INTERLEUKIN-27-producing B cells and their use

Abstract

To provide a pharmaceutical composition for use in suppressing the immune system of a mammal.SOLUTION: Provided is a pharmaceutical composition for use in suppressing the immune system of a mammal, wherein the mammal has an autoimmune disease selected from the group consisting of insulin-dependent diabetes, systemic lupus erythematosus (SLE), psoriasis, autoimmune hepatitis, thyroiditis, insulitis, orchitis, myasthenia gravis, idiopathic thrombocytopenic purpura, inflammatory bowel disease, rheumatoid arthritis (RA), scleroderma, and juvenile arthritis; and wherein the pharmaceutical composition comprises an isolated population of mammal cells comprising about 75% or more B-1a regulatory cells, the regulatory cells (a) expressing 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).SELECTED DRAWING: Figure 19

Description

[Technical Field]

[0001] Cross-reference of related applications This patent application claims the benefit of concurrently pending U.S. Provisional Patent Application No. 62 / 863,054, filed on 18 June 2019 (which is incorporated herein by reference in its entirety).

[0002] Statements concerning federally supported research and development This invention was made with federal government support by the National Eye Institute of the National Institutes of Health under project number Z01EY000350-18. The federal government reserves certain rights in this invention.

[0003] Incorporation by referencing electronically submitted properties A computer-readable nucleotide / amino acid sequence listing, submitted concurrently with this specification and identified as follows, is incorporated herein by reference in its entirety: one 2,692-byte ASCII (text) file named "749447_ST25.TXT" dated 12 June 2020. [Background technology]

[0004] Uveitis, age-related macular degeneration (AMD), graft-versus-host disease (GVHD), and multiple sclerosis (MS) are diseases that begin or progress as a result of adverse immunological activity. These diseases can cause blindness, paralysis, and significant pathological conditions that affect quality of life. Uveitis comprises a diverse group of potentially vision-threatening intraocular inflammatory diseases of infectious or autoimmune etiologies, in which autoreactive lymphocytes contribute to the pathology of the eye by attacking and damaging uveal tissue. Similarly, autoimmune processes contribute significantly to the progression of retinal degeneration associated with AMD, although the processes that initiate AMD are not clearly identified. MS is partially caused by lymphocytes that attack and / or destroy myelinated neurons, thereby disrupting synaptic transmission and interneuronal communication. In GVHD, allogeneic grafts are considered foreign in the recipient's body, and the grafted tissue attacks the host organism. Steroids are an effective treatment for uveitis and multiple sclerosis, but they 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 treatment for AMD, and existing treatments aim to slow progressive retinal degeneration. Therefore, there remains an unmet need for safe, effective, and long-term treatments for the aforementioned diseases. [Overview of the Initiative]

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

[0006] The present invention also provides a method for preparing a population of mammalian cells according to embodiments of the present invention, comprising: (a) isolating clusters of differentiated 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 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 for suppressing the immune system of a mammal, comprising administering a population of mammalian cells according to embodiments of the present invention to a mammal.

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

[0009] The present invention provides a method for preventing or mitigating the severity of graft-versus-host disease in mammals, comprising administering a population of mammalian cells according to embodiments of the present invention to a mammal before the mammal receives an allogeneic graft.

[0010] The present invention provides a method for preventing or reducing the severity of graft-versus-host disease in mammals, comprising (a) mixing a population of mammalian cells according to embodiments of the present invention with a graft material to form a graft mixture, and (b) administering the graft mixture to a mammal. [Brief explanation of the drawing]

[0011] [Figure 1]Figure 1 is a set of confocal microscope images showing CD19+ B cells sorted from C57BL / 6 mice. These cells were activated in vitro for 48 hours by stimulation with lipopolysaccharide (LPS) or anti-CD40 / anti-IgM antibody (BR). The cells were incubated with fluorescently labeled anti-p28 or anti-Ebi3 antibody. 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 peritoneal cavity 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] Figure 2B is a bar graph showing the percentage of B-1a and B2 cells from the peritoneal cavity that express IL-27, as shown in Figure 2A. [Figure 2C] Figure 2C is a bar graph showing the percentage of B-1a and B2 cells from the spleen expressing IL-27, as shown in Figure 2A. [Figure 2D] Figure 2D is a graph showing the results of the analysis of the culture supernatant from Figure 2A using enzyme immunosorbent assay (ELISA). [Figure 3] Figure 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 antibody (BCR) in or without IL-27. The plots show the frequencies of various cells in culture. The numbers in the quadrants indicate the percentage of B cells expressing p28, Ebi3, or p28 and Ebi3(IL-27). [Figure 4] Figure 4 is a bar graph showing the quantitative frequency of various cells in the culture shown in the plot in Figure 3. [Figure 5]Figure 5 is a graph showing the results of NanoString RNA analysis (NanoString Technologies, Inc., Seattle, Washington) of various cells in the culture shown in the plot in Figure 3, demonstrating that BCR / IL-27 synergistically upregulates the expression of IL-27 subunits p28 and IL-27Rα, altering 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 culture shown in the plot of Figure 3, demonstrating that BCR / IL-27 synergistically upregulates the expression of IL-27 subunits p28 and IL-27Rα, altering 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 selected CD19+ B cells from wild-type or IL-27RαKO mice activated in vitro for 48 hours by stimulation with anti-CD40 / anti-IgM antibody (BCR) in the presence or absence of IL-27. B cells expressing either p28, Ebi3, or both 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 various cultures. [Figure 8] Figure 8 is a graph showing the qPCR results for IL-27Rα expression in cells isolated from the peritoneal cavity and spleen of wild-type mice and classified as B-1a or B2 cells. [Figure 9] Figure 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] Figure 10 is a graph showing the CD19+ B cells in Figure 9 in the presence of IL-27. [Figure 11]Figure 11 is a graph showing the frequency of human B cells expressing either p28, Ebi3, or both p28 and Ebi3 (IL-27) after CD19+ B cells were isolated from human volunteer PBMCs and activated with PMA in the absence of IL-27. [Figure 12] Figure 12 is a flow cytometry plot showing the frequency of cells from Figure 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 with LPS (50 μg / mouse) (iv), and the frequency of IL-27-producing B-1a cells in the peritoneal cavity was evaluated daily until post-injection day 4. B-1a cells were isolated from the peritoneal cavity at various time points and analyzed by intracellular cytokine staining assays. [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 with LPS (50 μg / mouse) (iv), and the frequency of IL-27-producing B2 cells in the peritoneal cavity was evaluated daily until post-injection day 4. B2 cells were isolated from the peritoneal cavity at various time points and analyzed by intracellular cytokine staining assays. [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 with LPS (50 μg / mouse) (iv), and the frequency of IL-27-producing B-1a cells in the spleen was evaluated daily until post-injection day 4. B-1a cells were isolated from the spleen at various time points and analyzed by intracellular cytokine staining assays. [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 with LPS (50 μg / mouse) (iv), and the frequency of IL-27-producing B2 cells in the spleen was evaluated daily until post-injection day 4. B2 cells were isolated from the spleen at various time points and analyzed by intracellular cytokine staining assays. [Figure 15]Figure 15 is a flow cytometry bar graph showing the percentage of chemokine receptors in CXCR3+ cells. The values ​​in the bar graph represent the proportion of chemokine receptors expressed in CD19+ CD5+ CD23- B-1a B cells. The data represent 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 chemokine receptors in CXCR4+ cells. The values ​​in the bar graph represent the proportion of chemokine receptors expressed in CD19+ CD5+ CD23- B-1a B cells. The data represent 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 chemokine receptors in CXCR5+ cells. The numbers indicate the proportion of chemokine receptors expressed in CD19+ CD5+ CD23- B-1a B cells. The data represent at least three independent experiments (*P<0.05;**P<0.01;***P<0.001;****P<0.0001). [Figure 18] Figure 18 shows a set of fundus images of the retina demonstrating improvement in clinical scores after IL-27 injection. 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 with intraperitoneal injection of IL-27 (100 ng / mouse) or PBS on day (-1) of immunization and every other day until day 12 post-immunization. Eyes were analyzed 14 or 21 days after immunization by fundus examination, histology, optical coherence tomography (OCT), or electroretinography (ERG). [Figure 19] Figure 19 is a graph showing the EAU scores of the retina shown in Figure 18. The clinical EAU score and disease severity were assessed based on changes in the optic nerve head or retinal blood vessels, as well as retinal and choroidal infiltration. [Figure 20]Figure 20 is a set of images of hematoxylin and eosin histological sections of the retina from Figure 18. Scale bar = 200 mM; V = vitreous humor; GCL = ganglion cell layer; INL = inner granular layer; ONL = outer granular layer; RPE / CH = retinal pigment epithelium and choroid. [Figure 21] Figure 21 is a set of images showing the OCT analysis of the retina from Figure 18. It shows the layered structure of the retina. White arrows indicate inflammatory cells (white arrows) of the vitreous nerve or optic nerve. [Figure 22] Figure 22 is a graph showing the ERG analysis of the retina from Figure 18 20 days after EAU induction. The mean dark-adapted ERG wave amplitude is plotted as a function of flash brightness, and its value is the mean ± SEM from four animals in each group. [Figure 23] Figure 23 is a graph showing the ERG analysis of the retina from Figure 18 20 days after EAU induction. The mean dark-adapted ERGb wave amplitude is plotted as a function of flash intensity, and its value is the mean ± SEM from four animals in each group. [Figure 24] Figure 24 is a graph showing the ERG analysis of the retina from Figure 18 20 days after EAU induction. The mean of the light-adapted ERG wave amplitude is plotted as a function of flash brightness, and its value is the mean ± SEM from four animals in each group. [Figure 25] Figure 25 is a graph showing the ERG analysis of the retina from Figure 18 20 days after EAU induction. The mean of the light-adapted ERGb wave amplitude is plotted as a function of flash intensity, and its value is the mean ± SEM from four animals in each group. [Figure 26] Figure 26 is a graph showing the analysis of the cytokine IL-27 in the serum of the mouse from Figure 18. [Figure 27] Figure 18 is a graph showing the analysis of the cytokine IL-17 in the serum of mice. [Figure 28] Figure 28 is a graph showing the analysis of the cytokine IL-10 in the serum of the mouse from Figure 18. [Figure 29] Figure 29 is a graph showing the analysis of the cytokine IL-35 in the serum of the mouse from Figure 18. [Figure 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 spleen of control (PBS-treated) or IL-27-treated EAU mice. The gating strategy is as shown. [Figure 31] Figure 31 is a bar graph showing the percentage of IL27-expressing B cells in Figure 30. [Figure 32] Figure 32 is a flow cytometry plot showing the percentage of IL-27-expressing B cells in the spleen of control (PBS-treated) or IL-27-treated EAU mice. The gating strategy is as shown. The numbers in the quadrants indicate the percentage of CD19+ B cells expressing p28, Ebi3, p28 and Ebi3 (IL-27), CD19+ CD5+ CD1low B cells, or CD19+ CD5+ CD1low B cells. [Figure 33] Figure 33 is a bar graph showing the percentage of IL27-expressing B10 cells in the spleen of control (PBS-treated) or IL27-treated EAU mice from Figure 32. [Figure 34] Figure 34 is a bar graph showing the percentage of IL27-expressing B-1a cells in the spleen of control (PBS-treated) or IL27-treated EAU mice from Figure 32. [Figure 35] Figure 35 shows a set of fundus images of the retinas of mice 17 days after adoptive transplantation, as measured by fundus examination. Peritoneal B-1a cells (5 × 10⁵ cells / mouse; > 80% i27-Bregs) purified from wild-type donor CD45.2+ EAU mice were transferred to naive syngeneic wild-type or IL-27RαKOCD45.1+ mice. EAU was induced in recipient mice by immunization with IRBP651-670 24 hours after adoptive transplantation (n=12). Clinical disease was monitored by fundus examination up to 17 days post-adoptive transplantation. [Figure 36] Figure 36 is a graph showing the EAU score of the retina, as shown in Figure 35. [Figure 37]Figure 37 shows 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. The data represent at least three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 38A] Figure 38A shows 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] Figure 38B is a bar graph showing the percentage of CD4+ T cells expressing IFN-γ. [Figure 38C] Figure 38C is a bar graph showing the percentage of CD4+ T cells expressing IL-17. [Figure 38D] Figure 38D is a bar graph showing the percentage of CD4+ T cells expressing IFN-γ and IL-17. [Figure 38E] Figure 38E is a bar graph showing the percentage of CD4+ T cells expressing IL-10. [Figure 39] Figure 39 shows a set of flow cytometry plots from CD19+ T cells (ocular) 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). The data represent at least three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 40] Figure 40 shows a set of flow cytometry plots from CD19+ T cells (eyes) subjected to FACS. The numbers in the quadrants indicate the percentage of CD19+ CD5- CD23+ B2 cells expressing either p28 and Ebi3 (IL-27) or p35 and Ebi3 (IL-35). Data represent at least three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 41]Figure 41 is a bar graph showing the percentage of B-1a cells expressing p28 and Ebi3 in the eye shown in Figure 39. [Figure 42] Figure 42 is a bar graph showing the percentage of B2 cells expressing p28 and Ebi3 in the eye shown in Figure 40. [Figure 43] Figure 43 is a bar graph showing the percentage of B2 cells expressing p35 and Ebi3 in the eye shown in Figure 40. [Figure 44] Figure 44 shows a set of micrographs of hematoxylin and eosin-stained sections of the brain (top) and spinal cord (middle) of mice 17 days post-immunization (original magnification ×200). Arrows indicate inflammatory cells in the brain or spinal cord. The degree of EAE-induced demyelination was assessed by Luxol fast blue staining (bottom panel; Luxol fast blue is an alcohol-soluble copper phthalocyanine dye attracted to bases found in lipoproteins of the myelin sheath). Arrows indicate demyelination areas in the spinal cord. EAE was induced by immunization of C57BL / 6J mice with MOG35-55-peptide in CFA (n=12). Mice were treated with intraperitoneal injection of IL-27 (100 ng / mouse) or PBS every other day from day 0 of immunization to day 12 post-immunization. [Figure 45] Figure 45 is a graph showing the EAU scores for the spinal cord described in Figure 44. The clinical scores and disease assessments of the EAEs were verified by two masked researchers according to a well-established evaluation system. [Figure 46] Figure 46 shows 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 17 days post-immunization and digested with collagenase. 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 shows 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 17 days post-immunization and digested with collagenase. 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] Figure 48 is a bar graph showing the percentage of CD4 T cells expressing IFN-γ in Figures 46 and 47. [Figure 49] Figure 49 is a bar graph showing the percentage of CD4 T cells expressing IL-17 in Figures 46 and 47. [Figure 50] Figure 50 is a bar graph showing the percentage of CD4 T cells expressing IL-17 and IFN-γ as shown in Figures 46 and 47. [Figure 51] Figure 51 is a bar graph showing the percentage of CD4 T cells expressing IL-10, as shown in Figures 46 and 47. [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 immunized, PBS-treated, or IL-27-treated EAE mice, analyzed by intracellular cytokine staining assays 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 expressing either p28, Ebi3, p28 and Ebi3 (IL-27), or CD19+ CD5+ CD1dlow B cells in the spinal cord or brain. [Figure 53] Figure 53 is a bar graph showing the percentage of CD19 T cells expressing CD19+ CD5+ CD1dlow, as shown in Figure 52. [Figure 54]Figure 54 is a set of flow cytometry plots showing the percentage of IL-27-expressing B cells from the spleen of immunized, PBS-treated, or IL-27-treated EAE mice, with IL-27 (p28 and Ebi3) expression analyzed by intracellular cytokine staining assays. 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 either p28, Ebi3, or both p28 and Ebi3 (IL-27). [Figure 55] Figure 55 is a bar graph showing the percentage of CD19 T cells expressing p28 and Ebi3, as shown in Figure 54. [Figure 56] Figure 56 is a set of flow cytometry plots showing the analysis of spleen cells from PBS-treated or IL-27-treated EAE mice for IL-27 proliferation. The numbers in the quadrants represent the percentage of CD19+ CD5+ CD1dlow B-1a cells. [Figure 57] Figure 57 is a bar graph showing the percentage of CD19 T cells expressing CD19+ CD5+ CD1dhi, as shown in Figure 56. [Figure 58] Figure 58 is a bar graph showing the percentage of CD19 T cells expressing CD19+ CD5+ CD1dlow, as shown in Figure 56. [Figure 59] Figure 59 is a graph showing the EAU scores of spleen cells from MOG35-55 immunized (PBS-treated EAE or IL-27-treated) CD45.2+ mice, which were restimulated ex vivo and transplanted into naive CD45.1+ WT mice (1 × 10⁷ cells / mouse). Clinical scores and disease assessments of EAE were confirmed by two masked researchers. [Figure 60]Figure 60 shows a set of flow cytometry plots indicating 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 post-adoptive transplantation, digested with collagenase, and CD4+ T cells and IL-27-producing B-1a were analyzed by intracellular cytokine staining assays. The numbers in the quadrants indicate the percentage of CD4+ T cells expressing IL-17 or IFN-γ. Data represent more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 61] Figure 61 is a bar graph showing the percentage of cells in Figure 60 that express IL-17. [Figure 62] Figure 62 is a bar graph showing the percentage of cells in Figure 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 post-adoptive transplantation, digested with collagenase, and CD4+ T cells and IL-27-producing B-1a were analyzed by intracellular cytokine staining assays. The numbers in the quadrants show the percentage of CD19+ CD5+ CD11b+ B-1a cells expressing either p28, Ebi3, or both p28 and Ebi3 (IL-27). Data represent more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 64] Figure 64 is a bar graph showing the percentage of B-1a cells from the spinal cord expressing p28 and Ebi3 (IL-27), as shown in Figure 63. [Figure 65] Figure 65 is a bar graph showing the percentage of B-1a cells from the brain expressing p28 and Ebi3 (IL-27), as shown in Figure 63. [Figure 66] Figure 66 is a bar graph showing the percentage of B-1a cells from the spleen expressing p28 and Ebi3(IL-27), as shown in Figure 63. [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 (5 × 10⁵ cells / mouse; >80% i27-Bregs) from WT donor CD45.2+ mice, transplanted into naive syngeneic wild-type mice and induced in recipient mice by immunization with MOG35-55 24 hours after adaptive transplantation (n=12). EAE clinical scores and disease assessments were performed by two masked researchers. [Figure 68] Figure 68 shows a set of flow cytometry plots indicating the percentage of CD4+ T cells expressing IL-10, IL-17, or IFN-γ. Spinal cord and brain cells from PBS-treated or B-1a-treated mice were isolated 15 days post-immunization, digested with collagenase, and analyzed by intracellular cytokine staining assays. Data represent more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 69] Figure 69 is a bar graph showing the percentage of spinal cord and brain cells expressing IFN-γ, as shown in Figure 68. [Figure 70] Figure 70 is a bar graph showing the percentage of spinal cord and brain cells expressing IL-17, as shown in Figure 68. [Figure 71] Figure 71 is a bar graph showing the percentage of spinal cord and brain cells expressing IL-10, as shown in Figure 68. [Figure 72] Figure 72 is a set of flow cytometry plots showing the percentage of CD19+ CD5+ CD23- B-1a cells or CD19+ CD5- CD23+ B2 cells expressing either p28, Ebi3, or both p28 and Ebi3 (IL-27) in the spinal cord. Spinal cords of PBS-treated or B-1a-treated mice were isolated 15 days post-immunization, digested with collagenase, and analyzed by intracellular cytokine staining assays. Data represent more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 73] Figure 73 is a bar graph showing the percentage of spinal cord cells expressing p28 and Ebi3(IL-27) as shown in Figure 72. [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 either p28, Ebi3, or both p28 and Ebi3 (IL-27) in the brain. Brains of PBS-treated or B-1a-treated mice were isolated 15 days post-immunization, digested with collagenase, and analyzed by intracellular cytokine staining assays. Data represent more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 75] Figure 75 is a bar graph showing the percentage of brain cells expressing p28 and Ebi3(IL-27) as shown in Figure 74. [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 either p28, Ebi3, or p28 and Ebi3(IL-27) in the peritoneal cavity. Body fluids from the peritoneal cavity of PBS-treated or B-1a-treated mice were isolated 15 days after immunization, digested with collagenase, and analyzed by intracellular cytokine staining assays. Data represent more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 77] Figure 77 is a bar graph showing the percentage of cells from the peritoneal cavity expressing p28 and Ebi3 (IL-27) as shown in Figure 74. [Figure 78] Figure 78 is a schematic diagram showing macrophages from EAU mice cultured in a Transwell system containing B-1a cells from wild-type EAU mice in the lower well. The effect of macrophages on B-1a cell proliferation was evaluated by a [3H]-thymidine uptake assay. [Figure 79] Figure 79 is a set of flow cytometry plots showing the percentage of B-1a cells from Figure 78 expressing either p28, Ebi3, or both p28 and Ebi3 (IL-27). [Figure 80]Figure 80 is a bar graph showing the mean CPM of B-1a cells and macrophages in Figure 78. Proliferative responses were analyzed in five replicate cultures. Data represent at least three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 81] Figure 81 is a bar graph showing the percentage of B-1a cells and macrophages expressing p28 and Ebi3 (IL-27) as shown in Figure 78. [Figure 82] Figure 82 is a graph showing the ELISA analysis of IL-27 secretion from LPS-activated primary mouse peritoneal macrophages in the presence or absence of lentiviral guide RNA (sgp28-1 or spg28-2) that targets p28. [Figure 83] Figure 83 is a graph showing the ELISA analysis of IL-27 secretion from LPS-activated mouse primary peritoneal B-1a cells in the presence or absence of lentiviral guide RNAs (sgp28-1 or spg28-2) that target p28. [Figure 84] Figure 84 is a schematic diagram showing pathogenic (uveal) T cells derived from EAU mice cultured in a Transwell system containing B-1a cells infected with a lentiviral guide RNA (sgp28 / Ebi3) that targets IL-27 suppression. The effect of B-1a cells on the proliferation of uveal T cells was evaluated by a [3H]-thymidine uptake assay. [Figure 85] Figure 85 is a bar graph showing the average CPM values ​​for the cells in Figure 84. [Figure 86] Figure 86 is a set of flow cytometry plots showing the percentage of uveal CD4+ T cells expressing IL-10, IL-17, and / or IFN-γ as determined by intracellular cytokine staining assays. [Figure 87] Figure 87 is a bar graph showing the percentage of cells expressing IFN-γ as shown in Figure 86. [Figure 88] Figure 88 is a bar graph showing the percentage of cells in Figure 86 that express IL-17. [Figure 89]Figure 89 is a bar graph showing the percentage of cells in Figure 86 that express IFN-γ and IL-17. [Figure 90] Figure 90 is a bar graph showing the percentage of cells expressing IL-10, as shown in Figure 86. [Figure 91] Figure 91 is a schematic diagram showing pathogenic (uveal) T cells from EAU mice cultured in a Transwell system containing B-1a cells from wild-type EAU mice or B-1a cells infected with a lentiviral guide RNA (sgp28 / Ebi3) that targets IL-27 suppression. The effect of B-1a cells on the proliferation of uveal T cells was evaluated by a [3H]-thymidine uptake assay. [Figure 92] Figure 92 is a set of flow cytometry plots showing the percentage of LAG-3-expressing uveal-forming CD4+ T cells determined by intracellular cytokine staining assays. [Figure 93] Figure 93 is a bar graph showing the percentage of cells in Figure 92 that express LAG-3. [Figure 94] Figure 94 is a schematic diagram showing pathogenic (uveal) T cells from EAU mice cultured in a Transwell system containing B-1a cells from wild-type EAU mice or B-1a cells infected with lentiviral guide RNA (sgp28 / Ebi3) that targets IL-27 suppression. The effect of B-1a cells on the proliferation of uveal T cells was evaluated by a [3H]-thymidine uptake assay. [Figure 95] Figure 95 shows a set of flow cytometry plots of CD4+ CD25+ Foxp3+ and CD4+ CD25+ Foxp3- expressing p35, Ebi3, or IL-35(p35 / Ebi3). [Figure 96] Figure 96 is a bar graph showing the percentage of cells in Figure 95 that express p35 and Ebi3. [Figure 97]Figure 97 shows a set of flow cytometry plots of CD4+ CD25+ Foxp3+ and CD4+ CD25+ Foxp3- expressing p35, Ebi3, or IL-35(p35 / Ebi3). [Figure 98] Figure 98 is a bar graph showing the percentage of cells in Figure 97 that are CD4+, CD25+, and Foxp3+. [Figure 99] Figure 99 is a bar graph showing the percentage of cells in Figure 97 that are CD4+, CD25+, and Foxp3-. [Figure 100] Figure 100 is a set of flow cytometry plots and graphs showing the results of ELISA analysis (left flow cytometry plot) 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. Supernatant from the intraperitoneal culture was analyzed by qPCR (right). [Figure 101] Figure 101 is a bar graph showing the results of qPCR analysis of B-1a cells purified from the peritoneal cavity of C57BL / 6J mice that were injected with LPS 48 hours prior (iv). [Figure 102] Figure 102 is a flow cytometry plot showing B-1a or plasma cells (B2) from the peritoneal cavity or spleen of C57BL / 6J mice, respectively, after sorting using magnetic beads and activation with anti-CD40 / anti-IgM (BCR). [Figure 103] Figure 103 is a graph of qPCR analysis of RNA from the cells in Figure 102, quantifying the expression of Pd1 mRNA transcript. [Figure 104] Figure 104 is a graph of qPCR analysis of RNA from the cells in Figure 102, quantifying the expression of Lag3 mRNA transcripts. [Figure 105] Figure 105 is a set of graphs showing RNA isolated at various time points, analyzed by qRT-PCR of CD19+ B cells from the spleen of C57BL / 6J mice activated with anti-CD40 / anti-IgM, in or without IL-27. [Figure 106]Figure 106 shows the volcano plot analysis 24 hours after detecting genes differentially induced by IL-27 using a NanoString transcription factor panel. [Figure 107] Figure 107 shows images of Western blots. CD19+ B cells were isolated from the spleen 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 the IL-27-induced AICE complex. Transcription factors recruited to the CTLA4-AICE or p28-AICE loci were identified by supershift assay. Whole 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] Figure 108 is a bar graph showing the relative gene expression of B-1a cells from the peritoneal cavity. [Figure 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 either p28, Ebi3, or both p28 and Ebi3 (IL-27). These CD19+ B cells were derived from the spleen of C57BL / 6J (wild-type) mice after 3 days of activation with anti-CD40 / anti-IgM, or from mice with target irfH deficiency in B cells (CD19-IRF8KO). The gating strategy is as illustrated. Data represent more than three independent experiments (**P<0.01; ***P<0.001; ****P<0.0001). [Figure 110] Figure 110 is a bar graph showing the quantity of CD19+, CD27+, and CD383+ cells in Figure 109. [Figure 111] Figure 111 is a bar graph showing the amount of CD19+ CD5+ CD11b+ cells in Figure 109. [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, analyzing STAT1 binding to the p28 or ehi3 promoter region. 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 site. [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, where STAT3 binding to the p28 or ehi3 promoter region was analyzed. Cell lysates were immunoprecipitated with anti-STAT3 antibody or control IgG. Immunoprecipitation and input DNA were analyzed by qPCR using primers corresponding to the p28 or ehi3 promoter site. [Figure 114] Figure 114 shows a set of flow cytometry plots of FACS analysis of healthy human PBMCs cultured for 3 days with the TLR9 agonist CpG and BCR (anti-CD40 or anti-IgM). Gating with 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] Figure 115 is a bar graph showing the quantity of CD19+, CD20+, CD27+, CD43+, p28+, and Ebi3+ cells in Figure 114. [Figure 116] Figure 116 shows a set of flow cytometry plots of FACS analysis of healthy human PBMCs cultured for 3 days with the TLR9 agonist CpG and BCR (anti-CD40 or anti-IgM). Gating CD19+ CD20+ CD27+ CD43+ CD11b+ B-1 cells revealed that up to 35% of the cells were BCR-activated human B-1 cells. [Figure 117] Figure 117 is a bar graph showing the quantity of CD19+, CD20+, CD27+, CD43+, CD11 b+, p28+, and Ebi3+ cells in Figure 116. [Figure 118]Figure 118 is a bar graph showing the quantity of CD19+, CD20+, CD27+, CD43+, CD11b-, p28+, and Ebi3+ cells in Figure 116. [Figure 119] Figure 119 shows a set of flow cytometry plots of FACS analysis of human umbilical cord blood from healthy human donors. As many as 18.1% of resting B-1a cells constitutively produced IL-27, and IL-27-induced stimulation of BCR-activated umbilical cord blood B cells increased the percentage of umbilical cord blood i27-Breg to 73.9%. [Figure 120] Figure 120 is a bar graph showing the quantity of cells in Figure 119 (upper panel). [Figure 121] Figure 121 is a bar graph showing the quantity of cells in Figure 119 (bottom 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 Breg and i35-Breg). Activated human umbilical cord blood cells were grown for 6 days. The majority of Breg cells were i27-Breg (maximum slice in each pie chart, ranging from 61.2 + / - 5.3% to 87.1 + / - 3.1%), with much lower levels of IL-10-producing Breg (ranging from 2.6 + / - 0.3% to 6.7 + / - 1.1%) and i35-Breg (ranging from 10.2 + / - 2.7% to 32 + / - 6.8%) detected. [Figure 123] Figure 123 is a set of graphs showing the relative abundance of B-2 cells. These plots revealed that most i27-Breg cells are either naive or in the memory B cell pool. [Figure 124] Figure 124 is a graph showing that, similar to mouse species, human i27-Breg cells constitutively express the inhibitory receptors PD-1 and LAG3. [Figure 125] Figure 125 is a bar graph showing the amount of PD1+ cells in Figure 124. [Figure 126] Figure 126 is a bar graph showing the amount of LAG3+ cells in Figure 124. [Figure 127]Figure 127 shows a set of flow cytometry plots of FACS analysis of human i27-Breg cells. i27-Breg cells suppressed the proliferation response of inflammatory CD4+ T cells producing TNF-α, IL-17, and IFN-γ. [Figure 128] Figure 128 is a bar graph showing the average CPM values ​​for the cells in Figure 127. [Figure 129] Figure 129 is a bar graph showing the amount of TNFα+ CD4+ T cells in Figure 127. [Figure 130] Figure 130 is a bar graph showing the amount of IFNγ+ CD4+ T cells in Figure 127. [Figure 131] Figure 131 is a bar graph showing the amount of IL-17A+ CD4+ T cells in Figure 127. [Figure 132] Figure 132 shows the results of a proximity ligation assay (PLA) demonstrating the physical interaction between p28 and Ebi3. [Figure 133] Figure 133 is a gel showing that B cells produce the heterodimer (p28 / Ebi3) IL-27 cytokine. C57BL / 6J mice were injected (iv) with LPS or LPS+IL-27, and lysates or supernatants from cultured B-1a cells were subjected to cross-immunoprecipitation / Western blotting analysis 24 hours later. Antibodies used for IP or Western blotting are shown. [Figure 134] Figure 134 is a graph showing the results of NanoStringRNA analysis, which demonstrates that IL-27 altered the pattern of chemokine receptor expression in activated B cells. [Figure 135A] Figure 135A shows a flow cytometry plot illustrating the differences in the secretion of native IgM antibodies by unchallenged B-1a and i27-Breg cells in the peritoneal cavity. [Figure 135B] Figure 135B shows a set of graphs illustrating the differences in the secretion of native IgM antibodies by unchallenged intraperitoneal B-1a and i27-Breg cells. [Figure 136] Figure 136 shows a principal component analysis (PCA) plot illustrating the separation of cells into four different populations. [Figure 137] Figure 137 shows a gene ontology (GO) analysis illustrating the enrichment of functional pathways in i27-Breg cells. [Figure 138] Figure 138 is a heatmap of the gene signatures 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 are differentially expressed in i27-Bregs and B-1a cells, specifically genes encoding transcription factors, signaling proteins, cytokines, chemokines, or cell surface proteins. [Figure 140] Figure 140 shows a set of graphs from qPCR expression of genes encoding inhibitory receptors. [Figure 141A] Figure 141A is a set of representative flow cytometry plots showing a significant increase in CD19+ CD20+ CD27+ CD43+ B-1 cells that secrete IL-27 in response to anti-CD40 or BCR. [Figure 141B] Figure 141B is a scatter plot showing a significant increase in CD19+ CD20+ CD27+ CD43+ B-1 cells that secrete IL-27 in response to anti-CD40 or BCR. [Figure 142A] Figure 142A is a set of representative flow cytometry plots showing a significant increase in CD27+ CD43+ CD11+ or CD27+ CD43+ CD11- B-1 cells that secrete IL-27 in response to anti-CD40 or BCR. [Figure 142B] Figure 142B is a set of scatter plots showing a significant increase in CD27+ CD43+ CD11+ or CD27+ CD43+ CD11- B-1 cells that secrete IL-27 in response to anti-CD40 or BCR. [Figure 143A]Figure 143A shows a set of representative flow cytometry plots of human umbilical cord blood CD19+ B cells activated by BCR or BCR and IL-27 (top) or selected B-1a cells in blood (bottom), demonstrating a significant increase in IL-27-producing CD27+ CD43+ B-1a cells. [Figure 143B] Figure 143B is a set of scatter plots of human umbilical cord blood CD19+ B cells activated by BCR or BCR and IL-27 (top) or selected B-1a cells in blood (bottom), showing significant proliferation of IL-27-producing CD27+ CD43+ B-1a cells. [Figure 144] Figure 144 shows representative t-SNE clustering plots and flow cytometry pie charts illustrating the distribution and relative abundance of IL-27 (i27-Breg), IL-35 (i35-Breg), and IL-10 secreting Breg in the B-1 compartment of activated human umbilical cord blood. [Figure 145] Figure 145 shows graphs and pie charts illustrating the amounts of various Breg subsets (e.g., i27-Bregs, i35-Bregs, and B10 cells) in cultures after human CD19+ B cells in human blood have been activated for 6 days and analyzed by intracellular cytokine assays. [Figure 146] Figure 146 shows the results of RNA-Seq analysis using RNA from conventional CD19+ B-2, i27-Breg, i35-Breg, or B10 cells. [Figure 147] Figure 147 is a graph showing a heatmap analysis of genes differentially expressed between i27-Breg cells and i35-Breg cells. [Figure 148] Figure 148 is a graph showing a heatmap analysis illustrating the differences in gene expression between conventional CD19+ B-2 cells and i27-Breg cells. [Figure 149A] Figure 149A is a set of representative flow cytometry plots showing the percentage of IL-27-secreting CD11b+ B-1a cells after co-culture (1:1) of activated IL-27-producing B-1a cells with plasmacytoid dendritic cells. [Figure 149B] Figure 149B is a representative bar graph showing the percentage of IL-27-secreting CD11b+ B-1a cells after co-culture (1:1) of activated IL-27-producing B-1a cells with plasmacytoid dendritic cells. [Figure 150] Figure 150 is a scatter plot showing significant suppression of EAE after transplantation of IL-27 secreting peritoneal B-1a cells (>80% i27-Bregs) purified from WT donor CD45.2+ mice into naive syngeneic CD45.1+ mice, followed by immunization with MOG35-55 (n=12) 24 hours later. [Figure 151A] Figure 151A is a set of representative flow cytometry plots showing attenuation of EAE symptoms in i27-Breg-treated mice, indicated by the percentage of CD4+ T cells expressing IL-10, IL-17, or IFN-γ. [Figure 151B] Figure 151B is a set of scatter plots showing attenuation of EAE symptoms in i27-Breg-treated mice, indicated by the percentage of CD4+ T cells expressing IL-10, IL-17, or IFN-γ. [Figure 152A] Figure 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. [Figure 152B] Figure 152B is a set of scatter plots showing CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells that secrete IL-27 in the brain. [Figure 153A] Figure 153A is a set of representative flow cytometry plots showing CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells that secrete IL-27 in the brain. [Figure 153B] Figure 153B is a set of scatter plots showing CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells that secrete IL-27 in the brain. [Figure 154A]Figure 154A is a set of representative flow cytometry plots showing CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells that secrete IL-27 in the peritoneal cavity. [Figure 154B] Figure 154B is a set of scatter plots showing CD19+ CD5+ CD23- B-1a or CD19+ CD5- CD23+ B2 cells that secrete IL-27 in the peritoneal cavity. [Modes for carrying out the invention]

[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 to date (e.g., U.S. Patent No. 9,629,897) are antigen-specific and originate from the B2 lymphocyte lineage. The present invention provides an isolated population of human cells containing a high concentration of B-1a lineage regulatory B cells that produce and secrete interleukin-27 (i27-Bregs), a population that does not exist in nature.

[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-inducible gene 3) and IL-27p28. IL-27 is expressed by cells and interacts with the IL-27 receptor (IL-27R). IL-27R consists of two proteins: IL-27α (IL-27 alpha) and gpl30. IL-27 induces differentiation of diverse populations of T cells in the immune system. Intrinsic activation of B-1a regulatory cells by inflammatory stimuli triggers IL-27 production and simultaneous efflux of i27-Breg into the spleen, where conventional lymphocytes are reprogrammed to acquire immunomodulatory functions.

[0014] The cell population of the present invention contains approximately 25% or more B-1a regulatory cells (for example, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 55% or more, approximately 60% or more, approximately 65% ​​or more, approximately 70% or more, approximately 75% or more, approximately 80% or more, approximately 81% or more, approximately 82% or more, It may contain approximately 83% or more, approximately 84% or more, approximately 85% or more, approximately 86% or more, approximately 87% or more, approximately 88% or more, approximately 89% or more, or approximately 90% or more B-1a regulatory cells. Populations of B-1a cells at such relatively high proportions compared to other cell types in a cell population do 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 tissue. Within this minority population of less than 2%, i27-Breg is less than 2%, representing only about 4 / 10,000 of the naturally occurring human cell population (i.e., 0.02 × 0.02 = 0.0004).

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

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

[0017] PD-1 (or differentiation cluster 279 (CD279)) is a protein encoded by the human PDCD1 gene. 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, suppressive T cells).

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

[0019] Cell populations also express inhibitory cell surface receptors, glucocorticoid-inducible TNFR-related proteins (GITR or tumor necrosis factor receptor superfamily member 18 (TNFRSF18) or activation-inducible TNFR family receptor (AITR)), as needed. GITR is a protein encoded by the human TNFRSF18 gene. GITR expression has been shown to increase with T cell activation.

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

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

[0022] The cell populations of the present invention may originate from mammals. The term "mammal" includes rodents such as mice, lagomorphs such as rabbits, carnivores including cats and dogs, artiodactyls including cattle and pigs, odd-toed ungulates including equids, primates, cebioids, or silverfish. This includes, but is not limited to, simoids (monkeys) and anthropoids (humans and great apes). More preferably, the population of cells is of human origin.

[0023] The present invention provides a method for preparing a population of cells (e.g., human cells), comprising: (a) isolating differentiated cluster 5-positive (CD5+) expressing cells from mammalian tissue or a 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 (Tall-like receptor) agonist to provide activated cells; and (d) exposing the activated cells to IL-27. In this regard, the isolation of CD5+ (CD5 is expressed on the surface of T cells and B-1a cells) expressing cells can be carried out by any suitable method, for example, by using fluorescence-activated cell sorting (FACS), microfluidic cell sorting, or magnetic cell sorting.

[0024] Mammalian tissue or fluid samples may come from any suitable source, such as mammalian peripheral lymphoid tissue, mammalian umbilical cord blood, mammalian ascites, mammalian bone marrow, induced pluripotent stem cells (iPSCs), or other samples containing B-1a cells. In at least some embodiments, it may be preferable to use ascites or umbilical cord blood as a sample because these sources typically contain a higher proportion of B-1a cells than other samples (e.g., peripheral lymphoid tissue). In some embodiments, a preferred source of tissue or fluid may come from a donor subject treated with the cell population of the present invention.

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

[0026] The cultured cells are exposed to a BCR agonist or a TLR agonist. Any suitable BCR agonist or TLR agonist capable of activating 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 therefore CD5+ B-1a cells are activated with a BCR agonist or a TLR agonist. CD40 is a costimulatory protein found in antigen-presenting cells and is required for B cell activation following the interaction of the B cell receptor with an antibody against IgM. However, maximal IL-27 secretion by activated B-1a cells requires IL-27 signaling, provided by the binding of IL-27 to its homologous receptor on the B-1a cell and further upregulation of the IL-27 receptor.

[0027] As used herein, the terms “Tall-like receptor” and “TLR” refer to any member of a highly conserved family of mammalian proteins that recognize pathogen-associated molecular patterns and act as important 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 "Tall-like receptor 4" and "TLR4" refer to nucleic acids or polypeptides that have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity with publicly available TLR4 sequences. The appropriate TLR4 agonist is LPS.

[0029] The terms "Tall-like receptor 9" and "TLR9" refer to publicly available TLR9 sequences and at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, and 99% of them. This refers to nucleic acids or polypeptides with sequence identity or greater. A suitable TLR9 agonist is an oligonucleotide containing a CpG motif (CpG ODN).

[0030] Activated cells are exposed to IL-27. Exposure to IL-27 promotes the increase of i27-Bregs, efficiently and continuously increasing the proportion and quantity of i27-Bregs.

[0031] The method of the present invention is useful for treating diseases in mammals. The treatment may result in desirable suppression of the immune system.

[0032] The methods of the present invention are useful for the treatment, suppression, or prevention of GVHD. Patients can receive solid organs or allogeneic bone marrow or hematopoietic stem cell grafts. To prevent or reduce the severity of GVHD, populations of mammalian cells of the present invention are administered to the mammal before the mammal receives the allogeneic graft. Alternatively, GVHD can be prevented or suppressed by mixing the i27-Breg population of cells of the present invention with a graft material to form a graft mixture, and then administering the graft mixture to the mammal. In this regard, the graft material may 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 mammalian cell population of the present invention can be mixed with transplant material ex vivo. "Ex vivo" refers to a method performed in an artificial environment outside of a living organism, either within or on cells or tissues, with minimal alteration of natural conditions. In contrast, the term "in vivo" refers to a method performed within a living organism in its normal, intact state, while "in vitro" methods use components of an organism isolated from its normal biological context.

[0034] Populations of mammalian cells can be administered in the form of a pharmaceutically acceptable (e.g., physiologically acceptable) composition. The composition may comprise a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and populations 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 specific site to which the composition can be administered and the specific method used to administer the composition. The composition may optionally be sterile. The composition may be frozen or lyophilized for storage and reconstituted with a suitable sterile carrier before use. The composition may be prepared according to the prior art described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pennsylvania (2001).

[0035] Populations of mammalian cells can be administered to mammals (as defined herein). Preferably, the mammal is a mouse or a human.

[0036] The present invention provides a method for suppressing the immune system of a mammal, the method comprising suppressing the immune system of a mammal by administering a population of mammalian cells of the present invention to a mammal that requires such a population. Accordingly, 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 the mammal, thereby artificially increasing in vivo IL-27 production in the mammal to a high level, thereby suppressing autoimmunity in the mammal. Although IL-27 is rapidly cleared in vivo, administration of the isolated population of IL-27-producing B-1a cells enables the proliferation of i27-Breg cells and sustained in vivo IL-27 secretion. This offers a clear advantage over therapies that may rely on direct administration of IL-27.

[0037] IL-27 and IL-35 are two immunosuppressive cytokines of the IL-12 family. It is a member of the biologics. IL-35 or IL-27 hold great promise for suppressing autoimmune diseases, but the main drawbacks of using cytokines, especially heterodimeric cytokines, as biologics are their relatively short half-lives, transient bioactivity, and unpredictable pharmacokinetic characteristics. Another significant obstacle relates to dosage issues. Because the binding of IL-35 or IL-27 subunit proteins is not strong (non-covalent), IL-35 and IL-27 subunit proteins readily dissociate, making it difficult to determine the effective dose administered or required to improve disease with bioactive p35:Ebi3 or p28:Ebi3 heterodimers. The therapeutic use of i27-Breg offers several therapeutic advantages compared to 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-Breg proliferates in vivo, thereby sustaining IL-27 production in recipient host tissues. (ii) Ex vivo-generated i27-Breg proliferates in vivo and reprograms recipient entrained 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 autoantigens and offers potential therapeutic advantages over disease-specific Breg / Treg therapies used for autoimmune diseases.

[0038] As used herein, the term “autoimmunity” refers to a condition in which an organism (e.g., a mammal such as a human or mouse) is unable to recognize its own components as self, resulting in an immune response against the organism’s own cells and tissues. In other words, autoimmunity is an adaptive immune response to “self” antigens, characterized by the production of inflammatory cytokines that mediate disease by damaging host tissues, or the production of “autoantibodies” that may cause complement-mediated diseases.

[0039] "Autoimmune disease" refers to one of a group of diseases or disorders in which tissue damage is associated with humoral and / or cellular immune responses to the body's components, or, in a broader sense, immune responses against oneself. Pathological immune responses can be systemic or organ-specific. For example, immune responses against oneself can affect joints, skin, brain, myelin sheaths protecting neurons, kidneys, liver, pancreas, thyroid gland, 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 oneself (autoantibodies). The presence of autoantibodies may contribute to tissue inflammation, either as part of immune complexes or as unbound antibodies. In some autoimmune diseases, the presence of free autoantibodies significantly influences the pathogenesis 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 responses 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, including 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, for example, U.S. Patent No. 8,080,555).

[0040] The cell population and method of the present invention can be used to suppress autoimmunity associated with any autoimmune disease. There are more than 80 autoimmune diseases known in the art, examples of which include multiple sclerosis (MS), insulin-dependent diabetes mellitus, systemic lupus erythematosus (SLE), psoriasis, autoimmune hepatitis, thyroiditis, isletitis, uveitis, orchitis, myasthenia gravis, idiopathic thrombocytopenic purpura, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis). This includes colitis, encephalomyelitis, and systemic autoimmune diseases (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 mitigated in a mammal (e.g., human) affected by the autoimmune disease. 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 the symptoms of autoimmune diseases vary based on the disease and the location of the abnormal immune response. Symptoms common to several autoimmune diseases include, for example, fatigue, muscle and / or joint pain, muscle weakness, fever, glandular swelling, inflammation, susceptibility to infection, weight gain or loss, allergies, digestive problems, changes in blood pressure, and dizziness.

[0042] The cell population and method of the present invention can be used to reduce or suppress pancreatic inflammation.

[0043] The cell population and method of the present invention can be used to reduce or suppress the symptoms of AMD.

[0044] As used herein, terms such as “treatment” and “procedure” refer to obtaining a desired pharmacological and / or physiological effect.

[0045] Preferably, the pharmacological and / or physiological effects are therapeutic, i.e., the effects partially or completely cure the disease and / or adverse symptoms resulting from the disease. For this purpose, the method of the present invention comprises administering an isolated population of IL-27-producing B-1a cells in a “therapeutically effective amount.” “Therapeutically effective amount” means an amount that is effective in the dose and duration required to achieve the desired therapeutic outcome. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and body weight of the individual, as well as the ability of the population of IL-27-producing B-1a cells to induce the desired response in the individual.

[0046] Alternatively, the pharmacological and / or physiological effects may be prophylactic, that is, they may completely or partially prevent an autoimmune disease or its symptoms. In this regard, the method of the present invention comprises administering an isolated population of IL-27-producing B-1a cells in a “prophylactically effective amount” to mammals that are predisposed to or otherwise at risk of developing an autoimmune disease. A “prophylactically effective amount” means an amount that is effective in the required dosage and duration to achieve the desired prophylactic outcome (e.g., prevention of disease onset or prevention of disease recurrence).

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

[0048] If the method of the present invention involves administering an isolated population of IL-27-producing B-1a cells to a mammal, the isolated population of IL-27-producing B-1a cells is administered to the mammal in a dose sufficient to induce the production of B cells that produce IL-27 and suppress mammalian autoimmunity. The therapeutic or prophylactic effect can be monitored by regularly evaluating the treated patient. In the case of repeated administration for several days or more, treatment may be repeated depending on the condition until the desired suppression of disease symptoms occurs. It is returned. However, other dosing regimens may be useful and are within the scope of the present invention. The desired dose can be delivered by a single bolus administration of the composition, multiple bolus administrations of the composition, or continuous infusion administration of the composition.

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

[0050] The present invention can be used in combination with other existing treatments for autoimmune diseases. For example, the cell populations 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, antimalarial agents, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, cyclosporine A, tacrolimus, sirolimus, minocycline, leflunomide, and glucocorticoids).), calcineurin inhibitors (e.g., cyclosporine A or FK 506), lymphocyte recirculation modulators (e.g., FTY720 and FTY720 analogs), mTOR inhibitors (e.g., rapamycin, 40-0-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573, or TAFA-93), ascomycin with immunosuppressive properties (e.g., ABT-281, ASM981, etc.), corticosteroids, cyclophosphamide, azathioprene, methotrexate, leflunomide, mizoribine, mycophenolic acid, mycophenolic acid mofetil, 15-deoxysperguarin, or their immunosuppressive homologs, analogs or derivatives, 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, etc.) It can be used in combination with xorubicin (or 5-fluorouracil), anti-TNF agents (e.g., monoclonal antibodies against TNF such as infliximab, adalimumab, CDP870, or receptor constructs for TNF-RI or TNF-RII such as ENBREL® (Etanercept) or PEG-TNF-RI), pro-inflammatory cytokine blockers, IL-1 blockers (e.g., KINERET® (Anakinra) or IL-1 trap, AAL160, ACZ 885, and IL-6 blockers), chemokine blockers (e.g., protease inhibitors or activators), anti-IL-15 antibodies, anti-IL-6 antibodies, anti-CD20 antibodies, NSAIDs, and / or anti-infective agents.

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

[0052] Embodiments of the present invention may be useful individually 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 embodiments numbered 1 to 26. As will be apparent to those skilled in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or succeeding individually numbered embodiments. Accordingly, the present invention provides all combinations of these embodiments and is not limited to the combinations of embodiments expressly provided below.

[0053] (1) (a) Expressing inhibitory cell surface receptor lymphocyte activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and CXC chemokine receptor type 4 (CXCR4), (b) secretes interleukin-27 (IL-27), An isolated population of mammalian cells containing over 75% B-1a regulatory cells.

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

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

[0056] (4) A population of mammalian cells according to any one of embodiments (1) to (3), wherein regulatory cells further express 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), (a) Using fluorescence-activated cell sorting (FACS), differentiated cluster 5-positive (CD5+) expressing cells from a sample of mammalian peripheral lymphoid tissue, mammalian umbilical cord blood, mammalian ascites, induced pluripotent stem cells (iPSCs), or mammalian bone marrow to provide isolated CD5+ expressing cells. (b) To provide cultured cells by culturing the isolated CD5+-expressing cells in a cell culture medium. (c) Activating the cultured cells with a BCR (B cell receptor) or TLR (Toll-like receptor) agonist to provide activated cells, and (d) A method comprising exposing the activated cells to IL-27.

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

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

[0060] (8) The method according to embodiment (6) or (7), wherein the administration treats the disease of 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 according to 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 according to embodiment (9), wherein the autoimmune disease is uveitis.

[0066] (14) The method according to 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 according to embodiment (6) or (7), wherein the mammal has received a transplant of allogeneic bone marrow or hematopoietic stem cells.

[0070] (18) The method according to embodiment (6) or (7), wherein the mammal has received an allogeneic solid organ graft.

[0071] (19) The method according to 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 according to embodiment (21), wherein the mammal has received an allogeneic bone marrow or hematopoietic stem cell graft prior to the administration of the population of mammalian cells.

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

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

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

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

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

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

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

[0082] The following embodiments further illustrate the present invention, but should of course not be construed as 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, Maine). Female mice were used, and mice were randomized for all described studies. Human peripheral blood mononuclear cells (PBMCs) were obtained from the National Institutes of Health (NIH) blood bank managed by the NIH Transfusion Administration. Primary human umbilical cord blood CD19 + The 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 isolation medium (Mediatech Inc., Manassas, Virginia). Human CD19 +B cells were sorted using anti-CD19 antibody-conjugated magnetic beads (Miltenyl Biotec, Bergischgladbach, Germany). Mouse B2 cells were isolated from the spleen using a B cell isolation kit (130-090-862), CD19 microbeads (130-052-201), and 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 or out of the presence of IL-27. For B-1a cells, isolation was performed in a two-step procedure using the B-1a Cell Isolation Kit (catalog no. 130-097-413) as recommended by the manufacturer. In short, B-1a cells from the peritoneal cavity were negatively selected using a MACS® magnetic cell column consisting of magnetic beads labeled with a cocktail of biotin-conjugated non-B-1a antibodies and B-1a cells. Then, B-1a cells were positively selected with magnetic beads conjugated with B-1a-specific antibodies.

[0086] Immunofluorescence staining and confocal imaging analysis CD19 + B cells were activated in vitro for 48 hours by stimulation with LPS or anti-CD40 / anti-IgM antibody in or without IL-27. After fixing the cells and blocking with 5% goat serum, they were incubated with fluorescently labeled anti-p28 (Invitrogen, Waltham, Massachusetts) or anti-Ebi3 antibody (Santa Cruz Biotechnology, Dallas, Texas). The cells were washed and incubated with ALEXAFLUOR® 568-, ALEXAFLUOR® 488-, or ALEXAFLUOR® 647-labeled secondary antibodies (Invitrogen) containing 4',6-diamidino-2-phenylindole (DAPI), followed by laser scanning confocal imaging. Point 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 contains photoreceptor-retinoid-binding protein (IRBP) in a 0.2 ml emulsion (1:1 volume ratio with complete Freund's adjuvant (CFA) containing Mycobacterium tuberculosis H37RA strain (2.5 mg / ml)). 651-670 Active immunization was induced in C57BL / 6J and IL-27Rα KO mice using peptides. Mice were also administered Bordetella pertussis toxin (1 μg / mouse) concurrently with immunization. Mice were immunized on day -1 of immunization and post-immunization. Mice were treated with intraperitoneal injection of IL-27 (100 ng / mouse) or phosphate-buffered saline (PBS) every other day until day 12. Eight mice were used per group in each study, and the mice were matched for age and sex. 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 eye disease was examined using a binocular microscope with coaxial illumination. Eyes for histology were enucleated 21 days after immunization, fixed in 10% buffered formalin, and serially sectioned at the vertical pupillary optic nerve plane. All sections were stained with hematoxylin and eosin.

[0088] Fundus examination Fundus examinations were performed 10–21 days after EAU induction. Briefly, pupil dilation was achieved by topical administration of 1% tropicamide eye drops (Alcon Inc., Fort Worth, Texas) following systemic administration of general anesthesia (intraperitoneal injection of ketamine (1.4 mg / mouse) and xylazine (0.12 mg / mouse)). Fundus images were captured using a Micron III retinal imaging microscope for small rodents (Phoenix Research Labs, Pleasanton, California) or a modified KarlStorz veterinary ear endoscope combined with a Nikon D90 digital camera (see Oh et al., (2012), above, and Paques et al., Invest Ophthalmol.Vis.Sci., 48:2769–2774 (2007)). To avoid subjective bias, fundus photographs were evaluated by masked observers without knowledge of the mouse's identity. By positioning an endoscope and observing from superior, inferior, lateral, and medial fields of view, at least six images (two central posterior retinal images and four peripheral retinal images) were acquired from each eye to identify, map, and record individual lesions. A clinical assessment 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 of mouse retina using spectral-region optical coherence tomography (SD-OCT) Optical coherence tomography (OCT) is a non-invasive technique that enables visualization of the internal microstructure of various eye structures in living animals. An SD-OCT system (Bioptigen Inc., Morrisville, North Carolina) with a broadband light source at a central wavelength of 820 nm was used for in vivo non-contact imaging of the eyes of control or EAU mice. Mice were anesthetized and their pupils dilated as described above. The mice were then fixed using an easily rotatable, adjustable holder that allowed for horizontal or vertical scan scanning. Each scan was performed at least twice, readjusted each time. Scan dimensions (depth and lateral range) were adjusted until optimal signal intensity and contrast were achieved. Retinal thickness was measured and averaged from the central retinal region of all images obtained from both horizontal and vertical scans from the same eye using system software. The retinal thickness of the system software was determined using known methods (see Gabriele et al., Invest. Ophthalmol. Vis. Sci., 52: 2250-2254 (2011)).

[0090] Electroretinography (ERG) Prior to ERG recording, mice were dark-adapted overnight, and experiments were conducted 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 Pharmaceutical, Osaka, Japan) containing 0.5% tropicamide and 0.5% phenylephrine hydrochloride. ERGs were recorded using an electroretinography console (Espion E2; Diagnostics LLC, Lowell, Massachusetts) that generated and controlled light stimuli. Dark-adapted ERGs were recorded using a single flash with a Ganzfeld dome, with intensities of -4 to 1 log cd s / m. 2 It was delivered in 6 steps. The light-adapted ERG was 20 cd / m². 2 The light stimulation was obtained in the background, with 5 steps ranging from 0.3 to 30 cd / m². 2The procedure began with a Goni-O Copic prism solution (Alcon Labs, Fort Worth, Texas) used to provide good electrical contact and maintain corneal moisture. A reference electrode (gold wire) was placed in the mouth, and a ground electrode (subcutaneous stainless steel needle) was placed at the base of the tail. The signal was differentially amplified and digitized at a rate of 1 kHz. The amplitudes of the main ERG components (wave a and wave b) were measured using automatic and manual methods (Espion software; Diagnostics LLC, Lowell, Massachusetts). Fundus imaging was performed immediately after the ERG recording as described above.

[0091] Isolation of retinal cells To characterize inflammatory cells crossing the blood-retinal barrier in EAU, mice were anesthetized and perfused with 1×PBS. To immediately isolate the retina under a dissecting microscope, the enucleated eyes were placed in petri dishes containing culture medium (Roswell Park Memorial Laboratory medium (RPMI 1640)). The eyes were cut along the limbus, and the lens and cornea were carefully removed. The retina was then peeled off, the attached optic nerve was removed, and the freshly isolated retina was treated with 10 μg / ml DN. Cells were digested with collagenase (1 mg / ml) in RPMI 1640 medium containing ase (Sigma-Aldrich, St. Louis, Missouri) at 37°C for 2 hours. During incubation, cells were intermittently pipetted every 30 minutes, and the digestion reaction was stopped with 5 to 10 times the volume of RPMI 1640 medium of 10% fetal bovine serum (FBS). Cells were washed twice with complete RPMI 1640 medium, and cells were counted using a VI-CELL® XR cell viability analyzer (Beckman Coulter, Blair, California).

[0092] cell co-culture Retinal pigment epithelial cells isolated from the 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 (Miltenyi Biotech). Co-culture experiments were performed in a Transwell system (Corning Incorporated, Corning, New York) with RPMI 1640 medium containing 10% FBS. After seeding retinal pigment epithelial cells or B-1a cells (5×10 5 cells) in the lower well, macrophages or dendritic cells (5×10 5 cells) were seeded in the upper chamber (pore size: 0.4 pm) and restimulated with IRBP 651-670 (20 μg / ml). Cells were harvested for analysis by flow cytometry and thymidine incorporation assay 72 hours after co-culture. 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)<(此处似乎缺少内容) EAE was induced by subcutaneous immunization with 200 μg of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG 35-55 )(Sigma-Aldrich) in CFA emulsion containing 2.5 mg / ml of heat-killed and pulverized Mycobacterium tuberculosis H37RA strain. Mice were also injected intraperitoneally (i.p.) twice on day 0 and 2 days after immunization with 0.3 μg of pertussis toxin (Sigma-Aldrich) in 100 μl of RPMI-1640 medium containing 0.1% normal mouse serum. Some mice received MOG <(此处似乎缺少内容) 35-55Simultaneously with immunization, and every other day until day 12 post-immunization, IL-27 (100 ng / mouse) was administered. The control group or the IL-27 treatment group (n=12) was euthanized on day 17 post-immunization. Mice were monitored, and a masked observer assessed the severity of the disease daily. Clinical signs of EAE were graded according to the following scale: 0, no clinical symptoms; 1, fine motor impairment, incontinence or bladder weakness, drooping tail; 2, mild paraplegia (trouble initiating movement); 3, moderate paraplegia (hindlimb weakness); 4, complete forelimb and hindlimb paralysis; 5, mortally ill (see Liu et al., J.Immunol., 180:6070-6076 (2008)). Spinal cord and brain cells were collected 17 days after immunization and stained with hematoxylin and eosin (H&E). In the adoptive transplantation study, EAE mice treated with or without IL-27 were sacrificed 10 days after immunization and used as donors for passive induction of EAE by adoptive transplantation of encephalitis-inducing cells. Splenocytes were isolated and MOG 35-55 Mice were stimulated with peptide (20 μg / ml) and anti-CD40 antibody (10 μg / ml) in or without IL-27 for 3 days, and then intravenously (iv) transplanted into naive syngeneic recipients. (10 × 10) 6 (Individual cells / mouse; n=12). Twenty days after adoptive cell transplantation, disease was assessed, and brain or spinal cord tissue was collected from recipient mice, fixed in 10% buffered formalin, and sectioned for histopathological examination. Central nervous system (CNS) infiltrates were collected from the brain and spinal cord, and lymphocytes / mononuclear cells were separated by collagenase digestion and subsequent analysis using a Percoll gradient.

[0094] B-1a cell adoptive transplantation B-1a cells were isolated from the peritoneal cavity of donor mice and sorted using magnetic beads. B-1a cells were cultured for 48 hours in complete RPMI 1640 containing LPS (1 μg / ml). Wash (2x) to remove residual LPS, then transplant into C57BL / 6J and IL-27RαKO mice (5x 10x). 5 (Individual)

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

[0096] Growth assay Uveolar cells or B-1a cells were collected from IRBP-immunized C57BL / 6J or IL-27Rα KO mice on day 17 post-immunization. These cells were re-stimulated in vitro for 72 hours with IRBP peptide, either in the presence or absence of B-1a, dendritic cells, and macrophages. For in vitro studies, CD19 + B cells were stimulated with anti-CD40 antibody (10 μg / ml) and anti-IgM antibody (5 μg / ml) in the presence or absence of IL-27. During the last 24 hours of culture, the cells were... 3 H-thymidine (0.5 μCi / 10 μl / well) was pulsed. The data presented are the mean CPM ± SEM response 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 antibody (10 μg / ml) and anti-IgM antibody (5 μg / ml) as described above. For intracellular cytokine detection, cells were re-stimulated with phorbol myristate acetate (PMA) (50 ng / ml) / ionomycin (500 ng / ml) for 5 hours. GOLGIPLUG® (BD Pharmingen, San Diego, California) was added during the last 3 hours, 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) (Amadi-Obi). See 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 fluorescent dyes (including CD19, CD20, CD24, CD27, CD38, CD43, CD138, and CD11b). Cells were color-corrected, and quadrant gates were set using isotype controls with a background of less than 0.3%. Live cells were subjected to lateral scattering (SSC) and forward scattering (FSC) analysis.

[0098] Characterization of regulatory B cells (Bregs) and regulatory T cells (Tregs) Primary B cells isolated from the brain, spinal cord, retina, abdominal cavity, blood, spleen, or inflow area lymph nodes (LNs) of immunized EAE or EAU mice are CD19 + The cells were classified and used for surface and intracellular FACS analysis. Some cells were analyzed using LPS and IRBP. 651-670 - Peptides and anti-CD40 antibodies, MOG 35-55-Reactivated with peptides 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 hours. GOLGIPLUG® (BD Pharmingen) was added in the last hour, and intracellular cytokine staining was performed using the recommended BD CYTOFIX / CYTOPERM® kit (BD Pharmingen). FACS analysis was performed using a MACSQUANT® analyzer (Miltenyi Biotec) with protein-specific monoclonal antibodies and corresponding isotype control antibodies (BD Pharmingen) as described above. 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 the 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-corrected. Quadrant gates were set using isotype controls with a background of less than 0.5%.

[0099] CRISPRJCas9-mediated gene deletion sgRNAs were generated using known methods (see Sanjana et al., Nat. Med., 11:783-784 (2014)) and cloned into lentiCRISPR v2, pMD2.G. sgRNAs were selected using CRISPRSCAN, an online tool that racks sgRNA sites based on target binding efficiency and the probability of off-target hits. In the case of IL-27, three sgRNAs were selected and used to create a SpCas9 sgRNA scaffold driven by the U6 promoter. The sgRNAs were cloned into a lentiviral vector. 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 to clone three sgRNAs: 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 remove IL-27, the lentiviral clones expressing the sgRNAs were transduced into purified B-1a cells or macrophages. Two days after infection, cells were activated with LPS for 48 hours and analyzed by FACS or ELISA.

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

[0101] RNA extraction, nanostring analysis, and PCR Total RNA was isolated from the peritoneal cavity or spleen using the 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 spanned 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- 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), Cdk4(Mm_00726334). mRNA expression was normalized to the level of the GADPH(Mm_99999915) gene. NanoString nCounter analysis used a total of 100 ng of RNA per sample. A custom nCounter gene expression CodeSet immunology panel was used. The data were normalized using housekeeping genes and analyzed with nSolverAnalysis software version 3.

[0102] Immunoprecipitation and immunoblotting Whole cell lysates were prepared according to known techniques (see Li et al., Invest. Ophthalmol. Vis. Sci., 40:976-982 (1999)). The clarified lysates or cell supernatants were immunoprecipitated with antibodies pre-conjugated to Protein G-Sepharose beads according to known techniques (see Oh et al., J. Biol. Chem., 286:30888-30897 (2014)). The immunoprecipitates were separated 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 (Invitro gen), Ebi3, and β-actin (Santa Cruz Biotechnology). Preimmune serum was used in parallel as a control, and HRP conjugate secondary F(ab')2 (Zymed Labs, San Francisco, Ca) was used with the ECL system (Amersham, Arlington Heights, Illinois). A signal was detected in California.

[0103] Western blotting analysis The preparation of whole cell lysates and the performance of Western blot analysis were carried out 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 to pSTAT1, pSTAT3, STAT1, STAT3, p28, p35, Ebi3, IL-27Rα, GP130, IRF8, or β-actin (Santa Cruz Biotechnology and Cell Signaling Technology, Danvers, Massachusetts). Preimmune serum was used in parallel as a control, and HRP-conjugated secondary F(ab')2 Ab(Zymed Laboratories) was used with the ECL-PLUS system (Amersham). Signals were detected using ories. Each Western blotting analysis was repeated at least three times.

[0104] Chromatin immunoprecipitation (ChIP) analysis The ChIP assay was performed using the EZ-CHIP® chromatin immunoprecipitation kit (Millipore Sigma, Darmstadt, Germany). B cells were activated with LPS in or without IL-27, and the DNA-protein complexes were crosslinked for 10 minutes by adding fresh formaldehyde (Sigma-Aldrich) to the medium at a final concentration of 1%, followed by quenching with 135 mM glycine. The cells were then washed with cold PBS (2×) and lysed (EZ-CHIP® lysis buffer). They were sonicated (5×) in a 15-second burst (Sonic Dismembrator Model 1000, Thermo Fisher Scientific output 5). The lysates were then clarified with protein G-agarose for 1 hour, pelletized, and incubated overnight with control IgG or anti-STAT1 or STAT3 antibody (Cell Signaling Technology). Before incubation with antibodies, 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®). Immunoprecipitation and the input DNA were subjected to PCR and qPCR using primers to detect STAT1 and STAT3 binding activity. The primers for the IL-27p28 gene promoter (5'-CTGAAACCCCAGCTTCCTGCCA-3' (SEQ ID NO: 7) and 5'-CATCTCCTGGGTAGGGGGGTCTTATACT-3' (SEQ ID NO: 8)) were -134 to -303, and the STAT binding motif GGAAGGGAAA 77 This is ACGTT (SEQ ID NO: 9). The primers for the promoter region of the 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 a well-known procedure (see Yu et al., J.Immunol., 157:126-137 (1996)). A double-stranded oligonucleotide containing a motif from the AP1-IRF-1 complex element (AICE) 5'TGAnTCA / GAAA-3' (SEQ ID NO: 13) was injected using Klenow polymerase (New England). [α-P] is obtained by a fill-in reaction using BioLabs (Beverly, Massachusetts). 32 Labeled with dATP or (alpha-32P)dGTP (3000 Ci / mmol) (Perkin E labeled; Immer Inc., Waltham, Massachusetts). Selected CD19 + B cells treated with IL-27 (20 μg / ml) Stimulate with LPS (1 μg / ml) for 3 days in or without the presence of [the substance], using a known procedure (Yu See et al., J.Immunol., 157:126-137 (1996). Nuclear extracts were prepared in buffers 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-carboxyoxiran-2-carbonyl)-1-leupeptin]agmatine. Protein levels were measured by the recommended BCA method, and extracts were stored at -70°C until use. DNA-protein binding reactions were performed using 5 μg of nucleoprotein and 1 μg of double-stranded poly(dLC)(Bo). The test was performed in a 20 μl mixture containing 12 mM HEPES (pH 7.9), 60 mM KCl, 0.5 mM DTT, 12% glycerol, and 2.5 mM MgCl (Ehringer Mannheim, Barcelona, ​​Spain). After incubation on ice for 15 minutes, the sample was divided into 1 μl P solutions. 32 The sample was further incubated with a labeled probe (15,000 cpm) at room temperature for 20 minutes and fractionated on a 5% native polyacrylamide gel in 0.25x Tris-borate-EDTA buffer. Supershift analysis revealed that 32 Before adding the P-labeled probe, the extracts were pre-incubated with 1 μl of antibody specific to basic leucine zipper transcription factor (BATF) (Cell Signaling Technology), Jun B, Jun D, ​​IRF-4, IRF-8, or IRF-1 (Santa Cruz Biotechnology).

[0106] Proximity ligation assay Proximity ligation assay (PLA) was performed using the Duolink PLA kit (Sigma Aldrich, St. Louis, Missouri). Activated B cells were attached to slides and blocked with blocking solution for 1 hour, then incubated overnight with mouse anti-p28 (rabbit) and anti-Ebi3 (mouse) primary antibodies. Next, a pair of oligonucleotide-labeled secondary antibodies (PLA probes) bound to the primary antibodies were added and incubated for 1 hour, after which a ligation solution containing the connector oligos to hybridize was added. Proximity (within 40 nm) PLA probes then interacted and ligated to the connector oligos. The resulting closed circular DNA template was amplified by DNA polymerase. Then, complementary detection oligos bound to fluorescent dyes hybridized to the amplicon and p28:Ebi3 heterodimer repeat sequences were detected as distinct fluorescent spots by confocal microscopy (LSM 700, Carl Zeiss AG, Oberkochen, Germany).

[0107] RNA-Seq and analysis For RNA-Seq, mRNA was separated by oligo-dT beads, and the library followed the standard Illumina, Inc. library protocol (Kit RS-122-2101 TruSeq Stranded mRNA LT Sample prep kit, Illumina, Inc., San Diego, CA). The library was sequenced on a NovaSeq 6000 system (Illumina, Inc.). Relative gene abundance was measured by Read Count using StringTie. Statistical analysis was performed to identify differentially expressed genes using estimates of the abundance of each gene in the sample. Zeroed Read Count was used in the sample. Genes with one more count than their actual count were excluded. To facilitate log2 transformation, 1 was added to each Read Count value of the filtered genes. The filtered data was log2 transformed and subjected to trimmed mean (TMM) normalization of the M-values. The statistical significance of differential expression data was determined using an exact t-test with edgeR and multiplier change, which is the null hypothesis that there is no difference between groups. P-values ​​were adjusted for multiple testing using Benjamini and Hochberg false detection rate (FDR) correction. For heatmaps, counts were normalized using the R package Heatmap and BroadInstitute tool Morpheus. |Multiplier Change|> Hierarchical clustering analysis was performed using full linkage and Euclidean distance as measures of similarity to display the expression patterns of differentially expressed transcripts satisfying raw p-values ​​< 0.05 for 2 and independent t-tests.

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

[0109] Sample size is indicated in the figure or legend and refers to the number of animals. In vitro assays using human umbilical cord blood or PBMCs were independently replicated using cells from at least three unrelated donors. The results shown represent at least three independent experiments, as noted in the legend. Optical coherence tomography, ERG, and confocal imaging analyses were performed blindly. EAE and EAU scoring were performed by masked researchers. The intrinsic immunotherapeutic effect of i27-Breg in the EAU was validated and replicated in the EAE model. Mice were age / sex matched, randomized, and consisted of an equal number of males and females.

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

[0111] Immunohistochemical / confocal microscopy was performed on activated mouse CD19 +Co-localization of p28 and Ebi3 expression on B cells (Figure 1, white arrows) demonstrates that B lymphocytes produce IL-27. B1 lymphocytes (B-1a and B-lb) are innate immune B cells 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 from the 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 primary producers of IL-27 (Figure 2A), and IL-27 production by B-1a cells was confirmed by ELISA (Figure 2D). Mutual IP / Western analysis detected co-expression of p28 and Ebi3 in the lysates and supernatants of activated B-1a cells, providing further evidence that B-1a cells actually secrete heterodimeric IL-27 (p28 / Ebi3). PLA further demonstrated physical interactions between p28 and Ebi3 (Figure 132), providing direct evidence that B cells secrete heterodimeric IL-27 cytokines. Mutual 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 times) (Figure 3). Exposure to L-27 suggested that it could induce an increase in 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 and IL-27Rα, altering the pattern of chemokine receptor expression (Figure 5). Immunohistochemical / confocal microscopy analysis also detected upregulated expression of IL-27 by B cells in response to IL-27 / BCR signaling (white arrows) (Figure 6), suggesting that BCR and IL-27 signaling may be necessary for the optimal increase of i27-Bregs. Furthermore, chromatin immunoprecipitation assays demonstrated that IL-27 mediates its effect by inducing the binding of activated STAT1 and STAT3 to the proximal promoter of U27a (Figures 112 and 113). BCR / IL-27 induction signaling promoted the growth of IL-27-producing cells, but BCR / IL-27 induction signaling could not increase the growth of B cells lacking the IL-27 receptor (IL-27RαKO) in culture (Figure 7), clearly indicating that IL-27 signaling is necessary for the generation of IL-27-producing B cells. Consistent with the IL-27 requirement for 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, it was found that innate-like human B1 cells also produce IL-27 (Figures 9-11).

[0113] To investigate whether B cells can produce IL-27 in vivo, C57BL / 6J mice were injected with LPS (iv), and the percentage of IL-27-producing B-1a or B2 cells in the peritoneal cavity or spleen was measured. Approximately 19.4% of B-1a cells in the peritoneal cavity of PBS-treated mice were producing IL-27 at 24 hours, but this percentage increased to approximately 55.6% in LPS-injected mice (Figure 13A-14B). The rapid dynamics of this response indicate recruitment rather than proliferation. Interestingly, the percentage of IL-27-secreting B-1a cells in the peritoneal cavity rapidly decreased over time, eventually returning to basal levels by day 4 of inflammation (Figure 13A-14B). Similar analyses revealed different patterns of recruitment of IL-27-producing B-1a cells to the spleen. From day 1 after LPS injection, the percentage of B-1a cells recruited to the spleen gradually increased from 2.01%, peaking at 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 in the spleen or peritoneal cavity never exceeded 2% (Figures 13B and 14B). These results suggest that LPS injection induced a rapid increase in IL-27-producing B-1a cells, followed by their departure from the peritoneal cavity, and that these events transiently correlated with the subsequent recruitment of B-1a cells to the spleen.

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

[0115] In summary, these observations suggest that different regulation of chemokine receptor expression by B-1a cells in response to IL-27 promotes the release of B-1a cells from the peritoneal cavity and subsequent transport to the spleen.

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

[0117] EAU is an animal model of human uveitis and is associated with CFA retinal proteins / peptides. This is an intraocular inflammatory disease primarily mediated by T cells, induced by immunization. Using an EAU model, we investigated whether i27-Breg contributes to immunomodulation during uveitis. EAU is a photoreceptor-retinoid-binding protein (IRBP). 651-670 Immunization with peptides derived from IL-27 was induced in C57BL / 6J mice, and mice were treated with PBS (control) or IL-27 simultaneously with immunization. Fundus images of PBS-treated mice revealed features of uveitis, including blurring of the optic nerve head margin, enlargement of the perioptic area, moderate to severe retinal vasculitis, and cellular infiltration (Figure 18). In contrast, IL-27-treated mice were protected from EAU, exhibiting mild EAU with fewer cells and lower disease scores (Figure 19). Histological analysis of PBS-treated eyes showed vitreous, choroidal, photoreceptor cell damage, and inflammatory cells in the retinal folds, but 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 humor and optic disc in PBS-treated mice, but no inflammatory cells in IL-27-treated mice (Figure 21). Electroretinography (ERG) of IL-27-treated mice did not detect visual impairment in control mice (Figures 22-25). Consistent with improved EAU, increased IL-27 and decreased IL-17 were detected in the serum of IL-27-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 spleen of PBS-treated mice secreted IL-27, but the percentage of i27-Breg increased to over 15% in IL-27-treated mice (Figures 30 and 31), indicating a correlation between increased i27-Breg and improved EAU. B10 (CD 19+ CD5 + CD1d hi ) and B-1a (CD19 + CD5 + CD1d low Since the cells are CD5+ and exhibit innate immunity-like Breg function, we investigated whether the i27-Bregs induced in EAU originated from the B-1a or B10 pool. The spleens of PBS-treated mice contained moderate levels of IL-27-producing B10 cells (approximately 2.97%), but the proportion of these i27-Bregs did not increase in EAU in IL-27-treated mice (Figure 32-34). In contrast, more than 6% of B-1a cells in the spleens of PBS-treated mice were i27-Bregs, and this increased to more than 11% in IL-27-treated mice (Figure 32-34), indicating that in vivo exposure to IL-27 further induced an increase in i27-producing B-1a cells in EAU.

[0118] To investigate the potential therapeutic importance of i27-Bregs, peritoneal B-1a cells (>80% i27-Bregs) were used in WT donor CD45.2 + Purified from mice using EAU, 5 × 10 5 Individual / mouse naive same-type WT or IL-27RaKOCD45.1 + The cells were transplanted into mice, and EAU was induced 24 hours after prophylactic administration of B-1a cells. Fundus images at 17 days post-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 exhibited uveitis characteristics, albeit with less severe symptoms compared to IL-27Rα KO mice. In contrast, mice given prophylactic B-1a cells developed only mild EAU (Figures 35 and 36), correlated with a decrease in Thl / Thl7 cells (Figure 37) and an increase in IL-27-producing B-1a cells in the accompanying eye (approximately 10.7%) (Figure 39). This improvement was not observed in IL-27Rα recipients, demonstrating that the improvement was mediated by IL-27. Interestingly, B-1a therapy induced approximately 2.2 times proliferation of IL-35-producing Breg cells (i35-Breg) (Figure 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 with plasmacytoid dendritic cells (CD11b secreting IL-27). + Refer to Figures 149A-149B, which show the percentage of B-1a cells.

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

[0121] These studies used an EAE model that shares essential immunopathogenic characteristics with multiple sclerosis (MS) and exhibits a progressive and relapsing-remitting human disease pattern. EAE is a MOG model. 35-55-Immunization of C57BL / 6J mice with peptide / CFA was induced. Control PBS-treated mice developed EAE, characterized by inflammatory cell infiltration into the brain and spinal cord, flaccid tail, paraplegia, forelimb / hindlimb paralysis, and a near-fatal state (Figure 44). However, these prominent features of EAE were significantly reduced in IL-27-treated mice, as indicated by histology and lower EAE clinical scores (Figure 45). Disease attenuation was attributed to a significant decrease in the frequency of Thl7 or IFN-γ / IL-17 expressing Thl7 cells in the brain and spinal cord of IL-27-treated mice, and IL-10 expressing CD4 + This correlated with an increase in T cells (Figures 46-51). More importantly, i27-Breg cells were detected 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 significant levels of IL-27-producing B-1a cells in the spleen of IL-27-treated mice (Figures 54-58).

[0122] The role of i27-Breg cells in suppressing EAEs is CD45.1 + and CD45.2 + This was further demonstrated in adoption and transplantation studies using congenic mouse lineages. CD45.2 + Mouse, MOG 35-55 - Immunized with peptide / CFA and treated with PBS or IL-27. Encephalitis-induced cells were collected from the spleen and LN 21 days after immunization and treated with PBS or IL-27. CD45.2 + 10x10 from the mouse 6 Individual cells, unimmunized CD45.1 + Cells were transplanted into mice as adoptive cells, and the onset and severity of EAE were evaluated. Transplantation of cells from PBS-treated mice induced disease with EAE characteristics, while transplantation from IL-27-treated mice produced CD45.2 + CD45.1 cells administered +Mice developed mild, late-onset EAE (Figure 59). The reduction of EAE in recipient mice was partially due to suppression of the Th17 response (Figure 60) and co-proliferation of IL-27-producing B-1a cells (Figures 63-66). CD45.2 + It is noteworthy that the levels of IL-27-producing B-1a cells increased slightly in the spinal cord, brain, and spleen of recipient IL-27-treated mice (Figures 63-66), which indicates that the transplanted CD452 + i27-Bregs cells may have proliferated in vivo. Most notably, the recruitment of CD45.2 + Bregs into the CNS promoted the expansion of endogenous CD45.1 + Bregs (Figures 63-66). Thus, the expansion of transplanted i27-Bregs and endogenous CD45.1 + Bregs in the spinal cord and brain would have maintained long-term production of IL-27 in host tissues. Thus, i27-Breg therapy may offer therapeutic advantages 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 mainly antigen-specific and effective in suppressing diseases mediated by lymphocytes that recognize the same cognate self-antigen. Thus, we investigated whether IL-27-producing B-1a cells induced by unrelated stimuli such as LPS could suppress encephalitogenic lymphocytes that mediate EAE. CD4^5.2 + 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). Next, i27-Bregs were transferred into naive CD45.1 + congenic mice. EAE was induced by immunizing with MOG 35-55 (n = 7) 24 hours after adoptive transfer, in recipient CD45.1 +Induced in mice. Generated ex vivo Transplantation of generated B-1a i27-Bregs (5×10 5 cells / mouse) suppressed EAE (Figure 67), and disease improvement correlated with a decrease in IL-17-only positive and IL-17 / IFN-γ double-positive T cells in the brain and spinal cord, and an increase in IL-10-producing regulatory CD4 + T cells (Figures 68-71). 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), and most i27-Breg cells were observed to be B-1a cells. Similar results were obtained in the EAU model. Thus, consistent with their ontogenetic origin, suppression of CNS autoimmune diseases by innate immune i27-Breg cells does not require prior activation by the autoantigens that induced EAE or EAU. This result is in contrast to B2 Breg therapy, which mediates antigen-specific immunosuppression, suggesting that transplantation of autologous innate immune i27-Breg cells can be utilized for the treatment of a broader range of autoimmune diseases.

[0125] Example 5 This example shows that there is crosstalk between B-1a producing IL-27 and lymphocytes or bone marrow cells in the CNS.

[0126] This study investigated whether i27-Breg cells, which enter the CNS during EAE or EAU, may be a source of IL-27 contributing to the immunosuppressive environment of the CNS. B-1a cells and macrophages derived from IRBP-immunized wild-type cells were sorted, and 3 days of co-culture of these cells in a Transwell system was found to significantly increase IL-27-producing B-1a cells (Figures 78-81), suggesting that soluble mediators produced by myeloid cells may increase retinal IL-27 levels during uveitis by promoting the proliferation of i27-Breg cells. The data further demonstrate that macrophages, like B-1a cells, respond to inflammatory stimuli by producing IL-27 (Figures 82 and 83). However, infection of any cell type with lentiviruses 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 synergistically interact with myeloid cells to elevate CNS IL-27 levels during inflammation. Potential crosstalk between i27-Bregs and lymphocytes mediating CNS autoimmune diseases was also investigated. Co-culture with B-1a cells suppressed the proliferation of uveal T cells in the spleen and lymph nodes of EAU mice (Figures 84-90). When B-1a cells were deficient 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 in EAU through the paracrine effect of secreted IL-27.

[0127] The data shows that co-culture of B-1a cells and uveal-forming T cells inhibits LAG-3 (LAG-3) in an IL-27-dependent manner. + CD4 + CD4 expressing T cells +This shows that it induced proliferation of T cells (Figures 91-93) and i35-Bregs (Figures 94-96). Interestingly, the majority of 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 partially, by enabling effector T cells to acquire regulatory phenotypes and functions.

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

[0129] This study investigates whether B-1a cells, which suppress EAU and EAE through IL-27 production, also suppress inflammation by expressing inhibitory molecules. B-1a cells are, Cells were isolated from the mouse peritoneal cavity by sorting. Next, the cells were stimulated with LPS for 48 hours, and their ability to produce IgM demonstrated they were B-1a cells (Figure 100). Analysis of cDNA prepared from the cells by qPCR revealed that B-1a cells are indeed capable of expressing 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 challenges, as occurs during EAE, EAU, or sepsis. C57BL / 6J mice were injected with LPS (iv), and purified B-1a cells were isolated from the peritoneal cavity by magnetic bead sorting. qPCR analysis of cDNA derived from cells 48 hours after LPS administration confirmed that Lag3 and Pdl transcription is upregulated by intraperitoneal B-1a cells (Figure 101). Natural LAG-3 + CD138 +As regulatory plasma cells develop via antigen-specific mechanisms, B-1a and B2 cells sorted from mouse peritoneal cavity or spleen, and cells stimulated with anti-IgM / anti-CD40, showed that both B-1a and plasma cells upregulated Lag3 and Pd1 transcription in response to BCR signaling, as indicated by qPCR analysis (Figures 102-104). In summary, these observations suggest that B-1a cells can acquire the ability to express inhibitory molecules that enhance immunomodulatory activity in response to stimulation by pathogens (such as TLR agonists) or autoantigens.

[0130] Previous reports indicated that IL-35-producing B cells are primarily B2 CD138. + It had been shown that these were 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 by which activated B cells are biased towards the i27-Breg developmental program, we investigated IL-27-stimulated activated CD19 +We created transcriptome profiles of B cells. 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 important B cell pathways. Of particular interest were the different upregulations of IRF-8 and IRF-4, as these transcription factors are involved in B cell development and effector function. Considering reports that the mutual antagonism between IRF-4 and IRF-8 regulates B-, and that cell development accompanied by increased IL-4 favorable to plasma cell development is increased (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. Since IL-27 induces the proliferation of i27-Breg, we investigated whether IRF-8 activates the transcription of 1127a, which encodes the IL-27p28 subunit protein. Therefore, it should be noted that IRF-8 and IRF-4 activate transcription by heterodimerization with ETS / PU-1 or BATF family transcription factors and are recruited to the complex elements of the ETS-IRF(EICE) or AP1-IRF(AICE) immunomodulatory genes. EMSA and supershift analyses using validated AICE sites associated with the expression of Il27a or Ctla4 show that IL-27 induces the formation of the AICE complex 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 it was IRF-8, not IRF-4, that was recruited to the AICE of i / 27a. This suggests that IRF-8 promotes IL-27 expression 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 LPS-injected mice (Figure 108), suggesting an IRF-8 / IL-27 axis that may modulate a reciprocal autoregulatory loop promoting IRF-8 and IL-27 expression in B-1a cells. Significant reduction in IL-27-producing B-1a cells in CD19-IRF8KO mice (Figure 108-). 111) was also observed, which further highlights 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 towards the i27-Breg development program.

[0131] Example 7 This example demonstrates 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 of human B-1 cells (CD19 + CD20 + CD27 + CD43 + ) revealed that 19.9% ​​of BCR-activated B cells in human PBMCs produce IL-27 (Figures 114 and 115). CD19 + CD20 + CD27 + CD43 + CD11 +A cell population representing a subset of B-1a cells ready to migrate to the spleen and other antibody-producing sites in response to appropriate stimulation and gating revealed that as many as 35% of BCR-activated human B-1a cells could be recruited to the spleen and inflammatory sites during inflammatory diseases (Figures 116-118). Analysis of human umbilical cord blood from healthy human donors revealed that 18.1% of resting B-1a cells constitutively produce IL-27, and stimulating BCR-activated umbilical cord blood B cells with IL-27 increased the proportion of i27- in umbilical cord blood to 73.9% (Figures 119-121). To determine the relative abundance of i27-Bregs compared to other Breg subtypes (IL-10-producing Bregs and i35-Bregs), activated umbilical cord blood cells were grown for 6 days. The majority of Breg cells were i27-Breg, but low levels of IL-10-producing Breg and i35-Breg were detected, and their levels increased in a time-dependent manner (Figure 122). Similar analysis of B-2 cells revealed that most i27-Breg cells were either naive or in the 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 produce pro-inflammatory CD4, which produces TNF-α, IL-17, and / or IFN-γ. + The T cell proliferation response was suppressed (Figures 127-131). Since umbilical cord blood is a favorable source of hematopoietic stem cells for allotransplantation in patients with significant mismatches in human leukocyte antigens (HLA; gene complexes encoding major human histocompatibility complex (MHC) proteins), enrichment of umbilical cord blood i27-Breg is clinically interesting. Therefore, umbilical cord blood i27-Breg can be utilized to suppress the allogeneic response after allogeneic hematopoiesis and to protect against GVHD.

[0133] Example 8 This embodiment demonstrates that human i27-Breg cells can be used to successfully treat individuals who are suffering from or at risk of developing the disease.

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

[0135] Example 9 This embodiment demonstrates that i27-Breg has its own transcriptome.

[0136] Using peritoneal B-1a cells enriched with i27-Breg cells through activation with BCR and IL-27, we determined 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 B-1a cells constitutively secrete native IgM antibodies, while the development of the i27-Breg cell phenotype is consistent 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 spleen were used as comparators for RNA-seq analysis. Differentially regulated principal component analysis (PCA) 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 obtained from global RNA-Seq analysis identified 1,998 genes upregulated and 1,179 genes downregulated in i27-Breg (Figure 137). Genes differentially induced by i27-Breg (>2-fold higher expression) include genes encoding cytokines, cytokine receptors 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). Repressed genes include genes encoding 1112a, Notch2, Cxcr4, Ccr2, Ccr7), repressive receptors (Pdcd2, Cdldl, Ctla4), and transcription factors (Irf4, Ikzf3, Bach2, Pax5, Ebfl, Runxl, Foxol, Etsl) (Figure 139).To further verify that IL-27 is required for the maintenance of the i27-Breg transcriptome, IL-27-deficient B-1a cells express IL-35 (p35 and EBi3), but show defects in the expression of inhibitory receptor genes (Lag3, Pdl, as well as Pd-11, Pd-12) (Figure 140). Taken together, these results suggest that the i27-Breg transcriptome shows a significant increase in genes (Bhlhe40, Arid3a, and Cd5) necessary for B-1a development, highlighting the origin of i27-Breg development from innate immune B-1 cells. However, i27-Breg cells show the characteristic properties of transcription of differentiating germinal center B cells (Irf8↑, Batf↑, Pax5↑, Bach2↑, Ebfl↑), but do not show the characteristic properties of transcription of terminally differentiated plasma cells (Prdml↑, Bach2↑, Pax5↑, Ebfl↑). This proves that i27-Breg has its own transcriptome.

[0137] Example 10 This example proves that i27-Breg and i35-Breg of human umbilical cord blood and PBMC have different transcriptome profiles. <°

[0138] Human PBMC and umbilical cord blood (CB) B cells produce IL-27, and in PBMC, approximately 19.9% of activated B-1-like cells (CD19 + CD20 + CD27 + CD43 + ) are i27-bregs (Figures 141A and 141B). More than 40% of i27-Breg cells are CD19 + CD20 + CD27 + CD43 + CD11b +The phenotype shows an intracavitary B-1a subset known to redistribute to local lymph nodes in response to inflammation (Figure 142A-142C). On the other hand, approximately 18.1% of resting B-1a cells in CB constitutively secrete IL-27, and when activated in the presence of IL-27, the percentage of CB i27-Breg dramatically increases to 73.9% (Figure 143A-143C), suggesting that i27-Breg functions as the native Breg of human CB and is ready to be rapidly recruited to local lymph nodes in response to inflammation. t-SNE clustering analysis revealed three distinct spatially separated subsets of Breg cells within CB: B10, i27-Breg, and We grouped the cells into i27-Breg and i35-Breg. i27-Breg was the most abundant, containing over 85% Breg cells in culture on day 3, but decreased to less than 61% in culture on day 6 (Figure 144). B10 and i35-Breg cells were relatively sparse in culture on day 3, but i35-Breg increased substantially (32%) by day 6 (Figure 144). Interestingly, B cells at all stages of development were able to produce IL-10, IL-27, or IL-35, but i27-Breg was most abundant in immature and memory B cells (Figure 145). Principal component analysis and RNA-seq analysis revealed that i27-Breg and i35-Breg have different transcriptome profiles (Figure 146). Of the 3,744 differentially expressed genes, 1,575 were upregulated and 2,169 were downregulated in i27-Breg (Figure 147). CD19 + Similar comparisons between B cells and i27-Breg cells revealed that 3,207 of the 6,159 differentially expressed genes were upregulated by i27-Breg cells (Figure 148). Therefore, the results of human PBMC or CB analysis suggest that different Breg subsets are induced during the inflammatory response, and the relative abundance of each subset varies depending on the nature of the inflammatory challenge.

[0139] Example 11 This example demonstrates that i27-Bregs of innate immunity suppress CNS autoimmune diseases through a mechanism independent of BCR.

[0140] Intraperitoneal B-1 cells mostly do not respond to signals induced by BCR, but respond strongly to innate immune signals induced by pathogens or TLR agonists, suggesting the immunosuppressive activity of i27-Bregs. To clarify whether prior activation by IRBP or MOG autoantigens is required for the suppression of EAU or EAE via i27-Bregs, LPS injection was used to induce "sepsis" in CD45.2 + C57BL / 6J mice, and sorted B-1a cells (>83.5% i27-Bregs) derived from the peritoneal cavity and cells enriched with i27-Bregs (5×10 5 cells / mouse) were transplanted into naive CD45.1 + congenic mice. Twenty-four hours later, the mice were challenged by EAE induction. Clinical evaluation of the mice revealed significant suppression of EAE (Figure 150) or EAU compared to control mice administered an equal number of B-1a cells (<7% i27-Breg). The improvement of the disease was correlated with a decrease in IL-17 single-positive and IL-17 / IFN-γ double-positive Th17 cells, as well as an increase in Tregs in the brain and spinal cord (Figure 151A-151B), an increase in B-1a i27Breg cells in the spinal cord (Figure 152A-152B), brain (Figure 153A-153B) and peritoneal cavity (Figure 154A-154B). These results support the possibility that adoptive transfer i27-Breg therapy may be useful for the treatment of autoimmune diseases.

[0141] In summary, the above examples show that the natural immune IL-27-producing Breg population exists not only in the brains, spinal cords, retinas, and abdominal cavities of mice suffering from experimental autoimmune encephalomyelitis (EAE) or experimental autoimmune uveitis (EAU), which are models of multiple sclerosis and uveitis, respectively, but also in human umbilical cord blood and PBMC. In vitro experimental systems, including confocal microscopy, FACS-based cell sorting, RNA-seq, Chip assays, and immunohistochemistry, indicate that Bregs producing IL-27 have a unique transcriptome and are functionally different from other Bregs. The adoptive transfer of i27-Bregs was transported to the uvea, brain, and spinal cord, and by reprogramming resting B cells into i35-Breg cells that suppressed pathogenic T cells, it improved EAE and EAU, demonstrating the efficacy of i27-Breg immunotherapy.

[0142] All documents, including publications, patent applications, and patents cited herein, are incorporated herein by reference as if each document were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. to the same extent as if it were set forth in its entirety herein.

[0143] The use of the terms “a,” “an,” and “the” and similar references in the context describing the present invention (particularly in the context of the following claims) should be interpreted as covering both singular and plural forms. Otherwise, it would be clearly inconsistent with what is shown herein or by context. The terms “include,” “have,” “include,” and “contain” should be interpreted as unrestrictive terms (i.e., “include, but not limited to”) unless otherwise specified herein. The enumeration of numerical ranges herein is merely intended to serve as abbreviations for individually referring to each individual numerical within the range unless otherwise specified herein, and each individual numerical is incorporated into the specification as if it were individually specified herein. All methods described herein may be performed in any suitable order unless otherwise specified herein or by context. Any embodiments or use of exemplary language (e.g., “etc.”) provided herein is merely intended to better illustrate the present invention and, unless otherwise requested, does not limit the scope of the present invention. Nothing in the specification should be construed as indicating that any component not described in the claims is essential for carrying out the invention.

[0144] Preferred embodiments of the present invention are described herein, including the best modes known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will appropriately use such variations, and the inventors intend to carry out the invention in ways other than those specifically described herein. Thus, the invention includes all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is included in the invention unless otherwise indicated herein or unless it is clearly inconsistent with the context.

Claims

1. A pharmaceutical composition for use in suppressing the immune system of a mammal, The aforementioned mammal has an autoimmune disease selected from the group consisting of insulin-dependent diabetes mellitus, systemic lupus erythematosus (SLE), psoriasis, autoimmune hepatitis, thyroiditis, pancreatitis, orchitis, myasthenia gravis, idiopathic thrombocytopenic purpura, inflammatory bowel disease, rheumatoid arthritis (RA), scleroderma, and juvenile arthritis. The pharmaceutical composition comprises an isolated population of mammalian cells containing approximately 75% or more B-1a regulatory cells. The aforementioned regulatory cells, (a) Expressing inhibitory cell surface receptor lymphocyte activation gene 3 (LAG-3), programmed cell death protein 1 (PD-1), and C-X-C chemokine receptor type 4 (CXCR4), (b) Produces interleukin-27 (IL-27), Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the regulatory cells further express the inhibitory cell surface receptor glucocorticoid-inducible TNFR-related protein (GITR).

3. The pharmaceutical composition according to claim 1 or 2, wherein the regulatory cells further express the inhibitory cell surface receptor OX40.

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

5. The pharmaceutical composition according to any one of claims 1 to 4, further comprising administering interleukin-35 (IL-35)-producing B cells sequentially or simultaneously to the mammal.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is inflammatory bowel disease.

7. The pharmaceutical composition according to claim 6, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

8. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is insulin-dependent diabetes mellitus.

9. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is systemic erythematous lupus (SLE).

10. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is psoriasis.

11. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is autoimmune hepatitis.

12. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is thyroiditis.

13. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is islet inflammatory disease.

14. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is orchitis.

15. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is myasthenia gravis.

16. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is idiopathic thrombocytopenic purpura.

17. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is rheumatoid arthritis (RA) or juvenile arthritis.

18. The pharmaceutical composition according to any one of claims 1 to 5, wherein the autoimmune disease is scleroderma.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the population of mammalian cells suppresses inflammation.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the mammal is a human.

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