAU. This result is in contrast to B2 Breg therapy, which mediates antigen-specific immune suppression, and suggests that transplantation of autologous innate immune i27-Breg cells could be utilized as a treatment for a broader range of autoimmune diseases.

[0125] Example 5 This example shows that crosstalk exists between IL-27-producing B-1a and lymphoid or myeloid cells of the CNS.

[0126] In this study, we investigated whether i27-Bregs that enter the CNS during EAE or EAU could be a source of IL-27 that contributes to the immunosuppressive environment of the CNS. We sorted B-1a cells and macrophages from IRBP-immunized wild-type mice and found that co-culture of the cells for 3 days in a transwell system significantly increased IL-27-producing B-1a cells (Figures 78-81), suggesting that soluble mediators produced by bone marrow cells may increase retinal IL-27 levels during uveitis by promoting the proliferation of i27-Breg cells. The data further show that, like B-1a cells, macrophages respond to inflammatory stimuli by producing IL-27 (Figures 82 and 83). However, infection of either cell type with lentivirus expressing sgp28 / sgpEbi3 guide RNA targeting p28 and ehi3 expression suppressed the ability of macrophages or B-1a cells to produce IL-27 (Figures 82 and 83), suggesting that i27-Bregs may synergize with myeloid cells to elevate IL-27 levels in the CNS during inflammation. The potential crosstalk between i27-Bregs and lymphocytes mediating CNS autoimmune disease was also examined. Coculture with B-1a cells suppressed the proliferation of uveocyte-forming T cells in the spleen and lymph nodes of EAU mice (Figures 84-90). When B-1a cells were defective in IL-27 expression, their ability to suppress Thl7-induced inflammatory responses was reduced (Figures 86-90). These results suggest that B-1a cells entering the retina can suppress Thl7 cells during EAU via the paracrine effect of the IL-27 they secrete.

[0127] Data show that co-culture of B-1a cells with uveal T cells upregulates the inhibitory receptor LAG-3 (LAG-3) in an IL-27-dependent manner. + CD4 + T cells) expressing CD4 +We show that i27-Bregs induced the proliferation of T cells (Figures 91-93) and i35-Bregs (Figures 94-96). Interestingly, most of the IL-35-producing cells induced by IL-27 were Foxp3 negative (Figures 97-99). These results suggest that i27-Bregs can suppress intraocular inflammation, at least in part, by allowing effector T cells to acquire a regulatory phenotype and function.

[0128] Example 6 This example shows that IL-27 regulates B1 and B2 cells in different ways.

[0129] In this study, we investigate whether B-1a cells, which suppress EAU and EAE through the production of IL-27, also suppress inflammation by expressing inhibitory molecules. B-1a cells were isolated from mouse peritoneal cavities by sorting. We then stimulated the cells with LPS for 48 h and showed that they were B-1a cells by their ability to produce IgM (Figure 100). Analysis of cDNA prepared from the cells by qPCR revealed that B-1a cells could indeed express Lag3 and Pdl (Figure 100). Using an in vivo LPS model, we investigated whether innate immune B-1a cells also express these inhibitory receptors in response to inflammatory challenge, as occurs during EAE, EAU, or sepsis. C57BL / 6J mice were injected (iv) with LPS and purified B-1a cells were isolated from the peritoneal cavity by magnetic bead sorting. Results of qPCR analysis of cDNA derived from cells 48 hours after LPS administration confirmed that transcription of Lag3 and Pdl was upregulated by B-1a cells in the peritoneal cavity (Figure 101). + CD138 +As regulatory plasma cells develop through antigen-specific mechanisms, B-1a and B2 cells sorted from mouse peritoneal cavity or spleen and stimulated with anti-IgM / anti-CD40 showed that both B-1a and plasma cells up-regulate Lag3 and Pd1 transcription in response to BCR signaling as shown by qPCR analysis (Figures 102-104). Collectively, these observations suggest that in response to stimulation by pathogens (such as TLR agonists) or self-antigens, B-1a cells can acquire the ability to express inhibitory molecules that enhance their immunomodulatory activity.

[0130] Previous reports have shown that IL-35-producing B cells are exclusively B2 CD138 + It has been shown that IL-27-producing B cells originate from plasma cells (Shen et al., Nature, 507:366-370(2014)). However, this study shows that IL-27-producing B cells originate from the B1 compartment. To understand the mechanism that biases activated B cells toward the i27-Breg developmental program, we investigated the mechanism by which activated CD19 cells stimulated with IL-27 are derived from B1 compartment. +We profiled the B cell transcriptome. qPCR (Figure 105) and NanoString (Figure 106) RNA analysis identified several genes (Irf8, Irfl, Tbx21, Nfll3, Irf7, Xbpl, and Batf) that are specifically activated by IL-27, some of which are known to regulate key pathways in B cells. Of particular interest was the differential upregulation of IRF-8 and IRF-4, as these transcription factors are implicated in B cell development and effector function. Given reports that mutual antagonism between IRF-4 and IRF-8 regulates B-cell development with increased IL-4 in favor of plasma cell development (see, e.g., Xu et al., Nat. Immunol.,16:1274-1281(2015)), preferential upregulation of Irf8 by B-1a cells may promote the i27-Breg developmental program. Because IL-27 induces the proliferation of i27-Bregs, we investigated whether IRF-8 activates the transcription of IL-27p28 subunit protein encoding Il27a. It should be noted, therefore, that IRF-8 and IRF-4 activate transcription by heterodimerization with transcription factors of the ETS / PU-1 or BATF family, which are recruited to the complex elements of ETS-IRF (EICE) or AP1-IRF (AICE) immune regulatory genes. EMSA and supershift analysis using validated AICE sites associated with the expression of Il27a or Ctla4 indicate that IL-27 induces the formation of AICE complexes in activated B cells under in vivo or in vitro conditions. Furthermore, both IRF-4 and IRF-8 were recruited to the AICE of Ctla4, and IRF-8, but not IRF-4, was recruited to the AICE of i / 27a. This suggests that IRF-8 promotes the expression of IL-27 in B cells.Western blot analysis confirmed that IL-27 upregulates IRF-8 in B cells (Figure 107), and RNA analysis showed upregulated transcription of Irf8 by B-1a cells isolated from mice injected with LPS (Figure 108), suggesting an IRF-8 / IL-27 axis that may coordinate a mutual autoregulatory loop promoting the expression of IRF-8 and IL-27 in B-1a cells. A significant reduction in IL-27-producing B-1a cells in CD19-IRF8KO mice (Figures 108-111) was also observed, further highlighting the role of IRF-8 in promoting the proliferation of i27-Breg cells. These results suggest that preferential activation of the IRF-8 / IL-27 axis in the B1 compartment may bias activated B-1a cells toward the i27-Breg developmental program.

[0131] Example 7 This example demonstrated that i27-Breg cells exist in humans and can proliferate in response to inflammatory stimuli.

[0132] In this study, we investigated whether i27-Breg cells exist in humans and proliferate in response to inflammatory stimuli by culturing healthy human PBMCs with the TLR agonist CpG and BCR (anti-CD40 or anti-IgM) for 3 days. Gating on human B-1 cells (CD19 + CD20 + CD27 + CD43 + ) revealed that 19.9% ​​of BCR-activated B cells in human PBMCs produced IL-27 (Figures 114 and 115). + CD20 + CD27 + CD43 + CD11 +B-1 cells represent a subset of B-1a cells ready to migrate to the spleen and other sites of antibody production in response to appropriate stimuli and gating on this revealed that as many as 35% of BCR-activated human B-1a cells can be recruited to the spleen and sites of inflammation during inflammatory diseases (Figures 116-118). Analysis of human cord blood from healthy human donors revealed that 18.1% of resting B-1a cells constitutively produce IL-27 and stimulation of BCR-activated cord blood B cells with IL-27 increased the proportion of cord blood i27- to 73.9% (Figures 119-121). To determine the relative abundance of i27-Bregs versus other Breg subtypes (IL-10-producing Bres and i35-Bregs), activated cord blood cells were expanded for 6 days. Although the majority of Breg cells were i27-Breg, low levels of IL-10-producing Bregs and i35-Bregs were detected, and their levels increased in a time-dependent manner (Figure 122). Similar analysis of B-2 cells revealed that most i27-Bregs were in either the naive or memory B cell pool (Figure 123). Similar to mouse species, human i27-Breg cells constitutively express the inhibitory receptors PD-1 and LAG3 (Figures 124-126) and express the proinflammatory CD4+ receptors that produce TNF-α, IL-17, and / or IFN-γ. + They suppressed the T cell proliferative response (Figures 127-131). Enrichment of cord blood i27-Bregs is of clinical interest because cord blood is the preferred source of hematopoietic stem cells for allogeneic (non-autologous) transplantation for patients with significant human leukocyte antigen (HLA; the complex of genes that code for the human major histocompatibility complex (MHC) proteins) mismatches. Cord blood i27-Bregs can therefore be utilized to suppress alloreactive responses after allogeneic hematopoiesis and protect against GVHD.

[0133] Example 8 This example demonstrates that human i27-Breg cells can be used to successfully treat humans suffering from or at risk of suffering from a disease.

[0134] Human i27-Breg cells are administered by injection or intravenous administration to a human suffering from a disease such as uveitis, MS, AMD, and / or GVHD, or in need of prevention of a disease such as GVHD. After administration of the human i27-Breg cells, the severity and / or symptoms of the disease are reduced and / or prevented.

[0135] Example 9 This example shows that i27-Breg has its own transcriptome.

[0136] Peritoneal B-1a cells enriched for i27-Breg cells by activation with BCR and IL-27 were used to determine the gene expression program required for i27-Breg cell development. Characterization of highly enriched IL-27-producing B-1a cells (>83% i27-Bregs) revealed that while B-1a cells constitutively secrete natural IgM antibodies, the development into an i27-Breg cell phenotype coincides with the loss of IgM antibody production capacity (Figure 135A-135B). In addition to unchallenged B-1a cells, conventional B-2 and IL-35-producing B-2 cells (>57% i35-Breg) from mouse spleens were used as comparators in the RNA-seq analysis. Principal component analysis (PCA) of differentially regulated genes clearly separated B cells into four distinct populations (Figure 136). Gene Ontology (GO) analysis identified highly enriched genes encoding proteins that enhance molecular processes and pathways that further characterize the unique immunosuppressive activity of i27-Breg cells (Figure 137). Heatmaps derived from global RNA-Seq analysis identified 1,998 genes that were upregulated and 1,179 genes that were downregulated in i27-Breg (Figure 137). Differentially induced genes (>2-fold higher expression) in i27-Breg include genes encoding cytokines, cytokine and chemokine receptors (1127, Ebi3, 1110, 117r, I121r, Cxcr3, Cxcr5), inhibitory receptors (Pdcdl, Lag3) signaling molecules (Notch4, Statl, Stat3, Stat5, Aktl, Akt2), and transcription factors (Irf8, Irfl, Batf, Bhlhe40, Xbpl, Arid3a, Ikzfl, Ikzf2, Ikzf4). The repressed genes include genes encoding inhibitory receptors (Pdcd2, Cdldl, Ctla4) and transcription factors (Irf4, Ikzf3, Bach2, Pax5, Ebfl, Runxl, Foxol, Etsl) (Figure 139).To further validate that IL-27 is required for the maintenance of the i27-Breg transcriptome, we show that IL-27-deficient B-1a cells express IL-35 (p35 and EBi3) but are defective in the expression of inhibitory receptor genes (Lag3, Pdl, as well as Pd-11, Pd-12) (Figure 140). Collectively, these results suggest that the i27-Breg transcriptome shows a significant increase in genes required for B-1a development (Bhlhe40, Arid3a, and Cd5), highlighting the developmental origin of i27-Breg from innate immune B-1 cells. However, i27-Breg cells display transcriptional signatures of differentiating germinal center B cells (Irf8↑, Batf↑, Pax5↑, Bach2↑, Ebfl↑) but not of terminally differentiated plasma cells (Prdml↑, Bach2↑, Pax5↑, Ebfl↑). This demonstrates that i27-Breg has a unique transcriptome.

[0137] Example 10 This example demonstrates that i27-Breg and i35-Breg in human cord blood and PBMCs have distinct transcriptomic profiles.

[0138] Human PBMC and umbilical cord blood (CB) B cells produce IL-27, and in PBMC, activated B-1-like cells (CD19 + CD20 + CD27 + CD43 + Approximately 19.9% ​​of i27-Bregs are i27-bregs (Figures 141A and 141B). More than 40% of i27-Breg cells express CD19 + CD20 + CD27 + CD43 + CD11b +Phenotype, the B-1a subset in the CB known to redistribute to regional lymph nodes in response to inflammation (Figure 142A-142C). On the other hand, about 18.1% of resting B-1a cells in the CB constitutively secrete IL-27, and upon activation in the presence of IL-27, the percentage of CB i27-Breg dramatically increased to 73.9% (Figure 143A-143C), suggesting that i27-Breg function as natural Bregs in the human CB and are ready to be rapidly recruited to regional lymph nodes in response to inflammation. t-SNE clustering analysis grouped Breg cells in the CB into three distinct spatially separated subsets: B10, i27-Breg and i35-Breg. i27-Breg were the most abundant, containing more than 85% Bregs in day 3 culture, but decreased to less than 61% in day 6 culture (Figure 144). Although B10 and i35-Breg cells were relatively sparse in day 3 cultures, i35-Bregs were substantially increased (32%) by day 6 (Figure 144). Interestingly, while B cells at all stages of development were able to produce IL-10, IL-27, or IL-35, i27-Bregs were most abundant in immature and memory B cells (Figure 145). Principal component analysis and RNA-seq analysis revealed that i27-Bregs and i35-Bregs had distinct transcriptome profiles (Figure 146). Of the 3,744 differentially expressed genes, 1,575 were elevated in i27-Bregs and 2,169 were downregulated (Figure 147). CD19 + A similar comparison between B cells and i27-Bregs revealed that of 6,159 differentially expressed genes, 3,207 were upregulated in i27-Bregs (Figure 148). Thus, the results of the analysis of human PBMCs or CBs suggest that distinct Breg subsets are induced during the course of an inflammatory response and that the relative abundance of each subset varies depending on the nature of the inflammatory challenge.

[0139] Example 11 This example demonstrates that innate i27-Breg suppresses CNS autoimmune disease through a BCR-independent mechanism.

[0140] Peritoneal B-1 cells are largely unresponsive to BCR-induced signals but highly responsive to innate immune signals induced by pathogens and TLR agonists, suggesting an immunosuppressive activity of i27-Bregs. To determine whether i27-Breg-mediated suppression of EAU or EAE requires prior activation by IRBP or MOG autoantigens, we upregulated the expression of CD45.2 + Sepsis was induced in C57BL / 6J mice, and the peritoneal sorted B-1a cells (>83.5% i27-Bregs) and i27-Breg enriched cells (5 × l0 5 1 / mouse) and naive CD45.1 + The i27-Breg cells were adoptively transferred into congenic mice. 24 hours later, the mice were challenged with EAE induction. Clinical evaluation of the mice revealed significant suppression of EAE (Figure 150) or EAU compared to control mice receiving a comparable number of B-1a cells (<7% i27-Breg). Disease amelioration correlated with a decrease in IL-17-single positive and IL-17 / IFN-y-double positive Thl7 cells, and an increase in Tregs in the brain and spinal cord (Figures 151A-151B), spinal cord (Figures 152A-152B), brain (Figures 153A-153B), and peritoneal cavity (Figures 154A-154B). These results support that adoptive transfer i27-Breg therapy may be useful as a treatment for autoimmune diseases.

[0141] Taken together, the above examples demonstrated the presence of innate IL-27-producing Breg populations in human umbilical cord blood and PBMCs as well as in the brain, spinal cord, retina, and peritoneal cavity of mice with experimental autoimmune encephalomyelitis (EAE) or experimental autoimmune uveitis (EAU), which are models of multiple sclerosis and uveitis, respectively. In vitro experimental systems including confocal microscopy, FACS-based cell sorting, RNA-seq, Chip assay, and immunohistochemistry indicate that IL-27-producing Bregs have a unique transcriptome and are functionally distinct from other Bregs. Adoptive transfer of i27-Bregs ameliorated EAE and EAU by reprogramming resting B cells into i35-Breg cells that were transported to the uvea, brain, and spinal cord and suppressed pathogenic T cells, demonstrating the efficacy of i27-Breg immunotherapy.

[0142] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each individual reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0143] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the present invention (particularly in the context of the claims below) should be interpreted to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprises", "having", "including", and "including" should be interpreted as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The recitation of numerical ranges herein is merely intended to serve as a shorthand method of individually referring to each individual numerical value falling within the range, unless otherwise stated herein, and each individual numerical value is incorporated into the specification as if it were individually set forth herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "etc.") are merely intended to better illuminate the invention, and do not limit the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0144] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to make appropriate use of such variations, and the inventors do not intend to carry out the invention in ways other than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is included in the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. 1. An isolated population of mammalian cells comprising about 75% or more B-1a regulatory cells, the regulatory cells being: (a) expresses the inhibitory cell surface receptors lymphocyte activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and C-X-C chemokine receptor type 4 (CXCR4); and (b) producing interleukin-27 (IL-27); Group.

2. 2. The population of mammalian cells of claim 1, wherein the regulatory cells further express the inhibitory cell surface receptor glucocorticoid-inducible TNFR-related protein (GITR).

3. 3. The population of mammalian cells of claim 1 or 2, wherein the regulatory cells further express the inhibitory cell surface receptor OX40.

4. The population of mammalian cells of any one of claims 1 to 3, wherein the regulatory cells further express the inhibitory cell surface receptor cytotoxic T-lymphocyte-associated protein 4 (CTLA4).

5. A method for preparing a population of mammalian cells according to any one of claims 1 to 4, comprising: (a) isolating cluster of differentiation 5 positive (CD5+), CD19 positive (CD19+) and CD23 negative (CD23-) cells from a sample of mammalian peripheral lymphoid tissue, mammalian umbilical cord blood, mammalian ascites, or mammalian bone marrow using fluorescence activated cell sorting (FACS) to provide isolated CD5+CD19+CD23- cells; (b) culturing the isolated CD5+CD19+CD23− cells in a cell culture medium to provide cultured cells; (c) activating the cultured cells with a BCR (B cell receptor) or TLR (Toll-like receptor) agonist to provide activated cells; and (d) exposing the activated cells to IL-27. A method comprising:

6. A pharmaceutical composition comprising a population of mammalian cells according to any one of claims 1 to 4 for use in suppressing the immune system of a mammal.

7. 7. The pharmaceutical composition of claim 6, further comprising sequentially or simultaneously administering to said mammal B cells that produce interleukin-35 (IL-35).

8. The pharmaceutical composition of claim 6 or 7, wherein the mammal is treated for a disease.

9. The pharmaceutical composition according to any one of claims 6 to 8, wherein the mammal has an autoimmune disease.

10. The pharmaceutical composition of claim 9, wherein the autoimmune disease is an eye disease.

11. The pharmaceutical composition according to claim 9, wherein the autoimmune disease is a disease of the central nervous system.

12. The pharmaceutical composition according to claim 9, wherein the autoimmune disease is a brain disease.

13. The pharmaceutical composition of claim 9, wherein the autoimmune disease is uveitis.

14. The pharmaceutical composition according to claim 9, wherein the autoimmune disease is encephalomyelitis.

15. The pharmaceutical composition of any one of claims 6 to 8, wherein the mammal has multiple sclerosis.

16. The pharmaceutical composition according to any one of claims 6 to 8, the administration of which suppresses inflammation of the pancreas.

17. The pharmaceutical composition of claim 6 or 7, wherein the mammal has received an allogeneic bone marrow or hematopoietic stem cell transplant.

18. The pharmaceutical composition of claim 6 or 7, wherein the mammal is receiving an allogeneic solid organ transplant.

19. 19. The pharmaceutical composition of claim 17 or 18, wherein the mammal has graft-versus-host disease (GVHD).

20. The pharmaceutical composition of any one of claims 6 to 8, wherein the mammal has age-related macular degeneration (AMD).

21. A pharmaceutical composition comprising a population of mammalian cells according to any one of claims 1 to 4 for use in treating a mammal having graft-versus-host disease.

22. 22. The pharmaceutical composition of claim 21, wherein the mammal has received an allogeneic transplant prior to administration of the population of mammalian cells.

23. 22. The pharmaceutical composition of claim 21, wherein the mammal has received an allogeneic solid organ transplant prior to administration of the population of mammalian cells.

24. A pharmaceutical composition comprising a population of mammalian cells according to any one of claims 1 to 4 for use in preventing or reducing the severity of graft-versus-host disease in a mammal.

25. 25. The pharmaceutical composition of claim 24, wherein the allogeneic transplant is an allogeneic bone marrow or hematopoietic stem cell transplant.

26. 23. The pharmaceutical composition of claim 22, wherein the allogeneic transplant is an allogeneic solid organ transplant.

27. The population of mammalian cells according to any one of claims 1 to 4 or the pharmaceutical composition according to any one of claims 6 to 26, wherein the mammal is a human.

28. A pharmaceutical composition for treating or preventing a disease in a mammal, comprising the population of mammalian cells according to any one of claims 1 to 4 as an active ingredient.

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