Modified CD4+ T cells expressing IL-37 and methods of use thereof

Abstract

Disclosed herein are methods for generating populations of engineered CD4+ T cells that express nuclear IL-37. Also disclosed are methods of using engineered CD4+ T cells that express nuclear IL-37 for the treatment of immune or inflammatory diseases, disorders, or conditions.

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 390,259, filed July 18, 2022, the entire contents of which are incorporated herein by reference.

[0002] (Incorporated by reference to the sequence listing) The Sequence Listing XML associated with this application has been provided electronically in XML file format and is incorporated herein by reference in its entirety. The XML file containing the Sequence Listing XML is named "UNCO-047_001WO_SeqList_ST26.xml." The XML file was created on July 17, 2023 and is 178,344 bytes in size.

[0003] (Statement Regarding Federally Sponsored Research) This invention was made with government support under 1R01AI156534-01A1 awarded by the National Institute of Health and 5I01BX001228-10 awarded by the USDapartment of Veterans Affairs. The government has certain rights in this invention.

[0004] FIELD OF THE INVENTION The present disclosure is directed to modified CD4+ T cell compositions (i.e., engineered CD4+ T cells) and methods of making and using same for adoptive therapy and treatment of immune or inflammatory diseases, disorders, or conditions. [Background technology]

[0005] Immune tolerance is essential for preventing immune dysregulation, autoimmunity, and immune rejection. Among the cell populations known to be involved in immune tolerance are regulatory T (Treg) cells. Treg cells play crucial roles in suppressing immune responses, inducing tolerance, and maintaining homeostasis. Active suppression by Treg cells plays a key role in downregulating T cell responses to foreign and self-antigens. Because FOXP3+ Treg cells play an essential role in regulating immune responses and maintaining peripheral self-tolerance, these cells have the potential to be used as a tool for treating autoimmunity and preventing transplant rejection. Expression of the transcription factor forkhead box P3 (FOXP3) is important for Treg suppressive function. Continuous expression of FOXP3 stabilizes Treg cell populations and preserves tolerance, while loss of FOXP3 leads to Treg cell dysfunction. Loss of FOXP3 expression can lead to immune imbalance, tissue damage, and failure of autoimmune and transplant treatments.

[0006] FOXP3 expression in human Treg cells is dependent on the anti-inflammatory cytokine interleukin-37 (IL-37). Human Treg cells express the highest IL-37 mRNA levels among blood cells isolated from healthy human individuals. However, the instability of Treg cells poses a problem for current immunotherapies using adoptive transfer of Treg cells. After adoptive transfer, Treg cells can convert into inflammatory T cells, potentially exacerbating disease. Under inflammatory or pathogenic environments, some Treg cells lose FOXP3 expression and / or their suppressive function. Therefore, there is a long-standing unmet need in the art for improved methods for generating and maintaining stable immunosuppressive FOXP3+ Treg cells for improved immunotherapy.

[0007] Disclosed herein are methods for generating a population of modified CD4+ T cells that express IL-37, an anti-inflammatory cytokine important for maintaining the immunosuppressive function of Treg cells and inducing a Treg-like phenotype in non-Treg CD4+ T cells. High expression of IL-37 in the population of modified Treg cells disclosed herein generates stable Treg cells. High expression of IL-37 in a population of disease-associated CD4+ T cells disclosed herein generates non-pathogenic T cells or potentially Treg cells. The present disclosure provides methods for generating a population of modified CD4+ T cells that express IL-37. The present disclosure also provides methods for treating immune-mediated conditions or disorders using the population of modified CD4+ T cells. Summary of the Invention

[0008] The present disclosure provides a method for generating a population of modified CD4+ T cells, the method comprising introducing into a plurality of human T cells a composition comprising interleukin-37 (IL-37) or a nucleic acid sequence encoding IL-37 under conditions suitable for expressing IL-37 in the nuclei of the human T cells, thereby generating a plurality of modified CD4+ T cells.

[0009] The present disclosure also provides compositions comprising a population of modified CD4+ T cells produced by the methods described herein.

[0010] The present disclosure also provides a method of treating an immune disease or disorder or an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a population of engineered CD4+ T cells that express nuclear IL-37.

[0011] In some embodiments, the engineered population of CD4+ T cells are regulatory T cells or effector T cells. In some embodiments, the CD4+ cells are non-regulatory T cells. In some embodiments, the cells are T conv cells. In some embodiments, the engineered population of CD4+ T cells are regulatory T cells.

[0012] In some embodiments, nuclear expression of IL-37 in the plurality of engineered CD4+ T cells is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold greater than nuclear expression of IL-37 in a population of wild-type human T cells. In some embodiments, nuclear expression of IL-37 in the plurality of engineered CD4+ T cells is at least about 5-fold greater than nuclear expression of IL-37 in a population of wild-type human T cells.

[0013] In some embodiments, the nuclear expression of IL-37 in the plurality of engineered CD4+ T cells is about 1-2 fold, about 1-3 fold, about 1-4 fold, about 1-5 fold, about 1-6 fold, about 1-7 fold, about 1-8 fold, about 1-9 fold, about 1-10 fold, about 2-3 fold, about 2-4 fold, about 2-5 fold, about 2-6 fold, about 2-7 fold, about 2-8 fold, about 2-9 fold, about 2-10 fold, about 3-4 fold, about 4-5 fold, about 4-6 fold, about 4-7 fold, about 4-8 fold, about 4-9 fold, about 2-10 fold, about 3-4 fold, about 4-5 fold, about 4-6 fold, about 4-7 fold, about 4-8 fold, about 4-9 fold, about 5-10 fold, about 5-4 fold, about 5-6 fold, about 5-7 fold, about 5-8 fold, about 5-9 fold, about 5-10 fold, about 5-4 fold, about 5-8 fold, about 5-10 fold, about 5-4 fold, about 5-10 ... 3-5 fold, about 3-6 fold, about 3-7 fold, about 3-8 fold, about 3-9 fold, about 3-10 fold, about 4-5 fold, about 4-6 fold, about 4-7 fold, about 4-8 fold, about 4-9 fold, about 4-10 fold, about 5-6 fold, about 5-7 fold, about 5-8 fold, about 5-9 fold, about 5-10 fold, about 6-7 fold, about 6-8 fold, about 6-9 fold, about 6-10 fold, about 7-8 fold, about 7-9 fold, about 7-10 fold, about 8-9 fold, about 8-10 fold, about 9-10 fold. In some embodiments, nuclear expression of IL-37 in the plurality of engineered CD4+ T cells is about 5-10 fold greater than nuclear expression of IL-37 in a population of wild-type human T cells.

[0014] In some embodiments, nuclear IL-37 expression in the engineered population of CD4+ T cells is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% greater than nuclear IL-37 expression in a population of wild-type CD4+ T cells. In some embodiments, nuclear IL-37 expression in the engineered population of CD4+ T cells is at least about 50% greater than nuclear IL-37 expression in a population of wild-type CD4+ T cells. In some embodiments, nuclear IL-37 expression in the engineered population of CD4+ T cells is at least about 85% greater than nuclear IL-37 expression in a population of wild-type CD4+ T cells.

[0015] In some embodiments, the expression of nuclear IL-37 in the population of engineered CD4+ T cells is about 10% to 20%, about 10% to 30%, about 10% to 40%, about 10% to 50%, about 10% to 60%, about 10% to 70%, about 10% to 80%, about 10% to 90%, about 10% to 100%, about 20% to 30%, about 20% to 40%, about 20% to 50%, about 20% to 60%, about 20% to 70%, about 20% to 80%, about 20% to 90%, about 20% to 100%, about 30% to 40%, about 30% to 50%, about 40% to 50%, about 50% to 6 ... Approximately 30% to 60%, approximately 30% to 70%, approximately 30% to 80%, approximately 30% to 90%, approximately 30% to 100%, approximately 40% to 50%, approximately 40% to 60%, approximately 40% to 70%, approximately 40% to 80%, approximately 40% to 90%, approximately 40% to 100%, approximately 50% to 60%, approximately 50% to 70%, approximately 50% to 80%, approximately 50% to 90%, approximately 50% to 100%, approximately 60% to 70%, approximately 60% to 80%, approximately 60% to 90%, approximately 60% to 100%, approximately 70% to 80%, approximately 70% to 90%, approximately 70% to 100%, approximately 80% to 90%, approximately 80% to 100%, approximately 90% to 100% larger. In some embodiments, nuclear IL-37 expression in the population of engineered CD4+ T cells is about 50% to about 80% greater than nuclear IL-37 expression in a population of wild-type CD4+ T cells.

[0016] In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the plurality of engineered CD4+ T cells express at least one marker for regulatory T cells. In some embodiments, at least about 75% of the plurality of engineered CD4+ T cells express at least one marker for regulatory T cells. In some embodiments, at least about 95% of the plurality of engineered CD4+ T cells express at least one marker for regulatory T cells.

[0017] In some embodiments, at least one marker of regulatory T cells is selected from the group consisting of FOXP3, CD25, CD4, CTLA4, IL-10, GITR, TGF-beta, and CD127. In some embodiments, the minimum one marker is FOXP3. In some embodiments, the at least one marker is FOXP3 and CD25.

[0018] In some embodiments, the population of modified CD4+ T cells are allogeneic CD4+ T cells. In some embodiments, the population of modified CD4+ T cells are autologous CD4+ T cells.

[0019] In some embodiments, the immune disease or disorder is treated, the immune disease or disorder being selected from the group consisting of allergic contact hypersensitivity, graft-versus-host disease, transplant rejection, type 1 diabetes, systemic lupus erythematosus, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and multiple sclerosis. In some embodiments, the immune disease or disorder is allergic contact hypersensitivity. In some embodiments, the immune disease or disorder is graft-versus-host disease. In some embodiments, the immune disease or disorder is inflammatory bowel disease. In some embodiments, the immune disease or disorder is type 1 diabetes.

[0020] In some embodiments, treating an inflammatory disease or disorder selected from the group consisting of inflammatory diseases or disorders affecting the digestive system, joints, skin, respiratory system, and nervous system.

[0021] In some embodiments, the inflammatory disease or disorder is selected from the group consisting of psoriasis, traumatic brain injury, bronchitis, and pneumonitis. In some embodiments, the inflammatory disease or disorder is psoriasis. In some embodiments, the inflammatory disease or disorder is traumatic brain injury. In some embodiments, the inflammatory disease or disorder is bronchitis. In some embodiments, the inflammatory disease or disorder is pneumonitis. [Brief explanation of the drawings]

[0022] [Figure 1A]Figure 1A shows a series of graphs demonstrating that IL-37 is highly expressed in human T regulatory cells. Figure 1A shows a series of contour plots illustrating the flow cytometry gating strategy for analyzing cell surface protein and IL-37 mRNA expression in myeloid cells. [Figure 1B] Figure 1B shows a series of graphs demonstrating that IL-37 is highly expressed in human T regulatory cells. Figure 1B shows a series of contour plots illustrating the flow cytometry gating strategy for analyzing cell surface protein and IL-37 mRNA expression in lymphoid cells. [Figure 1C] Figure 1C shows a series of graphs demonstrating that IL-37 is highly expressed in human T regulatory cells. Figure 1C shows two pie charts showing the percentage of cell subsets among IL37-expressing cells from five healthy human donors. The left graph shows unstimulated cells. The right graph shows cells stimulated with 100 ng / ml LPS for 24 hours. Cell subsets and percentages are as indicated in the pie charts. mDC: bone marrow-derived dendritic cells, pDC: plasmacytoid dendritic cells, NK: natural killer cells. [Figure 1D] Figure 1D shows a series of graphs demonstrating that IL-37 is highly expressed in human T regulatory cells. Figure 1C shows graphs depicting the frequency of IL-37 mRNA-expressing cells in myeloid (left) and lymphoid (right) subsets in the absence (-) or presence (+) of LPS. Cell subsets are as indicated in the legend. mDC: bone marrow-derived dendritic cells, pDC: plasmacytoid dendritic cells, NK: natural killer cells. [Figure 1E] Figure 1E shows a series of graphs demonstrating that IL-37 is highly expressed in human T regulatory cells. Figure 1E shows a series of histograms showing IL-37 expression in myeloid (left) and lymphoid (right) cell subsets unstimulated (top) or treated with 100 ng / ml LPS for 24 hours (bottom). Cell subsets are as indicated in the legend. mDC: bone marrow-derived dendritic cells, pDC: plasmacytoid dendritic cells, NK: natural killer cells. [Figure 1F]Figure 1F shows a series of graphs demonstrating that IL-37 is highly expressed in human T regulatory cells. Figure 1F shows a graph depicting the mean fluorescent intensity (MFI) of IL37 mRNA expression in the unstimulated and LPS-treated cell subsets shown in Figure 1E. Cell subsets are as indicated in the legend. [Figure 2] A schematic diagram of the pLenti-IL37-C-Myc-DDK-P2A-Puro vector (IL37 OE) (Origene) transduced into Jurkat cells for expression of IL37 is shown. [Figure 3A]Figure 3 shows a series of graphs demonstrating that IL37 overexpression induces FOXP3 in Jurkat cells using the vector shown in Figure 2. Figure 3A shows a bar graph of IL37 mRNA expression in Jurkat cells transfected with an empty vector control or IL37 OE Jurkat cells (IL37 OE). GAPDH served as an internal control, and values represent IL37 gene expression as a ratio to GAPDH expression. Expression was measured using qRT-PCR. Figure 3B shows IL-37 and FOXP3 immunoblotting of empty vector control and IL37 OE Jurkat cells. Actin was used as an internal control. Gray bars represent IL37 OE Jurkat cells. Black bars represent vector control. Representative immunoblotting (left panel) and band quantification (right panel) of three immunoblot experiments. Protein band density was quantified using Image J, normalized to actin, and expressed as fold change compared to control Jurkat cells expressing an empty vector. Figure 3C shows a bar graph of FOXP3 mRNA expression in vector control and IL37OE Jurkat cells. GAPDH served as an internal control, and values represent FOXP3 gene expression as a ratio to GAPDH expression. Figures 3D-E show a histogram (Figure 3D) and a bar graph showing densitometric quantification (Figure 3E) of FOXP3 protein expression in vector control and IL37OE Jurkat cells. IgG was used for negative control staining (black-gray histogram). Expression is expressed as mean fluorescence intensity (MFI) in IL37OE Jurkat cells compared to the vector control. Small horizontal lines indicate the mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of three independent experiments. [Figure 3B]Figure 3 shows a series of graphs demonstrating that IL37 overexpression induces FOXP3 in Jurkat cells using the vector shown in Figure 2. Figure 3A shows a bar graph of IL37 mRNA expression in Jurkat cells transfected with an empty vector control or IL37 OE Jurkat cells (IL37 OE). GAPDH served as an internal control, and values represent IL37 gene expression as a ratio to GAPDH expression. Expression was measured using qRT-PCR. Figure 3B shows IL-37 and FOXP3 immunoblotting of empty vector control and IL37 OE Jurkat cells. Actin was used as an internal control. Gray bars represent IL37 OE Jurkat cells. Black bars represent vector control. Representative immunoblotting (left panel) and band quantification (right panel) of three immunoblot experiments. Protein band density was quantified using Image J, normalized to actin, and expressed as fold change compared to control Jurkat cells expressing an empty vector. Figure 3C shows a bar graph of FOXP3 mRNA expression in vector control and IL37OE Jurkat cells. GAPDH served as an internal control, and values represent FOXP3 gene expression as a ratio to GAPDH expression. Figures 3D-E show a histogram (Figure 3D) and a bar graph showing densitometric quantification (Figure 3E) of FOXP3 protein expression in vector control and IL37OE Jurkat cells. IgG was used for negative control staining (black-gray histogram). Expression is expressed as mean fluorescence intensity (MFI) in IL37OE Jurkat cells compared to the vector control. Small horizontal lines indicate the mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of three independent experiments. [Figure 3C]Figure 3 shows a series of graphs demonstrating that IL37 overexpression induces FOXP3 in Jurkat cells using the vector shown in Figure 2. Figure 3A shows a bar graph of IL37 mRNA expression in Jurkat cells transfected with an empty vector control or IL37 OE Jurkat cells (IL37 OE). GAPDH served as an internal control, and values represent IL37 gene expression as a ratio to GAPDH expression. Expression was measured using qRT-PCR. Figure 3B shows IL-37 and FOXP3 immunoblotting of empty vector control and IL37 OE Jurkat cells. Actin was used as an internal control. Gray bars represent IL37 OE Jurkat cells. Black bars represent vector control. Representative immunoblotting (left panel) and band quantification (right panel) of three immunoblot experiments. Protein band density was quantified using Image J, normalized to actin, and expressed as fold change compared to control Jurkat cells expressing an empty vector. Figure 3C shows a bar graph of FOXP3 mRNA expression in vector control and IL37OE Jurkat cells. GAPDH served as an internal control, and values represent FOXP3 gene expression as a ratio to GAPDH expression. Figures 3D-E show a histogram (Figure 3D) and a bar graph showing densitometric quantification (Figure 3E) of FOXP3 protein expression in vector control and IL37OE Jurkat cells. IgG was used for negative control staining (black-gray histogram). Expression is expressed as mean fluorescence intensity (MFI) in IL37OE Jurkat cells compared to the vector control. Small horizontal lines indicate the mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of three independent experiments. [Figure 3D]Figure 3 shows a series of graphs demonstrating that IL37 overexpression induces FOXP3 in Jurkat cells using the vector shown in Figure 2. Figure 3A shows a bar graph of IL37 mRNA expression in Jurkat cells transfected with an empty vector control or IL37 OE Jurkat cells (IL37 OE). GAPDH served as an internal control, and values represent IL37 gene expression as a ratio to GAPDH expression. Expression was measured using qRT-PCR. Figure 3B shows IL-37 and FOXP3 immunoblotting of empty vector control and IL37 OE Jurkat cells. Actin was used as an internal control. Gray bars represent IL37 OE Jurkat cells. Black bars represent vector control. Representative immunoblotting (left panel) and band quantification (right panel) of three immunoblot experiments. Protein band density was quantified using Image J, normalized to actin, and expressed as fold change compared to control Jurkat cells expressing an empty vector. Figure 3C shows a bar graph of FOXP3 mRNA expression in vector control and IL37OE Jurkat cells. GAPDH served as an internal control, and values represent FOXP3 gene expression as a ratio to GAPDH expression. Figures 3D-E show a histogram (Figure 3D) and a bar graph showing densitometric quantification (Figure 3E) of FOXP3 protein expression in vector control and IL37OE Jurkat cells. IgG was used for negative control staining (black-gray histogram). Expression is expressed as mean fluorescence intensity (MFI) in IL37OE Jurkat cells compared to the vector control. Small horizontal lines indicate the mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of three independent experiments. [Figure 3E]Figure 3 shows a series of graphs demonstrating that IL37 overexpression induces FOXP3 in Jurkat cells using the vector shown in Figure 2. Figure 3A shows a bar graph of IL37 mRNA expression in Jurkat cells transfected with an empty vector control or IL37 OE Jurkat cells (IL37 OE). GAPDH served as an internal control, and values represent IL37 gene expression as a ratio to GAPDH expression. Expression was measured using qRT-PCR. Figure 3B shows IL-37 and FOXP3 immunoblotting of empty vector control and IL37 OE Jurkat cells. Actin was used as an internal control. Gray bars represent IL37 OE Jurkat cells. Black bars represent vector control. Representative immunoblotting (left panel) and band quantification (right panel) of three immunoblot experiments. Protein band density was quantified using Image J, normalized to actin, and expressed as fold change compared to control Jurkat cells expressing an empty vector. Figure 3C shows a bar graph of FOXP3 mRNA expression in vector control and IL37OE Jurkat cells. GAPDH served as an internal control, and values represent FOXP3 gene expression as a ratio to GAPDH expression. Figures 3D-E show a histogram (Figure 3D) and a bar graph showing densitometric quantification (Figure 3E) of FOXP3 protein expression in vector control and IL37OE Jurkat cells. IgG was used for negative control staining (black-gray histogram). Expression is expressed as mean fluorescence intensity (MFI) in IL37OE Jurkat cells compared to the vector control. Small horizontal lines indicate the mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of three independent experiments. [Figure 4A]Figure 4 shows a series of graphs demonstrating that IL37-overexpressing (IL37 OE) Jurkat cells are phenotypically similar to T regulatory (Treg) cells. Figure 4A shows a bar graph depicting qRT-PCR analysis of CTLA-4 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4B shows a bar graph depicting qRT-PCR analysis of IL-10 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4C shows a bar graph depicting qRT-PCR analysis of GITR mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4D shows a bar graph depicting qRT-PCR analysis of TGFB mRNA expression in IL37 OE Jurkat cells compared to vector control. In each of Figures 4A-4D, GAPDH served as an internal control, and values represent gene expression levels as a ratio to GAPDH expression. Figure 4E shows a histogram depicting CTLA-4 expression in IL37 OE Jurkat cells compared to vector control. IgG was used as a negative control staining. Figure 4F shows quantification of CTLA-4 expression shown in Figure 4E by mean fluorescence intensity (MFI). Figure 4G shows a series of contour plots depicting IL-10+ cell gating in unstimulated and anti-CD3 / CD28-treated (+CD3 / CD28) IL37 OE Jurkat cells compared to vector control cells. IgG was used as a negative control staining. Figure 4H shows a bar graph depicting quantification of IL-10 expression in Jurkat cells shown in Figure 4G. Small horizontal lines indicate the mean ± sem. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of four independent experiments. [Figure 4B]Figure 4 shows a series of graphs demonstrating that IL37-overexpressing (IL37 OE) Jurkat cells are phenotypically similar to T regulatory (Treg) cells. Figure 4A shows a bar graph depicting qRT-PCR analysis of CTLA-4 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4B shows a bar graph depicting qRT-PCR analysis of IL-10 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4C shows a bar graph depicting qRT-PCR analysis of GITR mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4D shows a bar graph depicting qRT-PCR analysis of TGFB mRNA expression in IL37 OE Jurkat cells compared to vector control. In each of Figures 4A-4D, GAPDH served as an internal control, and values represent gene expression levels as a ratio to GAPDH expression. Figure 4E shows a histogram depicting CTLA-4 expression in IL37 OE Jurkat cells compared to vector control. IgG was used as a negative control staining. Figure 4F shows quantification of CTLA-4 expression shown in Figure 4E by mean fluorescence intensity (MFI). Figure 4G shows a series of contour plots depicting IL-10+ cell gating in unstimulated and anti-CD3 / CD28-treated (+CD3 / CD28) IL37 OE Jurkat cells compared to vector control cells. IgG was used as a negative control staining. Figure 4H shows a bar graph depicting quantification of IL-10 expression in Jurkat cells shown in Figure 4G. Small horizontal lines indicate the mean ± sem. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of four independent experiments. [Figure 4C]Figure 4 shows a series of graphs demonstrating that IL37-overexpressing (IL37 OE) Jurkat cells are phenotypically similar to T regulatory (Treg) cells. Figure 4A shows a bar graph depicting qRT-PCR analysis of CTLA-4 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4B shows a bar graph depicting qRT-PCR analysis of IL-10 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4C shows a bar graph depicting qRT-PCR analysis of GITR mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4D shows a bar graph depicting qRT-PCR analysis of TGFB mRNA expression in IL37 OE Jurkat cells compared to vector control. In each of Figures 4A-4D, GAPDH served as an internal control, and values represent gene expression levels as a ratio to GAPDH expression. Figure 4E shows a histogram depicting CTLA-4 expression in IL37 OE Jurkat cells compared to vector control. IgG was used as a negative control staining. Figure 4F shows quantification of CTLA-4 expression shown in Figure 4E by mean fluorescence intensity (MFI). Figure 4G shows a series of contour plots depicting IL-10+ cell gating in unstimulated and anti-CD3 / CD28-treated (+CD3 / CD28) IL37 OE Jurkat cells compared to vector control cells. IgG was used as a negative control staining. Figure 4H shows a bar graph depicting quantification of IL-10 expression in Jurkat cells shown in Figure 4G. Small horizontal lines indicate the mean ± sem. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of four independent experiments. [Figure 4D]Figure 4 shows a series of graphs demonstrating that IL37-overexpressing (IL37 OE) Jurkat cells are phenotypically similar to T regulatory (Treg) cells. Figure 4A shows a bar graph depicting qRT-PCR analysis of CTLA-4 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4B shows a bar graph depicting qRT-PCR analysis of IL-10 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4C shows a bar graph depicting qRT-PCR analysis of GITR mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4D shows a bar graph depicting qRT-PCR analysis of TGFB mRNA expression in IL37 OE Jurkat cells compared to vector control. In each of Figures 4A-4D, GAPDH served as an internal control, and values represent gene expression levels as a ratio to GAPDH expression. Figure 4E shows a histogram depicting CTLA-4 expression in IL37 OE Jurkat cells compared to vector control. IgG was used as a negative control staining. Figure 4F shows quantification of CTLA-4 expression shown in Figure 4E by mean fluorescence intensity (MFI). Figure 4G shows a series of contour plots depicting IL-10+ cell gating in unstimulated and anti-CD3 / CD28-treated (+CD3 / CD28) IL37 OE Jurkat cells compared to vector control cells. IgG was used as a negative control staining. Figure 4H shows a bar graph depicting quantification of IL-10 expression in Jurkat cells shown in Figure 4G. Small horizontal lines indicate the mean ± sem. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of four independent experiments. [Figure 4E]Figure 4 shows a series of graphs demonstrating that IL37-overexpressing (IL37 OE) Jurkat cells are phenotypically similar to T regulatory (Treg) cells. Figure 4A shows a bar graph depicting qRT-PCR analysis of CTLA-4 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4B shows a bar graph depicting qRT-PCR analysis of IL-10 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4C shows a bar graph depicting qRT-PCR analysis of GITR mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4D shows a bar graph depicting qRT-PCR analysis of TGFB mRNA expression in IL37 OE Jurkat cells compared to vector control. In each of Figures 4A-4D, GAPDH served as an internal control, and values represent gene expression levels as a ratio to GAPDH expression. Figure 4E shows a histogram depicting CTLA-4 expression in IL37 OE Jurkat cells compared to vector control. IgG was used as a negative control staining. Figure 4F shows quantification of CTLA-4 expression shown in Figure 4E by mean fluorescence intensity (MFI). Figure 4G shows a series of contour plots depicting IL-10+ cell gating in unstimulated and anti-CD3 / CD28-treated (+CD3 / CD28) IL37 OE Jurkat cells compared to vector control cells. IgG was used as a negative control staining. Figure 4H shows a bar graph depicting quantification of IL-10 expression in Jurkat cells shown in Figure 4G. Small horizontal lines indicate the mean ± sem. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of four independent experiments. [Figure 4F]Figure 4 shows a series of graphs demonstrating that IL37-overexpressing (IL37 OE) Jurkat cells are phenotypically similar to T regulatory (Treg) cells. Figure 4A shows a bar graph depicting qRT-PCR analysis of CTLA-4 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4B shows a bar graph depicting qRT-PCR analysis of IL-10 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4C shows a bar graph depicting qRT-PCR analysis of GITR mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4D shows a bar graph depicting qRT-PCR analysis of TGFB mRNA expression in IL37 OE Jurkat cells compared to vector control. In each of Figures 4A-4D, GAPDH served as an internal control, and values represent gene expression levels as a ratio to GAPDH expression. Figure 4E shows a histogram depicting CTLA-4 expression in IL37 OE Jurkat cells compared to vector control. IgG was used as a negative control staining. Figure 4F shows quantification of CTLA-4 expression shown in Figure 4E by mean fluorescence intensity (MFI). Figure 4G shows a series of contour plots depicting IL-10+ cell gating in unstimulated and anti-CD3 / CD28-treated (+CD3 / CD28) IL37 OE Jurkat cells compared to vector control cells. IgG was used as a negative control staining. Figure 4H shows a bar graph depicting quantification of IL-10 expression in Jurkat cells shown in Figure 4G. Small horizontal lines indicate the mean ± sem. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of four independent experiments. [Figure 4G]Figure 4 shows a series of graphs demonstrating that IL37-overexpressing (IL37 OE) Jurkat cells are phenotypically similar to T regulatory (Treg) cells. Figure 4A shows a bar graph depicting qRT-PCR analysis of CTLA-4 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4B shows a bar graph depicting qRT-PCR analysis of IL-10 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4C shows a bar graph depicting qRT-PCR analysis of GITR mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4D shows a bar graph depicting qRT-PCR analysis of TGFB mRNA expression in IL37 OE Jurkat cells compared to vector control. In each of Figures 4A-4D, GAPDH served as an internal control, and values represent gene expression levels as a ratio to GAPDH expression. Figure 4E shows a histogram depicting CTLA-4 expression in IL37 OE Jurkat cells compared to vector control. IgG was used as a negative control staining. Figure 4F shows quantification of CTLA-4 expression shown in Figure 4E by mean fluorescence intensity (MFI). Figure 4G shows a series of contour plots depicting IL-10+ cell gating in unstimulated and anti-CD3 / CD28-treated (+CD3 / CD28) IL37 OE Jurkat cells compared to vector control cells. IgG was used as a negative control staining. Figure 4H shows a bar graph depicting quantification of IL-10 expression in Jurkat cells shown in Figure 4G. Small horizontal lines indicate the mean ± sem. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of four independent experiments. [Figure 4H]Figure 4 shows a series of graphs demonstrating that IL37-overexpressing (IL37 OE) Jurkat cells are phenotypically similar to T regulatory (Treg) cells. Figure 4A shows a bar graph depicting qRT-PCR analysis of CTLA-4 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4B shows a bar graph depicting qRT-PCR analysis of IL-10 mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4C shows a bar graph depicting qRT-PCR analysis of GITR mRNA expression in IL37 OE Jurkat cells compared to vector control. Figure 4D shows a bar graph depicting qRT-PCR analysis of TGFB mRNA expression in IL37 OE Jurkat cells compared to vector control. In each of Figures 4A-4D, GAPDH served as an internal control, and values represent gene expression levels as a ratio to GAPDH expression. Figure 4E shows a histogram depicting CTLA-4 expression in IL37 OE Jurkat cells compared to vector control. IgG was used as a negative control staining. Figure 4F shows quantification of CTLA-4 expression shown in Figure 4E by mean fluorescence intensity (MFI). Figure 4G shows a series of contour plots depicting IL-10+ cell gating in unstimulated and anti-CD3 / CD28-treated (+CD3 / CD28) IL37 OE Jurkat cells compared to vector control cells. IgG was used as a negative control staining. Figure 4H shows a bar graph depicting quantification of IL-10 expression in Jurkat cells shown in Figure 4G. Small horizontal lines indicate the mean ± sem. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of four independent experiments. [Figure 5A]Figure 5A shows a series of graphs demonstrating that IL-37-overexpressing (IL37 OE) Jurkat cells are highly suppressive in an in vitro T cell suppression assay. Figures 5A and 5B show a series of graphs from an in vitro T cell suppression assay using vector control and IL37 OE Jurkat cells. Histograms (Figure 5A) and % proliferation (Figure 5B) show the division of CFSE-labeled CD4+CD25- T cell responders (Tresp) purified from healthy human donor PBMCs and cultured for 5 days with anti-CD3 and either vector control Jurkat cells (black bars), IL37 OE Jurkat cells (gray bars), or human primary Treg cells (white bars) at Treg:Tresp ratios ranging from 4:1 to 1:2. Purified Tresp cells (0:1) without Jurkat or Treg cells were used as a positive control for Tresp proliferation without suppression (orange bars). The x-axis shows the ratio of Jurkat cells to Tresp cells. The y-axis shows percent proliferation. Black bars represent vector control Jurkat cells. Figure 5C shows a bar graph of ELISA analysis of IL-37 in the supernatants of vector control and IL37 OE Jurkat cells. Recombinant human IL-37 controls at 2 ng / ml and 1 ng / ml were used as positive controls. Data represent the mean ± sem. NS, not significant (P>0.05), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of two independent experiments. [Figure 5B]Figure 5A shows a series of graphs demonstrating that IL-37-overexpressing (IL37 OE) Jurkat cells are highly suppressive in an in vitro T cell suppression assay. Figures 5A and 5B show a series of graphs from an in vitro T cell suppression assay using vector control and IL37 OE Jurkat cells. Histograms (Figure 5A) and % proliferation (Figure 5B) show the division of CFSE-labeled CD4+CD25- T cell responders (Tresp) purified from healthy human donor PBMCs and cultured for 5 days with anti-CD3 and either vector control Jurkat cells (black bars), IL37 OE Jurkat cells (gray bars), or human primary Treg cells (white bars) at Treg:Tresp ratios ranging from 4:1 to 1:2. Purified Tresp cells (0:1) without Jurkat or Treg cells were used as a positive control for Tresp proliferation without suppression (orange bars). The x-axis shows the ratio of Jurkat cells to Tresp cells. The y-axis shows percent proliferation. Black bars represent vector control Jurkat cells. Figure 5C shows a bar graph of ELISA analysis of IL-37 in the supernatants of vector control and IL37 OE Jurkat cells. Recombinant human IL-37 controls at 2 ng / ml and 1 ng / ml were used as positive controls. Data represent the mean ± sem. NS, not significant (P>0.05), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of two independent experiments. [Figure 5C]Figure 5A shows a series of graphs demonstrating that IL-37-overexpressing (IL37 OE) Jurkat cells are highly suppressive in an in vitro T cell suppression assay. Figures 5A and 5B show a series of graphs from an in vitro T cell suppression assay using vector control and IL37 OE Jurkat cells. Histograms (Figure 5A) and % proliferation (Figure 5B) show the division of CFSE-labeled CD4+CD25- T cell responders (Tresp) purified from healthy human donor PBMCs and cultured for 5 days with anti-CD3 and either vector control Jurkat cells (black bars), IL37 OE Jurkat cells (gray bars), or human primary Treg cells (white bars) at Treg:Tresp ratios ranging from 4:1 to 1:2. Purified Tresp cells (0:1) without Jurkat or Treg cells were used as a positive control for Tresp proliferation without suppression (orange bars). The x-axis shows the ratio of Jurkat cells to Tresp cells. The y-axis shows percent proliferation. Black bars represent vector control Jurkat cells. Figure 5C shows a bar graph of ELISA analysis of IL-37 in the supernatants of vector control and IL37 OE Jurkat cells. Recombinant human IL-37 controls at 2 ng / ml and 1 ng / ml were used as positive controls. Data represent the mean ± sem. NS, not significant (P>0.05), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). Data are representative of two independent experiments. [Figure 6A] Figure 6A shows a series of graphs depicting increased FOXP3 expression in transgenic (Tg) Treg cells expressing IL-37. Figure 6B shows a histogram depicting FOXP3 expression in IL-37 transgenic (Tg) Treg cells compared to wildtype (WT) and isotype controls. [Figure 6B]Figure 6B shows a series of graphs depicting increased FOXP3 expression in transgenic (Tg) Treg cells expressing IL-37. Figure 6B shows a bar graph depicting quantification of FOXP3 expression shown in Figure 6A. Expression is shown as mean fluorescence intensity (MFI). [Figure 7A] Figure 7A shows a series of graphs demonstrating the enhanced suppressive function of transgenic (Tg) Treg cells expressing IL-37 in an in vitro T cell suppression assay. Figure 7B shows a series of histograms depicting the division of CFSE-labeled T cell responder (Tresp) cells (CD4+CD25-) from WT mice co-incubated with Treg cells from either WT or IL37 transgenic (Tg) mice at the indicated ratios for 3 days. [Figure 7B] Figure 7B shows a series of graphs demonstrating the enhanced suppressive function of transgenic (Tg) Treg cells expressing IL-37 in an in vitro T cell suppression assay. Figure 7B shows a bar graph depicting the proliferation index of the T cell co-cultures shown in Figure 7A. [Figure 7C] Figure 7C shows a series of graphs demonstrating the enhanced suppressive function of transgenic (Tg) Treg cells expressing IL-37 in an in vitro T cell suppression assay. Figure 7C shows a bar graph depicting the percent (%) suppression of the T cell co-cultures analyzed in Figure 7A. [Figure 8A]Figure 8A shows a series of graphs and microscopy images demonstrating IL-37 transgenic Treg (IL37 Treg) cells suppressing skin inflammation in vivo using a mouse model. Figure 8A shows a schematic diagram illustrating an exemplary mouse contact hypersensitivity (CHS) model. Either donor IL-37 transgenic (Tg) Treg cells or donor WT Treg cells are adoptively transferred into WT mice (recipients) after hapten sensitization (DNFB) and before subsequent hapten challenge (DNFB). DNFB is dinitrofluorobenzene. Figure 8B shows a line graph depicting ear swelling in mice administered IL-37 Tg-Treg cells compared with WT Treg cells and vehicle controls in the CHS model shown in Figure 8A. Treatments are as indicated in the legend. Figure 8C shows a series of H&E microscopy images of ear specimens taken 48 hours after DNFB challenge in the mouse model shown in Figure 8A. Ear specimens were collected from mice that received either vehicle or DNFB treatment and adoptive transfer of cells as indicated. Scale bar, 100 μm. Figure 8D shows a series of contour plots (left panel) and quantification (right panel) of CFSE+CD3+ T cells present in the DNFB-treated ears of mice adoptively transferred with either PBS, WT Treg, or IL37 Tg Treg cells. Contour plots from ears of mice injected with PBS and challenged with DNFB were used as negative controls for CFSE gating. Numbers in the contour plots indicate the percentage of cells. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± s.e.m. (n = 5–8 mice per group) (Figures 8A–8D). Data represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. Statistics in (Figure 8B) show comparison with PBS-injected and DNFB-challenged mice (Student's t-test). Data are representative of two experiments. [Figure 8B]Figure 8A shows a series of graphs and microscopy images demonstrating IL-37 transgenic Treg (IL37 Treg) cells suppressing skin inflammation in vivo using a mouse model. Figure 8A shows a schematic diagram illustrating an exemplary mouse contact hypersensitivity (CHS) model. Either donor IL-37 transgenic (Tg) Treg cells or donor WT Treg cells are adoptively transferred into WT mice (recipients) after hapten sensitization (DNFB) and before subsequent hapten challenge (DNFB). DNFB is dinitrofluorobenzene. Figure 8B shows a line graph depicting ear swelling in mice administered IL-37 Tg-Treg cells compared with WT Treg cells and vehicle controls in the CHS model shown in Figure 8A. Treatments are as indicated in the legend. Figure 8C shows a series of H&E microscopy images of ear specimens taken 48 hours after DNFB challenge in the mouse model shown in Figure 8A. Ear specimens were collected from mice that received either vehicle or DNFB treatment and adoptive transfer of cells as indicated. Scale bar, 100 μm. Figure 8D shows a series of contour plots (left panel) and quantification (right panel) of CFSE+CD3+ T cells present in the DNFB-treated ears of mice adoptively transferred with either PBS, WT Treg, or IL37 Tg Treg cells. Contour plots from ears of mice injected with PBS and challenged with DNFB were used as negative controls for CFSE gating. Numbers in the contour plots indicate the percentage of cells. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± s.e.m. (n = 5–8 mice per group) (Figures 8A–8D). Data represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. Statistics in (Figure 8B) show comparison with PBS-injected and DNFB-challenged mice (Student's t-test). Data are representative of two experiments. [Figure 8C]Figure 8A shows a series of graphs and microscopy images demonstrating IL-37 transgenic Treg (IL37 Treg) cells suppressing skin inflammation in vivo using a mouse model. Figure 8A shows a schematic diagram illustrating an exemplary mouse contact hypersensitivity (CHS) model. Either donor IL-37 transgenic (Tg) Treg cells or donor WT Treg cells are adoptively transferred into WT mice (recipients) after hapten sensitization (DNFB) and before subsequent hapten challenge (DNFB). DNFB is dinitrofluorobenzene. Figure 8B shows a line graph depicting ear swelling in mice administered IL-37 Tg-Treg cells compared with WT Treg cells and vehicle controls in the CHS model shown in Figure 8A. Treatments are as indicated in the legend. Figure 8C shows a series of H&E microscopy images of ear specimens taken 48 hours after DNFB challenge in the mouse model shown in Figure 8A. Ear specimens were collected from mice that received either vehicle or DNFB treatment and adoptive transfer of cells as indicated. Scale bar, 100 μm. Figure 8D shows a series of contour plots (left panel) and quantification (right panel) of CFSE+CD3+ T cells present in the DNFB-treated ears of mice adoptively transferred with either PBS, WT Treg, or IL37 Tg Treg cells. Contour plots from ears of mice injected with PBS and challenged with DNFB were used as negative controls for CFSE gating. Numbers in the contour plots indicate the percentage of cells. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± s.e.m. (n = 5–8 mice per group) (Figures 8A–8D). Data represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. Statistics in (Figure 8B) show comparison with PBS-injected and DNFB-challenged mice (Student's t-test). Data are representative of two experiments. [Figure 8D]Figure 8A shows a series of graphs and microscopy images demonstrating IL-37 transgenic Treg (IL37 Treg) cells suppressing skin inflammation in vivo using a mouse model. Figure 8A shows a schematic diagram illustrating an exemplary mouse contact hypersensitivity (CHS) model. Either donor IL-37 transgenic (Tg) Treg cells or donor WT Treg cells are adoptively transferred into WT mice (recipients) after hapten sensitization (DNFB) and before subsequent hapten challenge (DNFB). DNFB is dinitrofluorobenzene. Figure 8B shows a line graph depicting ear swelling in mice administered IL-37 Tg-Treg cells compared with WT Treg cells and vehicle controls in the CHS model shown in Figure 8A. Treatments are as indicated in the legend. Figure 8C shows a series of H&E microscopy images of ear specimens taken 48 hours after DNFB challenge in the mouse model shown in Figure 8A. Ear specimens were collected from mice that received either vehicle or DNFB treatment and adoptive transfer of cells as indicated. Scale bar, 100 μm. Figure 8D shows a series of contour plots (left panel) and quantification (right panel) of CFSE+CD3+ T cells present in the DNFB-treated ears of mice adoptively transferred with either PBS, WT Treg, or IL37 Tg Treg cells. Contour plots from ears of mice injected with PBS and challenged with DNFB were used as negative controls for CFSE gating. Numbers in the contour plots indicate the percentage of cells. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± s.e.m. (n = 5–8 mice per group) (Figures 8A–8D). Data represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. Statistics in (Figure 8B) show comparison with PBS-injected and DNFB-challenged mice (Student's t-test). Data are representative of two experiments. [Figure 9A]Figure 9A shows a series of graphs illustrating the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vitro. Figure 9A shows a bar graph of Foxp3 mRNA expression analyzed via qRT-PCR in WT and IL37 Tg Treg cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of murine IL-6. GAPDH served as an internal control. Figure 9B shows a histogram (top) and quantification (bottom) of Foxp3 in WT and IL37 Tg CD4+ T cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of IL-6. Figure 9C shows a bar graph of IL37 mRNA in IL37 Tg Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of mouse IL-6. GAPDH served as an internal control. Figure 9D shows a contour plot (left) and quantification (right) of the percentage of CD25+FOXP3+ Treg cells in IL-37hi and IL-37lo Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of human IL-6. Figure 9E shows a bar graph showing Th lineage gene expression (TBET, GATA, RORγT) and Th17-specific gene expression (STAT3, IRF4, and IL17) in siIL37 human Tregs. GAPDH served as an internal control. Figure 9F shows a bar graph depicting Th lineage gene expression (Gata, Rorγt, Irf4, and Il17) in unstimulated or TCR-stimulated mouse Tregs + / - IL-6. Gapdh served as an internal control. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. Data represent the mean ± sem. (n = 5-8 mice per group or 3-6 donors per group). NS, not significant (p > 0.05), *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t-test). Data are representative of 2-3 independent experiments.Figure 9G shows a series of contour plots depicting Treg and Tconv cells from ear specimens taken from mice 48 hours after DFNB challenge in the CHS model shown in Figure 8 A. Figure 9H shows bar graphs depicting the quantification (frequency) of Treg and Tconv cells in the samples shown in Figure 9G. [Figure 9B]Figure 9A shows a series of graphs illustrating the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vitro. Figure 9A shows a bar graph of Foxp3 mRNA expression analyzed via qRT-PCR in WT and IL37 Tg Treg cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of murine IL-6. GAPDH served as an internal control. Figure 9B shows a histogram (top) and quantification (bottom) of Foxp3 in WT and IL37 Tg CD4+ T cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of IL-6. Figure 9C shows a bar graph of IL37 mRNA in IL37 Tg Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of mouse IL-6. GAPDH served as an internal control. Figure 9D shows a contour plot (left) and quantification (right) of the percentage of CD25+FOXP3+ Treg cells in IL-37hi and IL-37lo Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of human IL-6. Figure 9E shows a bar graph showing Th lineage gene expression (TBET, GATA, RORγT) and Th17-specific gene expression (STAT3, IRF4, and IL17) in siIL37 human Tregs. GAPDH served as an internal control. Figure 9F shows a bar graph depicting Th lineage gene expression (Gata, Rorγt, Irf4, and Il17) in unstimulated or TCR-stimulated mouse Tregs + / - IL-6. Gapdh served as an internal control. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. Data represent the mean ± sem. (n = 5-8 mice per group or 3-6 donors per group). NS, not significant (p > 0.05), *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t-test). Data are representative of 2-3 independent experiments.Figure 9G shows a series of contour plots depicting Treg and Tconv cells from ear specimens taken from mice 48 hours after DFNB challenge in the CHS model shown in Figure 8 A. Figure 9H shows bar graphs depicting the quantification (frequency) of Treg and Tconv cells in the samples shown in Figure 9G. [Figure 9C]Figure 9A shows a series of graphs illustrating the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vitro. Figure 9A shows a bar graph of Foxp3 mRNA expression analyzed via qRT-PCR in WT and IL37 Tg Treg cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of murine IL-6. GAPDH served as an internal control. Figure 9B shows a histogram (top) and quantification (bottom) of Foxp3 in WT and IL37 Tg CD4+ T cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of IL-6. Figure 9C shows a bar graph of IL37 mRNA in IL37 Tg Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of mouse IL-6. GAPDH served as an internal control. Figure 9D shows a contour plot (left) and quantification (right) of the percentage of CD25+FOXP3+ Treg cells in IL-37hi and IL-37lo Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of human IL-6. Figure 9E shows a bar graph showing Th lineage gene expression (TBET, GATA, RORγT) and Th17-specific gene expression (STAT3, IRF4, and IL17) in siIL37 human Tregs. GAPDH served as an internal control. Figure 9F shows a bar graph depicting Th lineage gene expression (Gata, Rorγt, Irf4, and Il17) in unstimulated or TCR-stimulated mouse Tregs + / - IL-6. Gapdh served as an internal control. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. Data represent the mean ± sem. (n = 5-8 mice per group or 3-6 donors per group). NS, not significant (p > 0.05), *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t-test). Data are representative of 2-3 independent experiments.Figure 9G shows a series of contour plots depicting Treg and Tconv cells from ear specimens taken from mice 48 hours after DFNB challenge in the CHS model shown in Figure 8 A. Figure 9H shows bar graphs depicting the quantification (frequency) of Treg and Tconv cells in the samples shown in Figure 9G. [Figure 9D]Figure 9A shows a series of graphs illustrating the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vitro. Figure 9A shows a bar graph of Foxp3 mRNA expression analyzed via qRT-PCR in WT and IL37 Tg Treg cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of murine IL-6. GAPDH served as an internal control. Figure 9B shows a histogram (top) and quantification (bottom) of Foxp3 in WT and IL37 Tg CD4+ T cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of IL-6. Figure 9C shows a bar graph of IL37 mRNA in IL37 Tg Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of mouse IL-6. GAPDH served as an internal control. Figure 9D shows a contour plot (left) and quantification (right) of the percentage of CD25+FOXP3+ Treg cells in IL-37hi and IL-37lo Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of human IL-6. Figure 9E shows a bar graph showing Th lineage gene expression (TBET, GATA, RORγT) and Th17-specific gene expression (STAT3, IRF4, and IL17) in siIL37 human Tregs. GAPDH served as an internal control. Figure 9F shows a bar graph depicting Th lineage gene expression (Gata, Rorγt, Irf4, and Il17) in unstimulated or TCR-stimulated mouse Tregs + / - IL-6. Gapdh served as an internal control. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. Data represent the mean ± sem. (n = 5-8 mice per group or 3-6 donors per group). NS, not significant (p > 0.05), *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t-test). Data are representative of 2-3 independent experiments.Figure 9G shows a series of contour plots depicting Treg and Tconv cells from ear specimens taken from mice 48 hours after DFNB challenge in the CHS model shown in Figure 8 A. Figure 9H shows bar graphs depicting the quantification (frequency) of Treg and Tconv cells in the samples shown in Figure 9G. [Figure 9E]Figure 9A shows a series of graphs illustrating the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vitro. Figure 9A shows a bar graph of Foxp3 mRNA expression analyzed via qRT-PCR in WT and IL37 Tg Treg cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of murine IL-6. GAPDH served as an internal control. Figure 9B shows a histogram (top) and quantification (bottom) of Foxp3 in WT and IL37 Tg CD4+ T cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of IL-6. Figure 9C shows a bar graph of IL37 mRNA in IL37 Tg Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of mouse IL-6. GAPDH served as an internal control. Figure 9D shows a contour plot (left) and quantification (right) of the percentage of CD25+FOXP3+ Treg cells in IL-37hi and IL-37lo Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of human IL-6. Figure 9E shows a bar graph showing Th lineage gene expression (TBET, GATA, RORγT) and Th17-specific gene expression (STAT3, IRF4, and IL17) in siIL37 human Tregs. GAPDH served as an internal control. Figure 9F shows a bar graph depicting Th lineage gene expression (Gata, Rorγt, Irf4, and Il17) in unstimulated or TCR-stimulated mouse Tregs + / - IL-6. Gapdh served as an internal control. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. Data represent the mean ± sem. (n = 5-8 mice per group or 3-6 donors per group). NS, not significant (p > 0.05), *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t-test). Data are representative of 2-3 independent experiments.Figure 9G shows a series of contour plots depicting Treg and Tconv cells from ear specimens taken from mice 48 hours after DFNB challenge in the CHS model shown in Figure 8 A. Figure 9H shows bar graphs depicting the quantification (frequency) of Treg and Tconv cells in the samples shown in Figure 9G. [Figure 9F]Figure 9A shows a series of graphs illustrating the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vitro. Figure 9A shows a bar graph of Foxp3 mRNA expression analyzed via qRT-PCR in WT and IL37 Tg Treg cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of murine IL-6. GAPDH served as an internal control. Figure 9B shows a histogram (top) and quantification (bottom) of Foxp3 in WT and IL37 Tg CD4+ T cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of IL-6. Figure 9C shows a bar graph of IL37 mRNA in IL37 Tg Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of mouse IL-6. GAPDH served as an internal control. Figure 9D shows a contour plot (left) and quantification (right) of the percentage of CD25+FOXP3+ Treg cells in IL-37hi and IL-37lo Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of human IL-6. Figure 9E shows a bar graph showing Th lineage gene expression (TBET, GATA, RORγT) and Th17-specific gene expression (STAT3, IRF4, and IL17) in siIL37 human Tregs. GAPDH served as an internal control. Figure 9F shows a bar graph depicting Th lineage gene expression (Gata, Rorγt, Irf4, and Il17) in unstimulated or TCR-stimulated mouse Tregs + / - IL-6. Gapdh served as an internal control. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. Data represent the mean ± sem. (n = 5-8 mice per group or 3-6 donors per group). NS, not significant (p > 0.05), *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t-test). Data are representative of 2-3 independent experiments.Figure 9G shows a series of contour plots depicting Treg and Tconv cells from ear specimens taken from mice 48 hours after DFNB challenge in the CHS model shown in Figure 8 A. Figure 9H shows bar graphs depicting the quantification (frequency) of Treg and Tconv cells in the samples shown in Figure 9G. [Figure 9G]Figure 9A shows a series of graphs illustrating the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vitro. Figure 9A shows a bar graph of Foxp3 mRNA expression analyzed via qRT-PCR in WT and IL37 Tg Treg cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of murine IL-6. GAPDH served as an internal control. Figure 9B shows a histogram (top) and quantification (bottom) of Foxp3 in WT and IL37 Tg CD4+ T cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of IL-6. Figure 9C shows a bar graph of IL37 mRNA in IL37 Tg Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of mouse IL-6. GAPDH served as an internal control. Figure 9D shows a contour plot (left) and quantification (right) of the percentage of CD25+FOXP3+ Treg cells in IL-37hi and IL-37lo Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of human IL-6. Figure 9E shows a bar graph showing Th lineage gene expression (TBET, GATA, RORγT) and Th17-specific gene expression (STAT3, IRF4, and IL17) in siIL37 human Tregs. GAPDH served as an internal control. Figure 9F shows a bar graph depicting Th lineage gene expression (Gata, Rorγt, Irf4, and Il17) in unstimulated or TCR-stimulated mouse Tregs + / - IL-6. Gapdh served as an internal control. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. Data represent the mean ± sem. (n = 5-8 mice per group or 3-6 donors per group). NS, not significant (p > 0.05), *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t-test). Data are representative of 2-3 independent experiments.Figure 9G shows a series of contour plots depicting Treg and Tconv cells from ear specimens taken from mice 48 hours after DFNB challenge in the CHS model shown in Figure 8 A. Figure 9H shows bar graphs depicting the quantification (frequency) of Treg and Tconv cells in the samples shown in Figure 9G. [Figure 9H]Figure 9A shows a series of graphs illustrating the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vitro. Figure 9A shows a bar graph of Foxp3 mRNA expression analyzed via qRT-PCR in WT and IL37 Tg Treg cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of murine IL-6. GAPDH served as an internal control. Figure 9B shows a histogram (top) and quantification (bottom) of Foxp3 in WT and IL37 Tg CD4+ T cells cultured with anti-CD3 / CD28 for 48 hours in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of IL-6. Figure 9C shows a bar graph of IL37 mRNA in IL37 Tg Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of mouse IL-6. GAPDH served as an internal control. Figure 9D shows a contour plot (left) and quantification (right) of the percentage of CD25+FOXP3+ Treg cells in IL-37hi and IL-37lo Treg cells cultured for 48 hours with anti-CD3 / CD28 in the absence (-IL-6) or presence (+IL-6) of 20 ng / ml of human IL-6. Figure 9E shows a bar graph showing Th lineage gene expression (TBET, GATA, RORγT) and Th17-specific gene expression (STAT3, IRF4, and IL17) in siIL37 human Tregs. GAPDH served as an internal control. Figure 9F shows a bar graph depicting Th lineage gene expression (Gata, Rorγt, Irf4, and Il17) in unstimulated or TCR-stimulated mouse Tregs + / - IL-6. Gapdh served as an internal control. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. Data represent the mean ± sem. (n = 5-8 mice per group or 3-6 donors per group). NS, not significant (p > 0.05), *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t-test). Data are representative of 2-3 independent experiments.Figure 9G shows a series of contour plots depicting Treg and Tconv cells from ear specimens taken from mice 48 hours after DFNB challenge in the CHS model shown in Figure 8 A. Figure 9H shows bar graphs depicting the quantification (frequency) of Treg and Tconv cells in the samples shown in Figure 9G. [Figure 10A] Figure 10A shows a series of bar graphs depicting gene expression knockdown in human Treg cells 24 hours after transfection with IL-37 siRNA (siIL37) compared to a scrambled siRNA control (siCtrl). Expression was analyzed by qRT-PCR. Figure 10B shows knockdown of IL-37 expression in Treg cells. [Figure 10B] Figure 10B shows a series of bar graphs showing gene expression knockdown in human Treg cells 24 hours after transfection with IL-37 siRNA (siIL37) compared to a scrambled siRNA control (siCtrl). Expression was analyzed by qRT-PCR. Figure 10B shows knockdown of FOXP3 expression in Treg cells. [Figure 11A]Figure 11A shows a series of images and graphs demonstrating that IL-37 interacts with p-SMAD3, translocates into the nucleus, and induces FOXP3 expression in human Treg cells. Figure 11A shows a series of confocal microscopy images demonstrating IL-37 localization in Treg compared to Tconv cells. Confocal images show cells stained with the DNA intercalating dye DAPI, IL-37, and an overlay of IL-37 and DAPI in Treg and Tconv cells. Right: Quantification of IL-37 intensity in over 100 individual Tconv and Treg cells. Right: Pearson's coefficient of IL-37 colocalization with DAPI counted using overlay images of IL-37 and DAPI in Tconv and Treg cells. Figure 11B shows images of immunoblotting of IL-37 against SMAD3 immunoprecipitates of resting Tconv and Treg cell lysates. Figure 11C shows a table listing Fullmoon® protein and phosphosite antibody arrays measuring fold changes between purified mouse Treg cells derived from wild-type and IL37 Tg. Figures 11D-11E show histograms (Figure 11D) and quantification (MFI) (Figure 11E) of CD4 and p-SMAD3 Ser208 from IL-37hi and IL-37lo human Treg cells. Figure 11F shows confocal microscopy images of immunofluorescence proximity ligation assays (PLA) of IL-37 and p-SMAD3 S208 (dots) in human Tconv and Treg cells. DNA intercalating dye DAPI. Scale bar, 10 μm. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± s.e.m. (n = 4–5 donors per group). NS, not significant (p > 0.05). **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two to four independent experiments. [Figure 11B]Figure 11A shows a series of images and graphs demonstrating that IL-37 interacts with p-SMAD3, translocates into the nucleus, and induces FOXP3 expression in human Treg cells. Figure 11A shows a series of confocal microscopy images demonstrating IL-37 localization in Treg compared to Tconv cells. Confocal images show cells stained with the DNA intercalating dye DAPI, IL-37, and an overlay of IL-37 and DAPI in Treg and Tconv cells. Right: Quantification of IL-37 intensity in over 100 individual Tconv and Treg cells. Right: Pearson's coefficient of IL-37 colocalization with DAPI counted using overlay images of IL-37 and DAPI in Tconv and Treg cells. Figure 11B shows images of immunoblotting of IL-37 against SMAD3 immunoprecipitates of resting Tconv and Treg cell lysates. Figure 11C shows a table listing Fullmoon® protein and phosphosite antibody arrays measuring fold changes between purified mouse Treg cells derived from wild-type and IL37 Tg. Figures 11D-11E show histograms (Figure 11D) and quantification (MFI) (Figure 11E) of CD4 and p-SMAD3 Ser208 from IL-37hi and IL-37lo human Treg cells. Figure 11F shows confocal microscopy images of immunofluorescence proximity ligation assays (PLA) of IL-37 and p-SMAD3 S208 (dots) in human Tconv and Treg cells. DNA intercalating dye DAPI. Scale bar, 10 μm. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± s.e.m. (n = 4–5 donors per group). NS, not significant (p > 0.05). **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two to four independent experiments. [Figure 11C]Figure 11A shows a series of images and graphs demonstrating that IL-37 interacts with p-SMAD3, translocates into the nucleus, and induces FOXP3 expression in human Treg cells. Figure 11A shows a series of confocal microscopy images demonstrating IL-37 localization in Treg compared to Tconv cells. Confocal images show cells stained with the DNA intercalating dye DAPI, IL-37, and an overlay of IL-37 and DAPI in Treg and Tconv cells. Right: Quantification of IL-37 intensity in over 100 individual Tconv and Treg cells. Right: Pearson's coefficient of IL-37 colocalization with DAPI counted using overlay images of IL-37 and DAPI in Tconv and Treg cells. Figure 11B shows images of immunoblotting of IL-37 against SMAD3 immunoprecipitates of resting Tconv and Treg cell lysates. Figure 11C shows a table listing Fullmoon® protein and phosphosite antibody arrays measuring fold changes between purified mouse Treg cells derived from wild-type and IL37 Tg. Figures 11D-11E show histograms (Figure 11D) and quantification (MFI) (Figure 11E) of CD4 and p-SMAD3 Ser208 from IL-37hi and IL-37lo human Treg cells. Figure 11F shows confocal microscopy images of immunofluorescence proximity ligation assays (PLA) of IL-37 and p-SMAD3 S208 (dots) in human Tconv and Treg cells. DNA intercalating dye DAPI. Scale bar, 10 μm. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± s.e.m. (n = 4–5 donors per group). NS, not significant (p > 0.05). **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two to four independent experiments. [Figure 11D]Figure 11A shows a series of images and graphs demonstrating that IL-37 interacts with p-SMAD3, translocates into the nucleus, and induces FOXP3 expression in human Treg cells. Figure 11A shows a series of confocal microscopy images demonstrating IL-37 localization in Treg compared to Tconv cells. Confocal images show cells stained with the DNA intercalating dye DAPI, IL-37, and an overlay of IL-37 and DAPI in Treg and Tconv cells. Right: Quantification of IL-37 intensity in over 100 individual Tconv and Treg cells. Right: Pearson's coefficient of IL-37 colocalization with DAPI counted using overlay images of IL-37 and DAPI in Tconv and Treg cells. Figure 11B shows images of immunoblotting of IL-37 against SMAD3 immunoprecipitates of resting Tconv and Treg cell lysates. Figure 11C shows a table listing Fullmoon® protein and phosphosite antibody arrays measuring fold changes between purified mouse Treg cells derived from wild-type and IL37 Tg. Figures 11D-11E show histograms (Figure 11D) and quantification (MFI) (Figure 11E) of CD4 and p-SMAD3 Ser208 from IL-37hi and IL-37lo human Treg cells. Figure 11F shows confocal microscopy images of immunofluorescence proximity ligation assays (PLA) of IL-37 and p-SMAD3 S208 (dots) in human Tconv and Treg cells. DNA intercalating dye DAPI. Scale bar, 10 μm. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± s.e.m. (n = 4–5 donors per group). NS, not significant (p > 0.05). **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two to four independent experiments. [Figure 11E]Figure 11A shows a series of images and graphs demonstrating that IL-37 interacts with p-SMAD3, translocates into the nucleus, and induces FOXP3 expression in human Treg cells. Figure 11A shows a series of confocal microscopy images demonstrating IL-37 localization in Treg compared to Tconv cells. Confocal images show cells stained with the DNA intercalating dye DAPI, IL-37, and an overlay of IL-37 and DAPI in Treg and Tconv cells. Right: Quantification of IL-37 intensity in over 100 individual Tconv and Treg cells. Right: Pearson's coefficient of IL-37 colocalization with DAPI counted using overlay images of IL-37 and DAPI in Tconv and Treg cells. Figure 11B shows images of immunoblotting of IL-37 against SMAD3 immunoprecipitates of resting Tconv and Treg cell lysates. Figure 11C shows a table listing Fullmoon® protein and phosphosite antibody arrays measuring fold changes between purified mouse Treg cells derived from wild-type and IL37 Tg. Figures 11D-11E show histograms (Figure 11D) and quantification (MFI) (Figure 11E) of CD4 and p-SMAD3 Ser208 from IL-37hi and IL-37lo human Treg cells. Figure 11F shows confocal microscopy images of immunofluorescence proximity ligation assays (PLA) of IL-37 and p-SMAD3 S208 (dots) in human Tconv and Treg cells. DNA intercalating dye DAPI. Scale bar, 10 μm. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± s.e.m. (n = 4–5 donors per group). NS, not significant (p > 0.05). **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two to four independent experiments. [Figure 11F]Figure 11A shows a series of images and graphs demonstrating that IL-37 interacts with p-SMAD3, translocates into the nucleus, and induces FOXP3 expression in human Treg cells. Figure 11A shows a series of confocal microscopy images demonstrating IL-37 localization in Treg compared to Tconv cells. Confocal images show cells stained with the DNA intercalating dye DAPI, IL-37, and an overlay of IL-37 and DAPI in Treg and Tconv cells. Right: Quantification of IL-37 intensity in over 100 individual Tconv and Treg cells. Right: Pearson's coefficient of IL-37 colocalization with DAPI counted using overlay images of IL-37 and DAPI in Tconv and Treg cells. Figure 11B shows images of immunoblotting of IL-37 against SMAD3 immunoprecipitates of resting Tconv and Treg cell lysates. Figure 11C shows a table listing Fullmoon® protein and phosphosite antibody arrays measuring fold changes between purified mouse Treg cells derived from wild-type and IL37 Tg. Figures 11D-11E show histograms (Figure 11D) and quantification (MFI) (Figure 11E) of CD4 and p-SMAD3 Ser208 from IL-37hi and IL-37lo human Treg cells. Figure 11F shows confocal microscopy images of immunofluorescence proximity ligation assays (PLA) of IL-37 and p-SMAD3 S208 (dots) in human Tconv and Treg cells. DNA intercalating dye DAPI. Scale bar, 10 μm. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± s.e.m. (n = 4–5 donors per group). NS, not significant (p > 0.05). **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two to four independent experiments. [Figure 12A]Figure 12A shows a series of images and graphs demonstrating the role of caspase-1 in regulating the nuclear localization of IL-37, as well as Foxp3 expression in mouse Treg cells and its function in maintaining immune suppression in vivo under inflammatory conditions. Figure 12A shows confocal microscopy images of unstimulated IL37 Tg and D20A mouse Treg cells. Images of cells stained with IL-37 (AF488), DAPI, and an overlay of IL-37 and DAPI. Quantification of IL-37 nuclear localization from the images in (A) is shown by the Pearson coefficient of IL-37 colocalization with DAPI (right) calculated using the overlay image of IL-37 and DAPI in Treg cells. Figure 12B shows a series of bar graphs of IL37 (left) and FOXP3 (right) in WT, IL37 Tg, and D20A Treg cells. Expression was analyzed by qRT-PCR. GAPDH served as an internal control. Each symbol represents an individual donor. Small horizontal lines indicate mean ± sem. (n ≥ 100 cells / group) (Figure 12A), (n = 4-6 mice per group) (Figure 12B). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 12B]Figure 12A shows a series of images and graphs demonstrating the role of caspase-1 in regulating the nuclear localization of IL-37, as well as Foxp3 expression in mouse Treg cells and its function in maintaining immune suppression in vivo under inflammatory conditions. Figure 12A shows confocal microscopy images of unstimulated IL37 Tg and D20A mouse Treg cells. Images of cells stained with IL-37 (AF488), DAPI, and an overlay of IL-37 and DAPI. Quantification of IL-37 nuclear localization from the images in (A) is shown by the Pearson coefficient of IL-37 colocalization with DAPI (right) calculated using the overlay image of IL-37 and DAPI in Treg cells. Figure 12B shows a series of bar graphs of IL37 (left) and FOXP3 (right) in WT, IL37 Tg, and D20A Treg cells. Expression was analyzed by qRT-PCR. GAPDH served as an internal control. Each symbol represents an individual donor. Small horizontal lines indicate mean ± sem. (n ≥ 100 cells / group) (Figure 12A), (n = 4-6 mice per group) (Figure 12B). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 13A]Figure 13A shows a series of images and graphs demonstrating that caspase-1 activity is required for immunosuppression. Figure 13A shows a series of H&E-stained microscopic images of ear specimens taken 48 hours after DNFB challenge from mice adoptively transferred with PBS, WT-Treg, IL37 Tg, or D20A Treg. Scale bar, 100 μm. Figure 13B shows a line graph depicting the CHS response, as measured by ear thickness, at 0, 6, 24, and 48 hours after vehicle (Vehicle + PBS) or DNFB challenge in WT mice adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), IL37 Tg Treg cells (DNFB + IL37 Tg-Treg), or D20A Treg cells (DNFB + D20ATreg). Figure 13C shows a series of bar graphs depicting the frequency of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) (left) and Tconv cells (CD4+CD25loCD45RBhi) (right) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg, IL37 Tg Treg, or D20A Treg cells. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± s.e.m. (n = 4-6 mice per group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 13B]Figure 13A shows a series of images and graphs demonstrating that caspase-1 activity is required for immunosuppression. Figure 13A shows a series of H&E-stained microscopic images of ear specimens taken 48 hours after DNFB challenge from mice adoptively transferred with PBS, WT-Treg, IL37 Tg, or D20A Treg. Scale bar, 100 μm. Figure 13B shows a line graph depicting the CHS response, as measured by ear thickness, at 0, 6, 24, and 48 hours after vehicle (Vehicle + PBS) or DNFB challenge in WT mice adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), IL37 Tg Treg cells (DNFB + IL37 Tg-Treg), or D20A Treg cells (DNFB + D20ATreg). Figure 13C shows a series of bar graphs depicting the frequency of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) (left) and Tconv cells (CD4+CD25loCD45RBhi) (right) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg, IL37 Tg Treg, or D20A Treg cells. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± s.e.m. (n = 4-6 mice per group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 13C]Figure 13A shows a series of images and graphs demonstrating that caspase-1 activity is required for immunosuppression. Figure 13A shows a series of H&E-stained microscopic images of ear specimens taken 48 hours after DNFB challenge from mice adoptively transferred with PBS, WT-Treg, IL37 Tg, or D20A Treg. Scale bar, 100 μm. Figure 13B shows a line graph depicting the CHS response, as measured by ear thickness, at 0, 6, 24, and 48 hours after vehicle (Vehicle + PBS) or DNFB challenge in WT mice adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), IL37 Tg Treg cells (DNFB + IL37 Tg-Treg), or D20A Treg cells (DNFB + D20ATreg). Figure 13C shows a series of bar graphs depicting the frequency of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) (left) and Tconv cells (CD4+CD25loCD45RBhi) (right) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg, IL37 Tg Treg, or D20A Treg cells. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± s.e.m. (n = 4-6 mice per group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 14A]Figure 14A-B show a series of graphs demonstrating that IL-37-overexpressing Jurkat cells are highly suppressive in vitro. Figures 14A-B show a series of graphs from an in vitro T cell suppression assay using vector control and IL37 OE Jurkat cells. Histograms (Figure 14A) and % proliferation (Figure 14B) show the division of CFSE-labeled CD4+CD25- T cell responders (Tresp) purified from healthy human donor PBMCs and cultured for 5 days with anti-CD3 and either vector control Jurkat cells (black bars), IL37 OE Jurkat cells (gray bars), or human primary Treg cells (white bars) at Treg:Tresp ratios ranging from 4:1 to 1:2. Purified Tresp cells (0:1) without Jurkat or Treg cells were used as a positive control for Tresp proliferation without suppression (orange bars). Figure 14C shows a bar graph of ELISA analysis of IL-37 in the supernatants of vector control and IL37 OE Jurkat cells. 2 ng / ml and 1 ng / ml recombinant human IL-37 controls were used as positive controls. Data represent mean ± sem. NS, not significant (P>0.05); *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (Student's t-test). Data are representative of two independent experiments. Figure 14D shows a series of confocal microscopy images and Western blots showing IL-37 expression in Jurkat cells transduced with either empty vector (Empty), IL-37 vector (IL37), or IL-37D20A vector (D20A). IL-37 was stained with AF488. Cells were stained with DAPI. Western blots were performed on the same cells (Empty, IL37, D20A) as indicated. Cell lysates were probed with IL-37 antibody. Actin was detected as a control. Figure 14E shows a series of graphs showing CTLA4 and FOXP3 gene expression by qRT-PCR and FOXP3 surface expression by flow cytometry in empty Jurkat cells (Empt) and IL-37-expressing Jurkat cells (IL37 or D20A) compared to an IgG control (IgG). [Figure 14B]Figure 14A-B show a series of graphs demonstrating that IL-37-overexpressing Jurkat cells are highly suppressive in vitro. Figures 14A-B show a series of graphs from an in vitro T cell suppression assay using vector control and IL37 OE Jurkat cells. Histograms (Figure 14A) and % proliferation (Figure 14B) show the division of CFSE-labeled CD4+CD25- T cell responders (Tresp) purified from healthy human donor PBMCs and cultured for 5 days with anti-CD3 and either vector control Jurkat cells (black bars), IL37 OE Jurkat cells (gray bars), or human primary Treg cells (white bars) at Treg:Tresp ratios ranging from 4:1 to 1:2. Purified Tresp cells (0:1) without Jurkat or Treg cells were used as a positive control for Tresp proliferation without suppression (orange bars). Figure 14C shows a bar graph of ELISA analysis of IL-37 in the supernatants of vector control and IL37 OE Jurkat cells. 2 ng / ml and 1 ng / ml recombinant human IL-37 controls were used as positive controls. Data represent mean ± sem. NS, not significant (P>0.05); *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (Student's t-test). Data are representative of two independent experiments. Figure 14D shows a series of confocal microscopy images and Western blots showing IL-37 expression in Jurkat cells transduced with either empty vector (Empty), IL-37 vector (IL37), or IL-37D20A vector (D20A). IL-37 was stained with AF488. Cells were stained with DAPI. Western blots were performed on the same cells (Empty, IL37, D20A) as indicated. Cell lysates were probed with IL-37 antibody. Actin was detected as a control. Figure 14E shows a series of graphs showing CTLA4 and FOXP3 gene expression by qRT-PCR and FOXP3 surface expression by flow cytometry in empty Jurkat cells (Empt) and IL-37-expressing Jurkat cells (IL37 or D20A) compared to an IgG control (IgG). [Figure 14C]Figure 14A-B show a series of graphs demonstrating that IL-37-overexpressing Jurkat cells are highly suppressive in vitro. Figures 14A-B show a series of graphs from an in vitro T cell suppression assay using vector control and IL37 OE Jurkat cells. Histograms (Figure 14A) and % proliferation (Figure 14B) show the division of CFSE-labeled CD4+CD25- T cell responders (Tresp) purified from healthy human donor PBMCs and cultured for 5 days with anti-CD3 and either vector control Jurkat cells (black bars), IL37 OE Jurkat cells (gray bars), or human primary Treg cells (white bars) at Treg:Tresp ratios ranging from 4:1 to 1:2. Purified Tresp cells (0:1) without Jurkat or Treg cells were used as a positive control for Tresp proliferation without suppression (orange bars). Figure 14C shows a bar graph of ELISA analysis of IL-37 in the supernatants of vector control and IL37 OE Jurkat cells. 2 ng / ml and 1 ng / ml recombinant human IL-37 controls were used as positive controls. Data represent mean ± sem. NS, not significant (P>0.05); *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (Student's t-test). Data are representative of two independent experiments. Figure 14D shows a series of confocal microscopy images and Western blots showing IL-37 expression in Jurkat cells transduced with either empty vector (Empty), IL-37 vector (IL37), or IL-37D20A vector (D20A). IL-37 was stained with AF488. Cells were stained with DAPI. Western blots were performed on the same cells (Empty, IL37, D20A) as indicated. Cell lysates were probed with IL-37 antibody. Actin was detected as a control. Figure 14E shows a series of graphs showing CTLA4 and FOXP3 gene expression by qRT-PCR and FOXP3 surface expression by flow cytometry in empty Jurkat cells (Empt) and IL-37-expressing Jurkat cells (IL37 or D20A) compared to an IgG control (IgG). [Figure 14D]Figure 14A-B show a series of graphs demonstrating that IL-37-overexpressing Jurkat cells are highly suppressive in vitro. Figures 14A-B show a series of graphs from an in vitro T cell suppression assay using vector control and IL37 OE Jurkat cells. Histograms (Figure 14A) and % proliferation (Figure 14B) show the division of CFSE-labeled CD4+CD25- T cell responders (Tresp) purified from healthy human donor PBMCs and cultured for 5 days with anti-CD3 and either vector control Jurkat cells (black bars), IL37 OE Jurkat cells (gray bars), or human primary Treg cells (white bars) at Treg:Tresp ratios ranging from 4:1 to 1:2. Purified Tresp cells (0:1) without Jurkat or Treg cells were used as a positive control for Tresp proliferation without suppression (orange bars). Figure 14C shows a bar graph of ELISA analysis of IL-37 in the supernatants of vector control and IL37 OE Jurkat cells. 2 ng / ml and 1 ng / ml recombinant human IL-37 controls were used as positive controls. Data represent mean ± sem. NS, not significant (P>0.05); *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (Student's t-test). Data are representative of two independent experiments. Figure 14D shows a series of confocal microscopy images and Western blots showing IL-37 expression in Jurkat cells transduced with either empty vector (Empty), IL-37 vector (IL37), or IL-37D20A vector (D20A). IL-37 was stained with AF488. Cells were stained with DAPI. Western blots were performed on the same cells (Empty, IL37, D20A) as indicated. Cell lysates were probed with IL-37 antibody. Actin was detected as a control. Figure 14E shows a series of graphs showing CTLA4 and FOXP3 gene expression by qRT-PCR and FOXP3 surface expression by flow cytometry in empty Jurkat cells (Empt) and IL-37-expressing Jurkat cells (IL37 or D20A) compared to an IgG control (IgG). [Figure 14E]Figure 14A-B show a series of graphs demonstrating that IL-37-overexpressing Jurkat cells are highly suppressive in vitro. Figures 14A-B show a series of graphs from an in vitro T cell suppression assay using vector control and IL37 OE Jurkat cells. Histograms (Figure 14A) and % proliferation (Figure 14B) show the division of CFSE-labeled CD4+CD25- T cell responders (Tresp) purified from healthy human donor PBMCs and cultured for 5 days with anti-CD3 and either vector control Jurkat cells (black bars), IL37 OE Jurkat cells (gray bars), or human primary Treg cells (white bars) at Treg:Tresp ratios ranging from 4:1 to 1:2. Purified Tresp cells (0:1) without Jurkat or Treg cells were used as a positive control for Tresp proliferation without suppression (orange bars). Figure 14C shows a bar graph of ELISA analysis of IL-37 in the supernatants of vector control and IL37 OE Jurkat cells. 2 ng / ml and 1 ng / ml recombinant human IL-37 controls were used as positive controls. Data represent mean ± sem. NS, not significant (P>0.05); *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 (Student's t-test). Data are representative of two independent experiments. Figure 14D shows a series of confocal microscopy images and Western blots showing IL-37 expression in Jurkat cells transduced with either empty vector (Empty), IL-37 vector (IL37), or IL-37D20A vector (D20A). IL-37 was stained with AF488. Cells were stained with DAPI. Western blots were performed on the same cells (Empty, IL37, D20A) as indicated. Cell lysates were probed with IL-37 antibody. Actin was detected as a control. Figure 14E shows a series of graphs showing CTLA4 and FOXP3 gene expression by qRT-PCR and FOXP3 surface expression by flow cytometry in empty Jurkat cells (Empt) and IL-37-expressing Jurkat cells (IL37 or D20A) compared to an IgG control (IgG). [Figure 15A]Figure 15 shows a series of graphs illustrating the role of caspase-1 in regulating the nuclear localization of IL-37 in human Treg cells and its function in maintaining FOXP3 expression. Figure 15A shows histograms (left) and quantification (MFI) (right) of p-caspase-1 Ser376 in Tconv and Treg cells. [Figure 15B] Figure 15B shows a series of graphs depicting the role of caspase-1 in regulating the nuclear localization of IL-37 in human Treg cells and its function in maintaining FOXP3 expression. Figure 15B shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in resting Tconv and Treg cells. [Figure 15C] Figure 15C shows a series of graphs depicting the role of caspase-1 in regulating the nuclear localization of IL-37 in human Treg cells and its function in maintaining FOXP3 expression. Figure 15D shows Western blot detection of cleaved caspase-1 (cleaved-casp-1) from Tconv and Treg cell lysates. Actin was used as a control. [Figure 15D] Figure 15A shows a series of graphs depicting the role of caspase-1 in regulating the nuclear localization of IL-37 in human Treg cells and its function in maintaining FOXP3 expression. Figure 15B shows a histogram of p-SMAD3 Ser208 in Treg cells + / - caspase-1 inhibitor Ac-YVAD-CMK. Tconv cells were used as a control. [Figure 15E] Figure 15E shows a series of graphs depicting the role of caspase-1 in regulating the nuclear localization of IL-37 in human Treg cells and its function in maintaining FOXP3 expression. Figure 15E shows confocal microscopy images of unstimulated human Treg cells cultured for 24 hours in the absence (-) or presence (+) of 50 μM caspase-1 inhibitor (Ac-YVAD-CMK). Images of cells stained with DAPI, IL-37 (AF488), and an overlay of IL-37 and DAPI. On the far right, fluorescence intensity profiles, presented in arbitrary units (AU), ranging from α to ω (white lines in merged images), with a line corresponding to the image staining. Scale bar, 5 μm. [Figure 15F] Figure 15F shows a series of graphs depicting the role of caspase-1 in regulating the nuclear localization of IL-37 in human Treg cells and its function in maintaining FOXP3 expression. Figure 15F shows histograms (left) and quantification (right) of IL-37 MFI in human Treg cells after 48 hours of culture in the absence (-) or presence (+) of 50 μM caspase-1 inhibitor (Ac-YVAD-CMK). [Figure 16A] Figure 16A shows FOXP3 mRNA (Figure 16A) and protein (Figure 16B) expression in human Treg cells unstimulated (Unstim or -) or stimulated with anti-CD3 / CD28 (CD3 / CD28 or -) for 48 hours in the absence or presence of 50 μM caspase-1 inhibitor (Ac-YVAD-CMK). mRNA expression was assessed by qRT-PCR (Figure 16A). Protein quantification (Figure 16B, right) is shown by the values on the contour plot (Figure 16B, left). GAPDH served as an internal control. Figure 16C shows a bar graph of FOXP3 gene expression after siRNA knockdown of caspase-1 in a population of human CD4+ T cells. Gene expression was analyzed by qRT-PCR. GAPDH served as an internal control. Figure 16D shows a contour plot of FOXP3+ cell gating (left) and quantification of the percentage of FOXP3+ T cells (right) in human CD4+ T cells transfected 48 hours prior with scrambled siRNA (Scramble) and siCasp-1. Cell populations on the contour plot are identified within boxes. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 4-6 donors per group). **P < 0.01, ***P < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to four independent experiments. [Figure 16B]Figure 16A shows FOXP3 mRNA (Figure 16A) and protein (Figure 16B) expression in human Treg cells unstimulated (Unstim or -) or stimulated with anti-CD3 / CD28 (CD3 / CD28 or -) for 48 hours in the absence or presence of 50 μM caspase-1 inhibitor (Ac-YVAD-CMK). mRNA expression was assessed by qRT-PCR (Figure 16A). Protein quantification (Figure 16B, right) is shown by the values on the contour plot (Figure 16B, left). GAPDH served as an internal control. Figure 16C shows a bar graph of FOXP3 gene expression after siRNA knockdown of caspase-1 in a population of human CD4+ T cells. Gene expression was analyzed by qRT-PCR. GAPDH served as an internal control. Figure 16D shows a contour plot of FOXP3+ cell gating (left) and quantification of the percentage of FOXP3+ T cells (right) in human CD4+ T cells transfected 48 hours prior with scrambled siRNA (Scramble) and siCasp-1. Cell populations on the contour plot are identified within boxes. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 4-6 donors per group). **P < 0.01, ***P < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to four independent experiments. [Figure 16C]Figure 16A shows FOXP3 mRNA (Figure 16A) and protein (Figure 16B) expression in human Treg cells unstimulated (Unstim or -) or stimulated with anti-CD3 / CD28 (CD3 / CD28 or -) for 48 hours in the absence or presence of 50 μM caspase-1 inhibitor (Ac-YVAD-CMK). mRNA expression was assessed by qRT-PCR (Figure 16A). Protein quantification (Figure 16B, right) is shown by the values on the contour plot (Figure 16B, left). GAPDH served as an internal control. Figure 16C shows a bar graph of FOXP3 gene expression after siRNA knockdown of caspase-1 in a population of human CD4+ T cells. Gene expression was analyzed by qRT-PCR. GAPDH served as an internal control. Figure 16D shows a contour plot of FOXP3+ cell gating (left) and quantification of the percentage of FOXP3+ T cells (right) in human CD4+ T cells transfected 48 hours prior with scrambled siRNA (Scramble) and siCasp-1. Cell populations on the contour plot are identified within boxes. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 4-6 donors per group). **P < 0.01, ***P < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to four independent experiments. [Figure 16D]Figure 16A shows FOXP3 mRNA (Figure 16A) and protein (Figure 16B) expression in human Treg cells unstimulated (Unstim or -) or stimulated with anti-CD3 / CD28 (CD3 / CD28 or -) for 48 hours in the absence or presence of 50 μM caspase-1 inhibitor (Ac-YVAD-CMK). mRNA expression was assessed by qRT-PCR (Figure 16A). Protein quantification (Figure 16B, right) is shown by the values on the contour plot (Figure 16B, left). GAPDH served as an internal control. Figure 16C shows a bar graph of FOXP3 gene expression after siRNA knockdown of caspase-1 in a population of human CD4+ T cells. Gene expression was analyzed by qRT-PCR. GAPDH served as an internal control. Figure 16D shows a contour plot of FOXP3+ cell gating (left) and quantification of the percentage of FOXP3+ T cells (right) in human CD4+ T cells transfected 48 hours prior with scrambled siRNA (Scramble) and siCasp-1. Cell populations on the contour plot are identified within boxes. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 4-6 donors per group). **P < 0.01, ***P < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to four independent experiments. [Figure 17] Confocal microscopy images showing IL-37 expression and localization in human Treg cells. IL-37 was stained with AF488 (green). Treg cells were stained with DAPI. [Figure 18A]Figure 18 shows a series of histograms and bar graphs demonstrating in vitro T cell suppression assays of CFSE-labeled CD4+CD25-CD127+ T cell responders (Tresp) cultured with CD4+CD25+CD127- human Treg cells transfected with scrambled siRNA (black) or siIL37 (gray). Figure 18A shows a series of histograms demonstrating the division of Tresp cells cultured with human Treg cells transfected with the indicated siRNAs at ratios ranging from 2:1 to 32:1. Tresp cells cultured alone with anti-CD3 for 3 days served as a positive control (Tresp:Treg ratio of 1:0). Figure 18B shows a bar graph demonstrating the proliferation index of Tresp cells in the cultures shown in Figure 18A. Tresp:Treg cell ratios are as indicated. Figure 18C shows a bar graph demonstrating the percent suppression (%) of Tresp cell cultures as shown in Figure 18B. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 3-5 donors per group). Data represent the mean ± sem. *p<0.05, **p<0.01, ***p<0.001. Data are representative of three independent experiments. [Figure 18B]Figure 18 shows a series of histograms and bar graphs demonstrating in vitro T cell suppression assays of CFSE-labeled CD4+CD25-CD127+ T cell responders (Tresp) cultured with CD4+CD25+CD127- human Treg cells transfected with scrambled siRNA (black) or siIL37 (gray). Figure 18A shows a series of histograms demonstrating the division of Tresp cells cultured with human Treg cells transfected with the indicated siRNAs at ratios ranging from 2:1 to 32:1. Tresp cells cultured alone with anti-CD3 for 3 days served as a positive control (Tresp:Treg ratio of 1:0). Figure 18B shows a bar graph demonstrating the proliferation index of Tresp cells in the cultures shown in Figure 18A. Tresp:Treg cell ratios are as indicated. Figure 18C shows a bar graph demonstrating the percent suppression (%) of Tresp cell cultures as shown in Figure 18B. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 3-5 donors per group). Data represent the mean ± sem. *p<0.05, **p<0.01, ***p<0.001. Data are representative of three independent experiments. [Figure 18C]Figure 18 shows a series of histograms and bar graphs demonstrating in vitro T cell suppression assays of CFSE-labeled CD4+CD25-CD127+ T cell responders (Tresp) cultured with CD4+CD25+CD127- human Treg cells transfected with scrambled siRNA (black) or siIL37 (gray). Figure 18A shows a series of histograms demonstrating the division of Tresp cells cultured with human Treg cells transfected with the indicated siRNAs at ratios ranging from 2:1 to 32:1. Tresp cells cultured alone with anti-CD3 for 3 days served as a positive control (Tresp:Treg ratio of 1:0). Figure 18B shows a bar graph demonstrating the proliferation index of Tresp cells in the cultures shown in Figure 18A. Tresp:Treg cell ratios are as indicated. Figure 18C shows a bar graph demonstrating the percent suppression (%) of Tresp cell cultures as shown in Figure 18B. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 3-5 donors per group). Data represent the mean ± sem. *p<0.05, **p<0.01, ***p<0.001. Data are representative of three independent experiments. [Figure 19A]Figure 19A shows a series of graphs depicting mouse T cells and Treg cells from wild-type (WT) and IL-37 transgenic (Tg) mice. Figure 19A shows pseudocolor plots (left) and quantification (right) of thymocytes from 6- to 8-week-old WT or IL-37 Tg mice stained with anti-CD4, anti-CD8, anti-CD25, and anti-CD45 RB antibodies. Numbers in the dot plots indicate the percentage of cells with CD8 single-positive (SP) cells (top left), CD4 and CD8 double-positive (DP) cells (top right), CD4 and CD8 double-negative (DN) cells (bottom left), and CD4 SP cells (bottom right). Figure 19B shows quantification of splenocytes, CD4+, and CD4+CD25+ T cells from purified WT or IL-37 Tg mice. Figure 19C shows a histogram (left) and bar graph (right) showing Foxp3 expression in IL37 Tg Treg cells compared with WT Treg cells and isotype controls. Expression is shown as mean fluorescence intensity (MFI). Figure 19D shows a series of histograms showing in vitro T cell suppression assays of CFSE-labeled CD4+CD25- T cell responders (Tresp) from WT mice cultured for 3 days with anti-CD3 and Treg cells from WT or IL37 Tg mice at Treg:Tresp ratios of 1:2, 1:3, and 1:8. Data represent the mean ± sem. (n = 5–8 mice per group) (Figures 19A–19D). NS, not significant (P > 0.05), *P < 0.05 (Student's t-test). Data are representative of two to three independent experiments. [Figure 19B]Figure 19A shows a series of graphs depicting mouse T cells and Treg cells from wild-type (WT) and IL-37 transgenic (Tg) mice. Figure 19A shows pseudocolor plots (left) and quantification (right) of thymocytes from 6- to 8-week-old WT or IL-37 Tg mice stained with anti-CD4, anti-CD8, anti-CD25, and anti-CD45 RB antibodies. Numbers in the dot plots indicate the percentage of cells with CD8 single-positive (SP) cells (top left), CD4 and CD8 double-positive (DP) cells (top right), CD4 and CD8 double-negative (DN) cells (bottom left), and CD4 SP cells (bottom right). Figure 19B shows quantification of splenocytes, CD4+, and CD4+CD25+ T cells from purified WT or IL-37 Tg mice. Figure 19C shows a histogram (left) and bar graph (right) showing Foxp3 expression in IL37 Tg Treg cells compared with WT Treg cells and isotype controls. Expression is shown as mean fluorescence intensity (MFI). Figure 19D shows a series of histograms showing in vitro T cell suppression assays of CFSE-labeled CD4+CD25- T cell responders (Tresp) from WT mice cultured for 3 days with anti-CD3 and Treg cells from WT or IL37 Tg mice at Treg:Tresp ratios of 1:2, 1:3, and 1:8. Data represent the mean ± sem. (n = 5–8 mice per group) (Figures 19A–19D). NS, not significant (P > 0.05), *P < 0.05 (Student's t-test). Data are representative of two to three independent experiments. [Figure 19C]Figure 19A shows a series of graphs depicting mouse T cells and Treg cells from wild-type (WT) and IL-37 transgenic (Tg) mice. Figure 19A shows pseudocolor plots (left) and quantification (right) of thymocytes from 6- to 8-week-old WT or IL-37 Tg mice stained with anti-CD4, anti-CD8, anti-CD25, and anti-CD45 RB antibodies. Numbers in the dot plots indicate the percentage of cells with CD8 single-positive (SP) cells (top left), CD4 and CD8 double-positive (DP) cells (top right), CD4 and CD8 double-negative (DN) cells (bottom left), and CD4 SP cells (bottom right). Figure 19B shows quantification of splenocytes, CD4+, and CD4+CD25+ T cells from purified WT or IL-37 Tg mice. Figure 19C shows a histogram (left) and bar graph (right) showing Foxp3 expression in IL37 Tg Treg cells compared with WT Treg cells and isotype controls. Expression is shown as mean fluorescence intensity (MFI). Figure 19D shows a series of histograms showing in vitro T cell suppression assays of CFSE-labeled CD4+CD25- T cell responders (Tresp) from WT mice cultured for 3 days with anti-CD3 and Treg cells from WT or IL37 Tg mice at Treg:Tresp ratios of 1:2, 1:3, and 1:8. Data represent the mean ± sem. (n = 5–8 mice per group) (Figures 19A–19D). NS, not significant (P > 0.05), *P < 0.05 (Student's t-test). Data are representative of two to three independent experiments. [Figure 19D]Figure 19A shows a series of graphs depicting mouse T cells and Treg cells from wild-type (WT) and IL-37 transgenic (Tg) mice. Figure 19A shows pseudocolor plots (left) and quantification (right) of thymocytes from 6- to 8-week-old WT or IL-37 Tg mice stained with anti-CD4, anti-CD8, anti-CD25, and anti-CD45 RB antibodies. Numbers in the dot plots indicate the percentage of cells with CD8 single-positive (SP) cells (top left), CD4 and CD8 double-positive (DP) cells (top right), CD4 and CD8 double-negative (DN) cells (bottom left), and CD4 SP cells (bottom right). Figure 19B shows quantification of splenocytes, CD4+, and CD4+CD25+ T cells from purified WT or IL-37 Tg mice. Figure 19C shows a histogram (left) and bar graph (right) showing Foxp3 expression in IL37 Tg Treg cells compared with WT Treg cells and isotype controls. Expression is shown as mean fluorescence intensity (MFI). Figure 19D shows a series of histograms showing in vitro T cell suppression assays of CFSE-labeled CD4+CD25- T cell responders (Tresp) from WT mice cultured for 3 days with anti-CD3 and Treg cells from WT or IL37 Tg mice at Treg:Tresp ratios of 1:2, 1:3, and 1:8. Data represent the mean ± sem. (n = 5–8 mice per group) (Figures 19A–19D). NS, not significant (P > 0.05), *P < 0.05 (Student's t-test). Data are representative of two to three independent experiments. [Figure 20A]Figure 20A shows a series of graphs depicting the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vivo. Figure 20A shows contour plots (top) and frequencies (bottom) of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) and Tconv cells (CD4+CD25loCD45RBhi) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg or IL37 Tg Treg cells. Numbers in the dot plots indicate the percentage of CFSE+ cells among Tconv or Treg cells. Figures 20B-20E show a series of analyses from ear specimens (Figures 20B, 20C) and cervical lymph nodes (Figures 20D, 20E) collected 48 hours after DNFB challenge from mice adoptively transferred with PBS (DNFB + PBS), WT-Tregs (DNFB + WT-Tregs), or IL37 Tg Tregs (DNFB + IL37 Tg-Tregs). Figures 20B and 20D show pie charts depicting the frequencies of CD8+, CD4+, and CD8-CD4- (DN) cells in CD3+ cells (top), and the frequencies of Tconv (CD4+CD25loCD45RBhi) and Treg (CD4+CD25hiCD45RBlo) cells in the CD4+ cell population (bottom). Figures 20C and 20E show bar graphs depicting cell counts and quantification of CD4+ and CD8+ T cells collected 48 hours after vehicle or DNFB challenge (+) from mice adoptively transferred with PBS, WT-Treg, or IL37 Tg Treg cells. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. (n = 3-5 mice / donor per group). *p<0.05, **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two independent experiments. [Figure 20B]Figure 20A shows a series of graphs depicting the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vivo. Figure 20A shows contour plots (top) and frequencies (bottom) of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) and Tconv cells (CD4+CD25loCD45RBhi) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg or IL37 Tg Treg cells. Numbers in the dot plots indicate the percentage of CFSE+ cells among Tconv or Treg cells. Figures 20B-20E show a series of analyses from ear specimens (Figures 20B, 20C) and cervical lymph nodes (Figures 20D, 20E) collected 48 hours after DNFB challenge from mice adoptively transferred with PBS (DNFB + PBS), WT-Tregs (DNFB + WT-Tregs), or IL37 Tg Tregs (DNFB + IL37 Tg-Tregs). Figures 20B and 20D show pie charts depicting the frequencies of CD8+, CD4+, and CD8-CD4- (DN) cells in CD3+ cells (top), and the frequencies of Tconv (CD4+CD25loCD45RBhi) and Treg (CD4+CD25hiCD45RBlo) cells in the CD4+ cell population (bottom). Figures 20C and 20E show bar graphs depicting cell counts and quantification of CD4+ and CD8+ T cells collected 48 hours after vehicle or DNFB challenge (+) from mice adoptively transferred with PBS, WT-Treg, or IL37 Tg Treg cells. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. (n = 3-5 mice / donor per group). *p<0.05, **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two independent experiments. [Figure 20C]Figure 20A shows a series of graphs depicting the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vivo. Figure 20A shows contour plots (top) and frequencies (bottom) of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) and Tconv cells (CD4+CD25loCD45RBhi) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg or IL37 Tg Treg cells. Numbers in the dot plots indicate the percentage of CFSE+ cells among Tconv or Treg cells. Figures 20B-20E show a series of analyses from ear specimens (Figures 20B, 20C) and cervical lymph nodes (Figures 20D, 20E) collected 48 hours after DNFB challenge from mice adoptively transferred with PBS (DNFB + PBS), WT-Tregs (DNFB + WT-Tregs), or IL37 Tg Tregs (DNFB + IL37 Tg-Tregs). Figures 20B and 20D show pie charts depicting the frequencies of CD8+, CD4+, and CD8-CD4- (DN) cells in CD3+ cells (top), and the frequencies of Tconv (CD4+CD25loCD45RBhi) and Treg (CD4+CD25hiCD45RBlo) cells in the CD4+ cell population (bottom). Figures 20C and 20E show bar graphs depicting cell counts and quantification of CD4+ and CD8+ T cells collected 48 hours after vehicle or DNFB challenge (+) from mice adoptively transferred with PBS, WT-Treg, or IL37 Tg Treg cells. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. (n = 3-5 mice / donor per group). *p<0.05, **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two independent experiments. [Figure 20D]Figure 20A shows a series of graphs depicting the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vivo. Figure 20A shows contour plots (top) and frequencies (bottom) of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) and Tconv cells (CD4+CD25loCD45RBhi) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg or IL37 Tg Treg cells. Numbers in the dot plots indicate the percentage of CFSE+ cells among Tconv or Treg cells. Figures 20B-20E show a series of analyses from ear specimens (Figures 20B, 20C) and cervical lymph nodes (Figures 20D, 20E) collected 48 hours after DNFB challenge from mice adoptively transferred with PBS (DNFB + PBS), WT-Tregs (DNFB + WT-Tregs), or IL37 Tg Tregs (DNFB + IL37 Tg-Tregs). Figures 20B and 20D show pie charts depicting the frequencies of CD8+, CD4+, and CD8-CD4- (DN) cells in CD3+ cells (top), and the frequencies of Tconv (CD4+CD25loCD45RBhi) and Treg (CD4+CD25hiCD45RBlo) cells in the CD4+ cell population (bottom). Figures 20C and 20E show bar graphs depicting cell counts and quantification of CD4+ and CD8+ T cells collected 48 hours after vehicle or DNFB challenge (+) from mice adoptively transferred with PBS, WT-Treg, or IL37 Tg Treg cells. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. (n = 3-5 mice / donor per group). *p<0.05, **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two independent experiments. [Figure 20E]Figure 20A shows a series of graphs depicting the role of elevated IL-37 in preventing the conversion of Treg cells to Tconv cells under inflammatory conditions in vivo. Figure 20A shows contour plots (top) and frequencies (bottom) of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) and Tconv cells (CD4+CD25loCD45RBhi) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg or IL37 Tg Treg cells. Numbers in the dot plots indicate the percentage of CFSE+ cells among Tconv or Treg cells. Figures 20B-20E show a series of analyses from ear specimens (Figures 20B, 20C) and cervical lymph nodes (Figures 20D, 20E) collected 48 hours after DNFB challenge from mice adoptively transferred with PBS (DNFB + PBS), WT-Tregs (DNFB + WT-Tregs), or IL37 Tg Tregs (DNFB + IL37 Tg-Tregs). Figures 20B and 20D show pie charts depicting the frequencies of CD8+, CD4+, and CD8-CD4- (DN) cells in CD3+ cells (top), and the frequencies of Tconv (CD4+CD25loCD45RBhi) and Treg (CD4+CD25hiCD45RBlo) cells in the CD4+ cell population (bottom). Figures 20C and 20E show bar graphs depicting cell counts and quantification of CD4+ and CD8+ T cells collected 48 hours after vehicle or DNFB challenge (+) from mice adoptively transferred with PBS, WT-Treg, or IL37 Tg Treg cells. Each symbol represents an individual donor / mouse. Small horizontal lines indicate the mean ± sem. (n = 3-5 mice / donor per group). *p<0.05, **p<0.01, ***p<0.001 (Student's t-test). Data are representative of two independent experiments. [Figure 21A]Figure 21A shows a series of graphs depicting the screening protocol and FOXP3 expression analysis in human Treg cells. Figure 21A illustrates the screening protocol to determine which proteins associate with FOXP3 transcription factors (NFAT, Smad3, and STAT5) and further analyze their role in FOXP3 expression and human Treg cell function. Figure 21B shows qRT-PCR analysis of the remaining 15 of 20 proteins associated with FOXP3 transcription factors (NFAT, Smad3, and STAT5) in resting Tconv and Treg cells. GAPDH served as an internal control. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem (n = 4 donors per group). NS, not significant (P > 0.05), *P < 0.05 (Student's t-test). Data are representative of four independent experiments. [Figure 21B] Figures 21B-1 and 21B-2 show a series of graphs depicting the screening protocol and FOXP3 expression analysis in human Treg cells. Figure 21A shows the screening protocol for determining which proteins associate with FOXP3 transcription factors (NFAT, Smad3, and STAT5) and further analyzing their role in FOXP3 expression and human Treg cell function. Figure 21B shows qRT-PCR analysis of the remaining 15 of 20 proteins associated with FOXP3 transcription factors (NFAT, Smad3, and STAT5) in resting Tconv and Treg cells. GAPDH served as an internal control. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem (n = 4 donors per group). NS, not significant (P > 0.05), *P < 0.05 (Student's t-test). Data are representative of four independent experiments. [Figure 22A]Figure 22A shows a series of graphs demonstrating that FOXP3 does not regulate IL-37 expression in human Treg cells. Figure 22A shows qRT-PCR analysis of FOXP3 (left) and IL-37 (right) in human Treg cells 24 hours after transfection with scrambled siRNA (Ctrl) or pooled siRNA against FOXP3. Human Tconv cells were used as a reference for negative FOXP3 expression. GAPDH served as an internal control. Figures 22B-C show the expression of CTLA-4 (Figure 22B) and IL-37 (Figure 22C) in human Treg cells transfected with scrambled siRNA (Ctrl) or pooled siRNA against FOXP3 48 hours prior and then cultured for 24 hours in the absence (Unstim or -) or presence (CD3 / CD28 or +) of anti-CD3 / CD28. Histograms and quantification (right) of unstimulated (left) and stimulated (center) Treg cells are shown. Tconv cells were used as a reference. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 3-5 donors per group) (Figures 22A-C). NS, not significant (P > 0.05), *P < 0.05, **P < 0.01 (Student's t-test). Data are representative of three independent experiments. [Figure 22B]Figure 22A shows a series of graphs demonstrating that FOXP3 does not regulate IL-37 expression in human Treg cells. Figure 22A shows qRT-PCR analysis of FOXP3 (left) and IL-37 (right) in human Treg cells 24 hours after transfection with scrambled siRNA (Ctrl) or pooled siRNA against FOXP3. Human Tconv cells were used as a reference for negative FOXP3 expression. GAPDH served as an internal control. Figures 22B-C show the expression of CTLA-4 (Figure 22B) and IL-37 (Figure 22C) in human Treg cells transfected with scrambled siRNA (Ctrl) or pooled siRNA against FOXP3 48 hours prior and then cultured for 24 hours in the absence (Unstim or -) or presence (CD3 / CD28 or +) of anti-CD3 / CD28. Histograms and quantification (right) of unstimulated (left) and stimulated (center) Treg cells are shown. Tconv cells were used as a reference. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 3-5 donors per group) (Figures 22A-C). NS, not significant (P > 0.05), *P < 0.05, **P < 0.01 (Student's t-test). Data are representative of three independent experiments. [Figure 22C]Figure 22A shows a series of graphs demonstrating that FOXP3 does not regulate IL-37 expression in human Treg cells. Figure 22A shows qRT-PCR analysis of FOXP3 (left) and IL-37 (right) in human Treg cells 24 hours after transfection with scrambled siRNA (Ctrl) or pooled siRNA against FOXP3. Human Tconv cells were used as a reference for negative FOXP3 expression. GAPDH served as an internal control. Figures 22B-C show the expression of CTLA-4 (Figure 22B) and IL-37 (Figure 22C) in human Treg cells transfected with scrambled siRNA (Ctrl) or pooled siRNA against FOXP3 48 hours prior and then cultured for 24 hours in the absence (Unstim or -) or presence (CD3 / CD28 or +) of anti-CD3 / CD28. Histograms and quantification (right) of unstimulated (left) and stimulated (center) Treg cells are shown. Tconv cells were used as a reference. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 3-5 donors per group) (Figures 22A-C). NS, not significant (P > 0.05), *P < 0.05, **P < 0.01 (Student's t-test). Data are representative of three independent experiments. [Figure 23A]A series of graphs depicting IL37-expressing cells in human PBMCs using the flow cytometry-based PrimeFlow® RNA assay. Figures 23A-23C show histograms (Figure 23A) and quantification by frequency (Figure 23B) and mean fluorescence intensity (MFI) (Figure 23C) of IL37 mRNA expression in myeloid and lymphoid cell subsets. PBMCs were treated for 24 hours in the absence (-, black line or filled symbols) or presence (+, gray line or open symbols) of 100 ng / ml LPS. Gray histograms represent the fluorescence of the labeled probe alone (background) for each immune cell subset as a negative control. Gating to determine positive cell frequency was established to exclude approximately 99% of control events as negative. RPL13a mRNA served as an internal control (not shown). Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem (n = 5 donors per group) (Figures 23A-C). NS, not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). Data are representative of two independent experiments. [Figure 23B]A series of graphs depicting IL37-expressing cells in human PBMCs using the flow cytometry-based PrimeFlow® RNA assay. Figures 23A-23C show histograms (Figure 23A) and quantification by frequency (Figure 23B) and mean fluorescence intensity (MFI) (Figure 23C) of IL37 mRNA expression in myeloid and lymphoid cell subsets. PBMCs were treated for 24 hours in the absence (-, black line or filled symbols) or presence (+, gray line or open symbols) of 100 ng / ml LPS. Gray histograms represent the fluorescence of the labeled probe alone (background) for each immune cell subset as a negative control. Gating to determine positive cell frequency was established to exclude approximately 99% of control events as negative. RPL13a mRNA served as an internal control (not shown). Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem (n = 5 donors per group) (Figures 23A-C). NS, not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). Data are representative of two independent experiments. [Figure 23C]A series of graphs depicting IL37-expressing cells in human PBMCs using the flow cytometry-based PrimeFlow® RNA assay. Figures 23A-23C show histograms (Figure 23A) and quantification by frequency (Figure 23B) and mean fluorescence intensity (MFI) (Figure 23C) of IL37 mRNA expression in myeloid and lymphoid cell subsets. PBMCs were treated for 24 hours in the absence (-, black line or filled symbols) or presence (+, gray line or open symbols) of 100 ng / ml LPS. Gray histograms represent the fluorescence of the labeled probe alone (background) for each immune cell subset as a negative control. Gating to determine positive cell frequency was established to exclude approximately 99% of control events as negative. RPL13a mRNA served as an internal control (not shown). Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem (n = 5 donors per group) (Figures 23A-C). NS, not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t-test). Data are representative of two independent experiments. [Figure 24A]Figure 24 shows a series of images and graphs demonstrating that IL-37 interacts with p-SMAD3 S208, translocates into the nucleus, and induces FOXP3 expression in human Treg cells. Figure 24A shows a schematic diagram of SMAD3 phosphorylation and IL-37 interaction. Figure 24B shows a bar graph of FOXP3 in purified human Treg cells treated with the Smad3 inhibitor SIS3 and flavopiridol. GAPDH served as an internal control. Figure 24C shows a pseudocolor plot of FOXP3 expression in purified human CD4+ T cells + / - TGF-β treated with control, SMAD3 inhibitor SIS3, and flavopiridol. Figure 24D shows Western blot analysis of SMAD3 phosphorylation, IL-37, and FOXP3 expression in the nucleus and cytoplasm of human Tregs treated with flavopiridol. Tubulin was used as a cytoplasmic control, and FOXP3 was used as a nuclear control. Each symbol represents an individual donor. Small horizontal lines indicate mean ± sem (n = 3-5 donors per group). **p<0.01. (Student's t-test). Data are representative of 2-4 independent experiments. [Figure 24B] Figure 24 shows a series of images and graphs demonstrating that IL-37 interacts with p-SMAD3 S208, translocates into the nucleus, and induces FOXP3 expression in human Treg cells. Figure 24A shows a schematic diagram of SMAD3 phosphorylation and IL-37 interaction. Figure 24B shows a bar graph of FOXP3 in purified human Treg cells treated with the Smad3 inhibitor SIS3 and flavopiridol. GAPDH served as an internal control. Figure 24C shows a pseudocolor plot of FOXP3 expression in purified human CD4+ T cells + / - TGF-β treated with control, SMAD3 inhibitor SIS3, and flavopiridol. Figure 24D shows Western blot analysis of SMAD3 phosphorylation, IL-37, and FOXP3 expression in the nucleus and cytoplasm of human Tregs treated with flavopiridol. Tubulin was used as a cytoplasmic control, and FOXP3 was used as a nuclear control. Each symbol represents an individual donor. Small horizontal lines indicate mean ± sem (n = 3-5 donors per group). **p<0.01. (Student's t-test). Data are representative of 2-4 independent experiments. [Figure 24C] Figure 24 shows a series of images and graphs demonstrating that IL-37 interacts with p-SMAD3 S208, translocates into the nucleus, and induces FOXP3 expression in human Treg cells. Figure 24A shows a schematic diagram of SMAD3 phosphorylation and IL-37 interaction. Figure 24B shows a bar graph of FOXP3 in purified human Treg cells treated with the Smad3 inhibitor SIS3 and flavopiridol. GAPDH served as an internal control. Figure 24C shows a pseudocolor plot of FOXP3 expression in purified human CD4+ T cells + / - TGF-β treated with control, SMAD3 inhibitor SIS3, and flavopiridol. Figure 24D shows Western blot analysis of SMAD3 phosphorylation, IL-37, and FOXP3 expression in the nucleus and cytoplasm of human Tregs treated with flavopiridol. Tubulin was used as a cytoplasmic control, and FOXP3 was used as a nuclear control. Each symbol represents an individual donor. Small horizontal lines indicate mean ± sem (n = 3-5 donors per group). **p<0.01. (Student's t-test). Data are representative of 2-4 independent experiments. [Figure 24D] Figure 24 shows a series of images and graphs demonstrating that IL-37 interacts with p-SMAD3 S208, translocates into the nucleus, and induces FOXP3 expression in human Treg cells. Figure 24A shows a schematic diagram of SMAD3 phosphorylation and IL-37 interaction. Figure 24B shows a bar graph of FOXP3 in purified human Treg cells treated with the Smad3 inhibitor SIS3 and flavopiridol. GAPDH served as an internal control. Figure 24C shows a pseudocolor plot of FOXP3 expression in purified human CD4+ T cells + / - TGF-β treated with control, SMAD3 inhibitor SIS3, and flavopiridol. Figure 24D shows Western blot analysis of SMAD3 phosphorylation, IL-37, and FOXP3 expression in the nucleus and cytoplasm of human Tregs treated with flavopiridol. Tubulin was used as a cytoplasmic control, and FOXP3 was used as a nuclear control. Each symbol represents an individual donor. Small horizontal lines indicate mean ± sem (n = 3-5 donors per group). **p<0.01. (Student's t-test). Data are representative of 2-4 independent experiments. [Figure 25A]A series of graphs showing the NLRC4 inflammasome and upstream signaling required for IL-37-directed FOXP3 expression in human Treg cells are shown. Figures 25A-25B show heatmaps / clustergrams (Figure 25A) and scatterplots (Figure 25B) of components of the inflammasome pathway and mRNA expression in purified human Tconv and Treg cells using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / . Expression of 84 genes related to the inflammasome was measured using a Qiagen RT2 Profiler PCR Inflammasome array according to the manufacturer's instructions. Scatterplots showing two-fold boundaries for the expression of inflammasome genes in Tconv and Treg cells. Figure 25C shows a bar graph of NAIP expression in unstimulated Tconv and Treg cells. Figure 25D shows histograms (top) and quantification (bottom) showing expression in Tconv (black) and Treg (gray) cells. Figure 25E shows immunoblotting of NLRC4 in Tconv and scrambled control and siNLRC4-transfected primary human CD4+CD25+CD127dim Treg cells. Figure 25F shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in Tconv and Treg cells. Figure 25G shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in scrambled control and siASC-, siNLRP2-, and siNLRC4-transfected CD4+CD25+CD127dim Treg cells, as well as the quantification of the % caspase-1+ Treg cells (far right). Figure 25H shows a bar graph of FOXP3 gene expression after siRNA knockdown of NLRC4 in purified CD4+CD25+CD127dim Treg cells. GAPDH served as an internal control.Figure 25I shows a pseudocolor plot (left) showing the percentage of CD4+FOXP3+ Treg cells in a population of purified CD4+ T cells 48 hours after NLRC4 siRNA knockdown, and its quantification of %FOXP3+CD4+ T cells (right). Figure 25J shows confocal microscopy images of proximity ligation assays of ASC and NLRC4 (dots) in Treg cells. DNA intercalating dye DAPI. Scale bar, 5 μm. Small horizontal lines indicate mean ± s.e.m. (n = 3 donors per group) (Figures 25A, 25B), (n = 5-6 donors per group) (Figures 25C-25I), and (n ≥ 100 cells / group (Figure 25J)). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 25B]A series of graphs showing the NLRC4 inflammasome and upstream signaling required for IL-37-directed FOXP3 expression in human Treg cells are shown. Figures 25A-25B show heatmaps / clustergrams (Figure 25A) and scatterplots (Figure 25B) of components of the inflammasome pathway and mRNA expression in purified human Tconv and Treg cells using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / . Expression of 84 genes related to the inflammasome was measured using a Qiagen RT2 Profiler PCR Inflammasome array according to the manufacturer's instructions. Scatterplots showing two-fold boundaries for the expression of inflammasome genes in Tconv and Treg cells. Figure 25C shows a bar graph of NAIP expression in unstimulated Tconv and Treg cells. Figure 25D shows histograms (top) and quantification (bottom) showing expression in Tconv (black) and Treg (gray) cells. Figure 25E shows immunoblotting of NLRC4 in Tconv and scrambled control and siNLRC4-transfected primary human CD4+CD25+CD127dim Treg cells. Figure 25F shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in Tconv and Treg cells. Figure 25G shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in scrambled control and siASC-, siNLRP2-, and siNLRC4-transfected CD4+CD25+CD127dim Treg cells, as well as the quantification of the % caspase-1+ Treg cells (far right). Figure 25H shows a bar graph of FOXP3 gene expression after siRNA knockdown of NLRC4 in purified CD4+CD25+CD127dim Treg cells. GAPDH served as an internal control.Figure 25I shows a pseudocolor plot (left) showing the percentage of CD4+FOXP3+ Treg cells in a population of purified CD4+ T cells 48 hours after NLRC4 siRNA knockdown, and its quantification of %FOXP3+CD4+ T cells (right). Figure 25J shows confocal microscopy images of proximity ligation assays of ASC and NLRC4 (dots) in Treg cells. DNA intercalating dye DAPI. Scale bar, 5 μm. Small horizontal lines indicate mean ± s.e.m. (n = 3 donors per group) (Figures 25A, 25B), (n = 5-6 donors per group) (Figures 25C-25I), and (n ≥ 100 cells / group (Figure 25J)). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 25C]A series of graphs showing the NLRC4 inflammasome and upstream signaling required for IL-37-directed FOXP3 expression in human Treg cells are shown. Figures 25A-25B show heatmaps / clustergrams (Figure 25A) and scatterplots (Figure 25B) of components of the inflammasome pathway and mRNA expression in purified human Tconv and Treg cells using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / . Expression of 84 genes related to the inflammasome was measured using a Qiagen RT2 Profiler PCR Inflammasome array according to the manufacturer's instructions. Scatterplots showing two-fold boundaries for the expression of inflammasome genes in Tconv and Treg cells. Figure 25C shows a bar graph of NAIP expression in unstimulated Tconv and Treg cells. Figure 25D shows histograms (top) and quantification (bottom) showing expression in Tconv (black) and Treg (gray) cells. Figure 25E shows immunoblotting of NLRC4 in Tconv and scrambled control and siNLRC4-transfected primary human CD4+CD25+CD127dim Treg cells. Figure 25F shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in Tconv and Treg cells. Figure 25G shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in scrambled control and siASC-, siNLRP2-, and siNLRC4-transfected CD4+CD25+CD127dim Treg cells, as well as the quantification of the % caspase-1+ Treg cells (far right). Figure 25H shows a bar graph of FOXP3 gene expression after siRNA knockdown of NLRC4 in purified CD4+CD25+CD127dim Treg cells. GAPDH served as an internal control.Figure 25I shows a pseudocolor plot (left) showing the percentage of CD4+FOXP3+ Treg cells in a population of purified CD4+ T cells 48 hours after NLRC4 siRNA knockdown, and its quantification of %FOXP3+CD4+ T cells (right). Figure 25J shows confocal microscopy images of proximity ligation assays of ASC and NLRC4 (dots) in Treg cells. DNA intercalating dye DAPI. Scale bar, 5 μm. Small horizontal lines indicate mean ± s.e.m. (n = 3 donors per group) (Figures 25A, 25B), (n = 5-6 donors per group) (Figures 25C-25I), and (n ≥ 100 cells / group (Figure 25J)). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 25D]A series of graphs showing the NLRC4 inflammasome and upstream signaling required for IL-37-directed FOXP3 expression in human Treg cells are shown. Figures 25A-25B show heatmaps / clustergrams (Figure 25A) and scatterplots (Figure 25B) of components of the inflammasome pathway and mRNA expression in purified human Tconv and Treg cells using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / . Expression of 84 genes related to the inflammasome was measured using a Qiagen RT2 Profiler PCR Inflammasome array according to the manufacturer's instructions. Scatterplots showing two-fold boundaries for the expression of inflammasome genes in Tconv and Treg cells. Figure 25C shows a bar graph of NAIP expression in unstimulated Tconv and Treg cells. Figure 25D shows histograms (top) and quantification (bottom) showing expression in Tconv (black) and Treg (gray) cells. Figure 25E shows immunoblotting of NLRC4 in Tconv and scrambled control and siNLRC4-transfected primary human CD4+CD25+CD127dim Treg cells. Figure 25F shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in Tconv and Treg cells. Figure 25G shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in scrambled control and siASC-, siNLRP2-, and siNLRC4-transfected CD4+CD25+CD127dim Treg cells, as well as the quantification of the % caspase-1+ Treg cells (far right). Figure 25H shows a bar graph of FOXP3 gene expression after siRNA knockdown of NLRC4 in purified CD4+CD25+CD127dim Treg cells. GAPDH served as an internal control.Figure 25I shows a pseudocolor plot (left) showing the percentage of CD4+FOXP3+ Treg cells in a population of purified CD4+ T cells 48 hours after NLRC4 siRNA knockdown, and its quantification of %FOXP3+CD4+ T cells (right). Figure 25J shows confocal microscopy images of proximity ligation assays of ASC and NLRC4 (dots) in Treg cells. DNA intercalating dye DAPI. Scale bar, 5 μm. Small horizontal lines indicate mean ± s.e.m. (n = 3 donors per group) (Figures 25A, 25B), (n = 5-6 donors per group) (Figures 25C-25I), and (n ≥ 100 cells / group (Figure 25J)). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 25E]A series of graphs showing the NLRC4 inflammasome and upstream signaling required for IL-37-directed FOXP3 expression in human Treg cells are shown. Figures 25A-25B show heatmaps / clustergrams (Figure 25A) and scatterplots (Figure 25B) of components of the inflammasome pathway and mRNA expression in purified human Tconv and Treg cells using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / . Expression of 84 genes related to the inflammasome was measured using a Qiagen RT2 Profiler PCR Inflammasome array according to the manufacturer's instructions. Scatterplots showing two-fold boundaries for the expression of inflammasome genes in Tconv and Treg cells. Figure 25C shows a bar graph of NAIP expression in unstimulated Tconv and Treg cells. Figure 25D shows histograms (top) and quantification (bottom) showing expression in Tconv (black) and Treg (gray) cells. Figure 25E shows immunoblotting of NLRC4 in Tconv and scrambled control and siNLRC4-transfected primary human CD4+CD25+CD127dim Treg cells. Figure 25F shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in Tconv and Treg cells. Figure 25G shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in scrambled control and siASC-, siNLRP2-, and siNLRC4-transfected CD4+CD25+CD127dim Treg cells, as well as the quantification of the % caspase-1+ Treg cells (far right). Figure 25H shows a bar graph of FOXP3 gene expression after siRNA knockdown of NLRC4 in purified CD4+CD25+CD127dim Treg cells. GAPDH served as an internal control.Figure 25I shows a pseudocolor plot (left) showing the percentage of CD4+FOXP3+ Treg cells in a population of purified CD4+ T cells 48 hours after NLRC4 siRNA knockdown, and its quantification of %FOXP3+CD4+ T cells (right). Figure 25J shows confocal microscopy images of proximity ligation assays of ASC and NLRC4 (dots) in Treg cells. DNA intercalating dye DAPI. Scale bar, 5 μm. Small horizontal lines indicate mean ± s.e.m. (n = 3 donors per group) (Figures 25A, 25B), (n = 5-6 donors per group) (Figures 25C-25I), and (n ≥ 100 cells / group (Figure 25J)). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 25F]A series of graphs showing the NLRC4 inflammasome and upstream signaling required for IL-37-directed FOXP3 expression in human Treg cells are shown. Figures 25A-25B show heatmaps / clustergrams (Figure 25A) and scatterplots (Figure 25B) of components of the inflammasome pathway and mRNA expression in purified human Tconv and Treg cells using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / . Expression of 84 genes related to the inflammasome was measured using a Qiagen RT2 Profiler PCR Inflammasome array according to the manufacturer's instructions. Scatterplots showing two-fold boundaries for the expression of inflammasome genes in Tconv and Treg cells. Figure 25C shows a bar graph of NAIP expression in unstimulated Tconv and Treg cells. Figure 25D shows histograms (top) and quantification (bottom) showing expression in Tconv (black) and Treg (gray) cells. Figure 25E shows immunoblotting of NLRC4 in Tconv and scrambled control and siNLRC4-transfected primary human CD4+CD25+CD127dim Treg cells. Figure 25F shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in Tconv and Treg cells. Figure 25G shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in scrambled control and siASC-, siNLRP2-, and siNLRC4-transfected CD4+CD25+CD127dim Treg cells, as well as the quantification of the % caspase-1+ Treg cells (far right). Figure 25H shows a bar graph of FOXP3 gene expression after siRNA knockdown of NLRC4 in purified CD4+CD25+CD127dim Treg cells. GAPDH served as an internal control.Figure 25I shows a pseudocolor plot (left) showing the percentage of CD4+FOXP3+ Treg cells in a population of purified CD4+ T cells 48 hours after NLRC4 siRNA knockdown, and its quantification of %FOXP3+CD4+ T cells (right). Figure 25J shows confocal microscopy images of proximity ligation assays of ASC and NLRC4 (dots) in Treg cells. DNA intercalating dye DAPI. Scale bar, 5 μm. Small horizontal lines indicate mean ± s.e.m. (n = 3 donors per group) (Figures 25A, 25B), (n = 5-6 donors per group) (Figures 25C-25I), and (n ≥ 100 cells / group (Figure 25J)). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 25G]A series of graphs showing the NLRC4 inflammasome and upstream signaling required for IL-37-directed FOXP3 expression in human Treg cells are shown. Figures 25A-25B show heatmaps / clustergrams (Figure 25A) and scatterplots (Figure 25B) of components of the inflammasome pathway and mRNA expression in purified human Tconv and Treg cells using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / . Expression of 84 genes related to the inflammasome was measured using a Qiagen RT2 Profiler PCR Inflammasome array according to the manufacturer's instructions. Scatterplots showing two-fold boundaries for the expression of inflammasome genes in Tconv and Treg cells. Figure 25C shows a bar graph of NAIP expression in unstimulated Tconv and Treg cells. Figure 25D shows histograms (top) and quantification (bottom) showing expression in Tconv (black) and Treg (gray) cells. Figure 25E shows immunoblotting of NLRC4 in Tconv and scrambled control and siNLRC4-transfected primary human CD4+CD25+CD127dim Treg cells. Figure 25F shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in Tconv and Treg cells. Figure 25G shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in scrambled control and siASC-, siNLRP2-, and siNLRC4-transfected CD4+CD25+CD127dim Treg cells, as well as the quantification of the % caspase-1+ Treg cells (far right). Figure 25H shows a bar graph of FOXP3 gene expression after siRNA knockdown of NLRC4 in purified CD4+CD25+CD127dim Treg cells. GAPDH served as an internal control.Figure 25I shows a pseudocolor plot (left) showing the percentage of CD4+FOXP3+ Treg cells in a population of purified CD4+ T cells 48 hours after NLRC4 siRNA knockdown, and its quantification of %FOXP3+CD4+ T cells (right). Figure 25J shows confocal microscopy images of proximity ligation assays of ASC and NLRC4 (dots) in Treg cells. DNA intercalating dye DAPI. Scale bar, 5 μm. Small horizontal lines indicate mean ± s.e.m. (n = 3 donors per group) (Figures 25A, 25B), (n = 5-6 donors per group) (Figures 25C-25I), and (n ≥ 100 cells / group (Figure 25J)). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 25H]A series of graphs showing the NLRC4 inflammasome and upstream signaling required for IL-37-directed FOXP3 expression in human Treg cells are shown. Figures 25A-25B show heatmaps / clustergrams (Figure 25A) and scatterplots (Figure 25B) of components of the inflammasome pathway and mRNA expression in purified human Tconv and Treg cells using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / . Expression of 84 genes related to the inflammasome was measured using a Qiagen RT2 Profiler PCR Inflammasome array according to the manufacturer's instructions. Scatterplots showing two-fold boundaries for the expression of inflammasome genes in Tconv and Treg cells. Figure 25C shows a bar graph of NAIP expression in unstimulated Tconv and Treg cells. Figure 25D shows histograms (top) and quantification (bottom) showing expression in Tconv (black) and Treg (gray) cells. Figure 25E shows immunoblotting of NLRC4 in Tconv and scrambled control and siNLRC4-transfected primary human CD4+CD25+CD127dim Treg cells. Figure 25F shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in Tconv and Treg cells. Figure 25G shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in scrambled control and siASC-, siNLRP2-, and siNLRC4-transfected CD4+CD25+CD127dim Treg cells, as well as the quantification of the % caspase-1+ Treg cells (far right). Figure 25H shows a bar graph of FOXP3 gene expression after siRNA knockdown of NLRC4 in purified CD4+CD25+CD127dim Treg cells. GAPDH served as an internal control.Figure 25I shows a pseudocolor plot (left) showing the percentage of CD4+FOXP3+ Treg cells in a population of purified CD4+ T cells 48 hours after NLRC4 siRNA knockdown, and its quantification of %FOXP3+CD4+ T cells (right). Figure 25J shows confocal microscopy images of proximity ligation assays of ASC and NLRC4 (dots) in Treg cells. DNA intercalating dye DAPI. Scale bar, 5 μm. Small horizontal lines indicate mean ± s.e.m. (n = 3 donors per group) (Figures 25A, 25B), (n = 5-6 donors per group) (Figures 25C-25I), and (n ≥ 100 cells / group (Figure 25J)). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 25I]A series of graphs showing the NLRC4 inflammasome and upstream signaling required for IL-37-directed FOXP3 expression in human Treg cells are shown. Figures 25A-25B show heatmaps / clustergrams (Figure 25A) and scatterplots (Figure 25B) of components of the inflammasome pathway and mRNA expression in purified human Tconv and Treg cells using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / . Expression of 84 genes related to the inflammasome was measured using a Qiagen RT2 Profiler PCR Inflammasome array according to the manufacturer's instructions. Scatterplots showing two-fold boundaries for the expression of inflammasome genes in Tconv and Treg cells. Figure 25C shows a bar graph of NAIP expression in unstimulated Tconv and Treg cells. Figure 25D shows histograms (top) and quantification (bottom) showing expression in Tconv (black) and Treg (gray) cells. Figure 25E shows immunoblotting of NLRC4 in Tconv and scrambled control and siNLRC4-transfected primary human CD4+CD25+CD127dim Treg cells. Figure 25F shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in Tconv and Treg cells. Figure 25G shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in scrambled control and siASC-, siNLRP2-, and siNLRC4-transfected CD4+CD25+CD127dim Treg cells, as well as the quantification of the % caspase-1+ Treg cells (far right). Figure 25H shows a bar graph of FOXP3 gene expression after siRNA knockdown of NLRC4 in purified CD4+CD25+CD127dim Treg cells. GAPDH served as an internal control.Figure 25I shows a pseudocolor plot (left) showing the percentage of CD4+FOXP3+ Treg cells in a population of purified CD4+ T cells 48 hours after NLRC4 siRNA knockdown, and its quantification of %FOXP3+CD4+ T cells (right). Figure 25J shows confocal microscopy images of proximity ligation assays of ASC and NLRC4 (dots) in Treg cells. DNA intercalating dye DAPI. Scale bar, 5 μm. Small horizontal lines indicate mean ± s.e.m. (n = 3 donors per group) (Figures 25A, 25B), (n = 5-6 donors per group) (Figures 25C-25I), and (n ≥ 100 cells / group (Figure 25J)). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 25J]A series of graphs showing the NLRC4 inflammasome and upstream signaling required for IL-37-directed FOXP3 expression in human Treg cells are shown. Figures 25A-25B show heatmaps / clustergrams (Figure 25A) and scatterplots (Figure 25B) of components of the inflammasome pathway and mRNA expression in purified human Tconv and Treg cells using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / . Expression of 84 genes related to the inflammasome was measured using a Qiagen RT2 Profiler PCR Inflammasome array according to the manufacturer's instructions. Scatterplots showing two-fold boundaries for the expression of inflammasome genes in Tconv and Treg cells. Figure 25C shows a bar graph of NAIP expression in unstimulated Tconv and Treg cells. Figure 25D shows histograms (top) and quantification (bottom) showing expression in Tconv (black) and Treg (gray) cells. Figure 25E shows immunoblotting of NLRC4 in Tconv and scrambled control and siNLRC4-transfected primary human CD4+CD25+CD127dim Treg cells. Figure 25F shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in Tconv and Treg cells. Figure 25G shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in scrambled control and siASC-, siNLRP2-, and siNLRC4-transfected CD4+CD25+CD127dim Treg cells, as well as the quantification of the % caspase-1+ Treg cells (far right). Figure 25H shows a bar graph of FOXP3 gene expression after siRNA knockdown of NLRC4 in purified CD4+CD25+CD127dim Treg cells. GAPDH served as an internal control.Figure 25I shows a pseudocolor plot (left) showing the percentage of CD4+FOXP3+ Treg cells in a population of purified CD4+ T cells 48 hours after NLRC4 siRNA knockdown, and its quantification of %FOXP3+CD4+ T cells (right). Figure 25J shows confocal microscopy images of proximity ligation assays of ASC and NLRC4 (dots) in Treg cells. DNA intercalating dye DAPI. Scale bar, 5 μm. Small horizontal lines indicate mean ± s.e.m. (n = 3 donors per group) (Figures 25A, 25B), (n = 5-6 donors per group) (Figures 25C-25I), and (n ≥ 100 cells / group (Figure 25J)). *p < 0.05, ***p < 0.001, ****p < 0.0001 (Student's t-test). Data are representative of two to three independent experiments. [Figure 26A]Figure 26A shows a series of graphs demonstrating that basal PAK1 / 2 / 3 signaling promotes NLRC4 inflammasome activation in human Treg cells. Figure 26A shows histograms (left) and quantification (MFI) (right) of p-PAK1 / 2 / 3 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26B shows immunoblotting images of activated group 1 PAK in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Figure 26C shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26D shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26E shows confocal microscopy images of IL-37 localization in Treg cells treated with DMSO (top) or 1 μM FRAX 597 (bottom) for 24 hours. Scale bar, 10 μm. Figure 26F shows a bar graph of FOXP3 gene expression in Tconv and Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. GAPDH served as an internal control. Figure 26G shows immunoblotting images of FOXP3 in Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Small horizontal lines indicate the mean ± sem (n = 4-6 donors per group) (n ≥ 100 cells / group) (Figure 26E). *p < 0.05, **p < 0.01, ****p < 0.0001 (Student's t-test). Data are representative of 3-4 independent experiments. [Figure 26B]Figure 26A shows a series of graphs demonstrating that basal PAK1 / 2 / 3 signaling promotes NLRC4 inflammasome activation in human Treg cells. Figure 26A shows histograms (left) and quantification (MFI) (right) of p-PAK1 / 2 / 3 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26B shows immunoblotting images of activated group 1 PAK in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Figure 26C shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26D shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26E shows confocal microscopy images of IL-37 localization in Treg cells treated with DMSO (top) or 1 μM FRAX 597 (bottom) for 24 hours. Scale bar, 10 μm. Figure 26F shows a bar graph of FOXP3 gene expression in Tconv and Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. GAPDH served as an internal control. Figure 26G shows immunoblotting images of FOXP3 in Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Small horizontal lines indicate the mean ± sem (n = 4-6 donors per group) (n ≥ 100 cells / group) (Figure 26E). *p < 0.05, **p < 0.01, ****p < 0.0001 (Student's t-test). Data are representative of 3-4 independent experiments. [Figure 26C]Figure 26A shows a series of graphs demonstrating that basal PAK1 / 2 / 3 signaling promotes NLRC4 inflammasome activation in human Treg cells. Figure 26A shows histograms (left) and quantification (MFI) (right) of p-PAK1 / 2 / 3 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26B shows immunoblotting images of activated group 1 PAK in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Figure 26C shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26D shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26E shows confocal microscopy images of IL-37 localization in Treg cells treated with DMSO (top) or 1 μM FRAX 597 (bottom) for 24 hours. Scale bar, 10 μm. Figure 26F shows a bar graph of FOXP3 gene expression in Tconv and Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. GAPDH served as an internal control. Figure 26G shows immunoblotting images of FOXP3 in Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Small horizontal lines indicate the mean ± sem (n = 4-6 donors per group) (n ≥ 100 cells / group) (Figure 26E). *p < 0.05, **p < 0.01, ****p < 0.0001 (Student's t-test). Data are representative of 3-4 independent experiments. [Figure 26D]Figure 26A shows a series of graphs demonstrating that basal PAK1 / 2 / 3 signaling promotes NLRC4 inflammasome activation in human Treg cells. Figure 26A shows histograms (left) and quantification (MFI) (right) of p-PAK1 / 2 / 3 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26B shows immunoblotting images of activated group 1 PAK in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Figure 26C shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26D shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26E shows confocal microscopy images of IL-37 localization in Treg cells treated with DMSO (top) or 1 μM FRAX 597 (bottom) for 24 hours. Scale bar, 10 μm. Figure 26F shows a bar graph of FOXP3 gene expression in Tconv and Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. GAPDH served as an internal control. Figure 26G shows immunoblotting images of FOXP3 in Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Small horizontal lines indicate the mean ± sem (n = 4-6 donors per group) (n ≥ 100 cells / group) (Figure 26E). *p < 0.05, **p < 0.01, ****p < 0.0001 (Student's t-test). Data are representative of 3-4 independent experiments. [Figure 26E]Figure 26A shows a series of graphs demonstrating that basal PAK1 / 2 / 3 signaling promotes NLRC4 inflammasome activation in human Treg cells. Figure 26A shows histograms (left) and quantification (MFI) (right) of p-PAK1 / 2 / 3 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26B shows immunoblotting images of activated group 1 PAK in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Figure 26C shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26D shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26E shows confocal microscopy images of IL-37 localization in Treg cells treated with DMSO (top) or 1 μM FRAX 597 (bottom) for 24 hours. Scale bar, 10 μm. Figure 26F shows a bar graph of FOXP3 gene expression in Tconv and Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. GAPDH served as an internal control. Figure 26G shows immunoblotting images of FOXP3 in Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Small horizontal lines indicate the mean ± sem (n = 4-6 donors per group) (n ≥ 100 cells / group) (Figure 26E). *p < 0.05, **p < 0.01, ****p < 0.0001 (Student's t-test). Data are representative of 3-4 independent experiments. [Figure 26F]Figure 26A shows a series of graphs demonstrating that basal PAK1 / 2 / 3 signaling promotes NLRC4 inflammasome activation in human Treg cells. Figure 26A shows histograms (left) and quantification (MFI) (right) of p-PAK1 / 2 / 3 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26B shows immunoblotting images of activated group 1 PAK in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Figure 26C shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26D shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26E shows confocal microscopy images of IL-37 localization in Treg cells treated with DMSO (top) or 1 μM FRAX 597 (bottom) for 24 hours. Scale bar, 10 μm. Figure 26F shows a bar graph of FOXP3 gene expression in Tconv and Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. GAPDH served as an internal control. Figure 26G shows immunoblotting images of FOXP3 in Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Small horizontal lines indicate the mean ± sem (n = 4-6 donors per group) (n ≥ 100 cells / group) (Figure 26E). *p < 0.05, **p < 0.01, ****p < 0.0001 (Student's t-test). Data are representative of 3-4 independent experiments. [Figure 26G]Figure 26A shows a series of graphs demonstrating that basal PAK1 / 2 / 3 signaling promotes NLRC4 inflammasome activation in human Treg cells. Figure 26A shows histograms (left) and quantification (MFI) (right) of p-PAK1 / 2 / 3 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26B shows immunoblotting images of activated group 1 PAK in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Figure 26C shows histograms (left) and quantification (MFI) (right) of p-NLRC4 S533 in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26D shows a flow cytometry-based FAM-FLICA® assay measuring caspase-1 activation in purified CD4+CD25+CD127dim Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. Figure 26E shows confocal microscopy images of IL-37 localization in Treg cells treated with DMSO (top) or 1 μM FRAX 597 (bottom) for 24 hours. Scale bar, 10 μm. Figure 26F shows a bar graph of FOXP3 gene expression in Tconv and Treg cells treated with DMSO or 1 μM FRAX 597 for 24 hours. GAPDH served as an internal control. Figure 26G shows immunoblotting images of FOXP3 in Treg cells treated with DMSO or 1 μM FRAX 597 for 48 hours. Small horizontal lines indicate the mean ± sem (n = 4-6 donors per group) (n ≥ 100 cells / group) (Figure 26E). *p < 0.05, **p < 0.01, ****p < 0.0001 (Student's t-test). Data are representative of 3-4 independent experiments. [Figure 27A]Figure 27A shows a series of graphs illustrating the differences between expanded WT Treg cells and expanded IL37 Treg cells. Figure 27A shows a line graph depicting the number of WT and IL37 Treg cells over the expansion assay time scale. Figure 27B shows a series of graphs depicting an in vitro T cell suppression assay using expanded WT or IL37 primary murine CD4+CD25+CD127- Treg cells. Histograms (top), proliferation index (left, bottom), and % suppression (right, bottom) showing the division of CFSE-labeled CD4+CD25-CD127+ T cell responders (Tresp) cultured for 3 hours with irradiated APCs and anti-CD3 and Treg cells at Tresp:Treg ratios of 1:1 to 8:1. PI; proliferation index. "Tresp only" (left) are Tresp cells cultured with anti-CD3. Figure 27C shows qRT-PCR analysis of IL37, Foxp3, and Il17 expression in expanded WT and stimulated IL37Tg Treg cells + / - anti-CD3 / CD28. Gapdh served as an internal control. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± s.e.m. (n = 1 mouse per group) (B), (n = 3 mice per group) (Figure 27C). Data represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. (Student's t-test). Data are representative of one (Figures 27A-B) or three (Figure 27C) independent experiments. [Figure 27B]Figure 27A shows a series of graphs illustrating the differences between expanded WT Treg cells and expanded IL37 Treg cells. Figure 27A shows a line graph depicting the number of WT and IL37 Treg cells over the expansion assay time scale. Figure 27B shows a series of graphs depicting an in vitro T cell suppression assay using expanded WT or IL37 primary murine CD4+CD25+CD127- Treg cells. Histograms (top), proliferation index (left, bottom), and % suppression (right, bottom) showing the division of CFSE-labeled CD4+CD25-CD127+ T cell responders (Tresp) cultured for 3 hours with irradiated APCs and anti-CD3 and Treg cells at Tresp:Treg ratios of 1:1 to 8:1. PI; proliferation index. "Tresp only" (left) are Tresp cells cultured with anti-CD3. Figure 27C shows qRT-PCR analysis of IL37, Foxp3, and Il17 expression in expanded WT and stimulated IL37Tg Treg cells + / - anti-CD3 / CD28. Gapdh served as an internal control. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± s.e.m. (n = 1 mouse per group) (B), (n = 3 mice per group) (Figure 27C). Data represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. (Student's t-test). Data are representative of one (Figures 27A-B) or three (Figure 27C) independent experiments. [Figure 27C]Figure 27A shows a series of graphs illustrating the differences between expanded WT Treg cells and expanded IL37 Treg cells. Figure 27A shows a line graph depicting the number of WT and IL37 Treg cells over the expansion assay time scale. Figure 27B shows a series of graphs depicting an in vitro T cell suppression assay using expanded WT or IL37 primary murine CD4+CD25+CD127- Treg cells. Histograms (top), proliferation index (left, bottom), and % suppression (right, bottom) showing the division of CFSE-labeled CD4+CD25-CD127+ T cell responders (Tresp) cultured for 3 hours with irradiated APCs and anti-CD3 and Treg cells at Tresp:Treg ratios of 1:1 to 8:1. PI; proliferation index. "Tresp only" (left) are Tresp cells cultured with anti-CD3. Figure 27C shows qRT-PCR analysis of IL37, Foxp3, and Il17 expression in expanded WT and stimulated IL37Tg Treg cells + / - anti-CD3 / CD28. Gapdh served as an internal control. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± s.e.m. (n = 1 mouse per group) (B), (n = 3 mice per group) (Figure 27C). Data represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. (Student's t-test). Data are representative of one (Figures 27A-B) or three (Figure 27C) independent experiments. [Figure 28A]Figure 28A shows a series of graphs and microscopic images demonstrating the effect of ex vivo-expanded IL37 mouse Treg cells in suppressing contact hypersensitivity (CHS) in vivo. Figure 28A shows a schematic diagram of a CHS experiment using adoptive transfer of expanded Treg cells from WT or IL37 Tg mice (donors) to WT mice (recipients). Figure 28B shows a line graph depicting ear swelling in mice adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), or IL37 Tg Treg cells (DNFB + IL37 Tg-Treg). Figure 28C shows a series of H&E microscopic images of ear specimens taken 48 hours after DNFB challenge from mice adoptively transferred with PBS, WT-Treg, or IL37 Tg-Treg. Scale bar, 100 μm. Figure 28D shows contour plots of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) and Tconv cells (CD4+CD25loCD45RBhi) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg or IL37 Tg Treg cells. Numbers in the contour plots indicate the percentage of CFSE+ cells among Tconv or Treg cells. Data represent mean ± sem. *p<0.05. Data are representative of two experiments. [Figure 28B]Figure 28A shows a series of graphs and microscopic images demonstrating the effect of ex vivo-expanded IL37 mouse Treg cells in suppressing contact hypersensitivity (CHS) in vivo. Figure 28A shows a schematic diagram of a CHS experiment using adoptive transfer of expanded Treg cells from WT or IL37 Tg mice (donors) to WT mice (recipients). Figure 28B shows a line graph depicting ear swelling in mice adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), or IL37 Tg Treg cells (DNFB + IL37 Tg-Treg). Figure 28C shows a series of H&E microscopic images of ear specimens taken 48 hours after DNFB challenge from mice adoptively transferred with PBS, WT-Treg, or IL37 Tg-Treg. Scale bar, 100 μm. Figure 28D shows contour plots of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) and Tconv cells (CD4+CD25loCD45RBhi) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg or IL37 Tg Treg cells. Numbers in the contour plots indicate the percentage of CFSE+ cells among Tconv or Treg cells. Data represent mean ± sem. *p<0.05. Data are representative of two experiments. [Figure 28C]Figure 28A shows a series of graphs and microscopic images demonstrating the effect of ex vivo-expanded IL37 mouse Treg cells in suppressing contact hypersensitivity (CHS) in vivo. Figure 28A shows a schematic diagram of a CHS experiment using adoptive transfer of expanded Treg cells from WT or IL37 Tg mice (donors) to WT mice (recipients). Figure 28B shows a line graph depicting ear swelling in mice adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), or IL37 Tg Treg cells (DNFB + IL37 Tg-Treg). Figure 28C shows a series of H&E microscopic images of ear specimens taken 48 hours after DNFB challenge from mice adoptively transferred with PBS, WT-Treg, or IL37 Tg-Treg. Scale bar, 100 μm. Figure 28D shows contour plots of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) and Tconv cells (CD4+CD25loCD45RBhi) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg or IL37 Tg Treg cells. Numbers in the contour plots indicate the percentage of CFSE+ cells among Tconv or Treg cells. Data represent mean ± sem. *p<0.05. Data are representative of two experiments. [Figure 28D]Figure 28A shows a series of graphs and microscopic images demonstrating the effect of ex vivo-expanded IL37 mouse Treg cells in suppressing contact hypersensitivity (CHS) in vivo. Figure 28A shows a schematic diagram of a CHS experiment using adoptive transfer of expanded Treg cells from WT or IL37 Tg mice (donors) to WT mice (recipients). Figure 28B shows a line graph depicting ear swelling in mice adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), or IL37 Tg Treg cells (DNFB + IL37 Tg-Treg). Figure 28C shows a series of H&E microscopic images of ear specimens taken 48 hours after DNFB challenge from mice adoptively transferred with PBS, WT-Treg, or IL37 Tg-Treg. Scale bar, 100 μm. Figure 28D shows contour plots of CFSE-labeled cells among Treg cells (CD4+CD25hiCD45RBlo) and Tconv cells (CD4+CD25loCD45RBhi) from ear specimens collected 48 hours after DNFB challenge from mice adoptively transferred with WT-Treg or IL37 Tg Treg cells. Numbers in the contour plots indicate the percentage of CFSE+ cells among Tconv or Treg cells. Data represent mean ± sem. *p<0.05. Data are representative of two experiments. [Figure 29A]Figure 29A shows a series of graphs and microscopy images demonstrating the effect of ex vivo-expanded IL37 mouse Treg cells in suppressing established inflammation in a mouse model of psoriasis. Figure 29A shows a schematic diagram of a psoriasis experiment using adoptive transfer of expanded Treg cells from WT or IL37 Tg mice (donors) to WT mice (recipients). Figure 29B shows a line graph depicting ear swelling in mice stimulated daily with imiquimod and adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), or IL37 Tg Treg cells (DNFB + IL37 Tg-Treg) on D0. Figure 29C shows a series of H&E microscopy images of ear specimens taken on D5 after adoptive transfer with PBS, WT-Treg, or IL37 Tg-Treg. Scale bar, 100 μm. Figure 29D shows clinical images of mouse ears from WT mice that received no treatment (-CTL, -control), PBS (+CTL, +control), expanded WT Treg cells (+WT Treg), or adoptive transfer of IL37 Tg Treg cells (+IL37 Treg). Data represent mean ± sem. ****p<0.0001. Data are representative of two experiments. [Figure 29B]Figure 29A shows a series of graphs and microscopy images demonstrating the effect of ex vivo-expanded IL37 mouse Treg cells in suppressing established inflammation in a mouse model of psoriasis. Figure 29A shows a schematic diagram of a psoriasis experiment using adoptive transfer of expanded Treg cells from WT or IL37 Tg mice (donors) to WT mice (recipients). Figure 29B shows a line graph depicting ear swelling in mice stimulated daily with imiquimod and adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), or IL37 Tg Treg cells (DNFB + IL37 Tg-Treg) on D0. Figure 29C shows a series of H&E microscopy images of ear specimens taken on D5 after adoptive transfer with PBS, WT-Treg, or IL37 Tg-Treg. Scale bar, 100 μm. Figure 29D shows clinical images of mouse ears from WT mice that received no treatment (-CTL, -control), PBS (+CTL, +control), expanded WT Treg cells (+WT Treg), or adoptive transfer of IL37 Tg Treg cells (+IL37 Treg). Data represent mean ± sem. ****p<0.0001. Data are representative of two experiments. [Figure 29C]Figure 29A shows a series of graphs and microscopy images demonstrating the effect of ex vivo-expanded IL37 mouse Treg cells in suppressing established inflammation in a mouse model of psoriasis. Figure 29A shows a schematic diagram of a psoriasis experiment using adoptive transfer of expanded Treg cells from WT or IL37 Tg mice (donors) to WT mice (recipients). Figure 29B shows a line graph depicting ear swelling in mice stimulated daily with imiquimod and adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), or IL37 Tg Treg cells (DNFB + IL37 Tg-Treg) on D0. Figure 29C shows a series of H&E microscopy images of ear specimens taken on D5 after adoptive transfer with PBS, WT-Treg, or IL37 Tg-Treg. Scale bar, 100 μm. Figure 29D shows clinical images of mouse ears from WT mice that received no treatment (-CTL, -control), PBS (+CTL, +control), expanded WT Treg cells (+WT Treg), or adoptive transfer of IL37 Tg Treg cells (+IL37 Treg). Data represent mean ± sem. ****p<0.0001. Data are representative of two experiments. [Figure 29D]Figure 29A shows a series of graphs and microscopy images demonstrating the effect of ex vivo-expanded IL37 mouse Treg cells in suppressing established inflammation in a mouse model of psoriasis. Figure 29A shows a schematic diagram of a psoriasis experiment using adoptive transfer of expanded Treg cells from WT or IL37 Tg mice (donors) to WT mice (recipients). Figure 29B shows a line graph depicting ear swelling in mice stimulated daily with imiquimod and adoptively transferred with PBS (DNFB + PBS), WT Treg cells (DNFB + WT-Treg), or IL37 Tg Treg cells (DNFB + IL37 Tg-Treg) on D0. Figure 29C shows a series of H&E microscopy images of ear specimens taken on D5 after adoptive transfer with PBS, WT-Treg, or IL37 Tg-Treg. Scale bar, 100 μm. Figure 29D shows clinical images of mouse ears from WT mice that received no treatment (-CTL, -control), PBS (+CTL, +control), expanded WT Treg cells (+WT Treg), or adoptive transfer of IL37 Tg Treg cells (+IL37 Treg). Data represent mean ± sem. ****p<0.0001. Data are representative of two experiments. [Figure 30A] Figure 30A shows a series of graphs demonstrating the effect of ex vivo expanded IL37 mouse Treg cells in suppressing neuroinflammation in a mouse model of traumatic brain injury (TBI). Figure 30A shows a schematic diagram of a TBI experiment using adoptive transfer of expanded Treg cells from WT or IL37 Tg mice (donors) to WT mice (recipients). [Figure 30B] Figure 30A shows a series of graphs depicting the effect of ex vivo expanded IL37 mouse Treg cells in suppressing neuroinflammation in a mouse model of traumatic brain injury (TBI). Figure 30B shows a line graph depicting neurological severity scores after TBI injury in mice adoptively transferred with PBS (DNFB+PBS), WT Treg cells (DNFB+WT-Treg), or IL37 Tg Treg cells (DNFB+IL37 Tg-Treg) 3 hours after TBI. [Figure 30C]Figure 30G shows a series of graphs demonstrating the effect of ex vivo expanded IL37 mouse Treg cells in suppressing neuroinflammation in a mouse model of traumatic brain injury (TBI). Figure 30C shows a series of graphs demonstrating analysis of cervical lymph nodes from mice with TBI for CD3+ (Figure 30C), CD4+ (Figure 30D), CD8+ (Figure 30E), and Treg cell counts (Figure 30F). Figure 30G shows a surface histogram (left) and mean fluorescence intensity (MFI) quantification (right) of CD8+ T cells from cervical lymph nodes. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± s.e.m. (n = 6 mice per group). Data represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. (Student's t-test). Data are representative of one independent experiment. [Figure 30D] Figure 30A shows a series of graphs depicting analysis of cervical lymph nodes from mice with TBI for CD3+ (Figure 30C), CD4+ (Figure 30D), CD8+ (Figure 30E), and Treg cell counts (Figure 30F). Figure 30G shows a surface histogram (left) and mean fluorescence intensity (MFI) quantification (right) of CD8+ T cells from cervical lymph nodes. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± sem. (n = 6 mice per group). Data represent the mean ± sem. *p<0.05, **p<0.01, ***p<0.001. (Student's t-test). Data are representative of one independent experiment. [Figure 30E]Figure 30A shows a series of graphs depicting analysis of cervical lymph nodes from mice with TBI for CD3+ (Figure 30C), CD4+ (Figure 30D), CD8+ (Figure 30E), and Treg cell counts (Figure 30F). Figure 30G shows a surface histogram (left) and mean fluorescence intensity (MFI) quantification (right) of CD8+ T cells from cervical lymph nodes. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± sem. (n = 6 mice per group). Data represent the mean ± sem. *p<0.05, **p<0.01, ***p<0.001. (Student's t-test). Data are representative of one independent experiment. [Figure 30F] Figure 30A shows a series of graphs depicting analysis of cervical lymph nodes from mice with TBI for CD3+ (Figure 30C), CD4+ (Figure 30D), CD8+ (Figure 30E), and Treg cell counts (Figure 30F). Figure 30G shows a surface histogram (left) and mean fluorescence intensity (MFI) quantification (right) of CD8+ T cells from cervical lymph nodes. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± sem. (n = 6 mice per group). Data represent the mean ± sem. *p<0.05, **p<0.01, ***p<0.001. (Student's t-test). Data are representative of one independent experiment. [Figure 30G] Figure 30A shows a series of graphs depicting analysis of cervical lymph nodes from mice with TBI for CD3+ (Figure 30C), CD4+ (Figure 30D), CD8+ (Figure 30E), and Treg cell counts (Figure 30F). Figure 30G shows a surface histogram (left) and mean fluorescence intensity (MFI) quantification (right) of CD8+ T cells from cervical lymph nodes. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± sem. (n = 6 mice per group). Data represent the mean ± sem. *p<0.05, **p<0.01, ***p<0.001. (Student's t-test). Data are representative of one independent experiment. [Figure 31A]Figure 31A shows a series of graphs depicting Treg and IL37-expressing cells in PBMCs from healthy donors and type 1 diabetes (T1D) patients using the flow cytometry-based PrimeFlow® RNA assay. Figure 31A shows contour plots of CD25+CD127lo cell gating on human CD3+CD4+ T cells from healthy donors (left) and T1D patients (center), as well as quantification of the percentage of CD3+CD4+CD25hiCD127lo cells (right). Figure 31B shows bar graphs of CD3+, CD4+, CD8+, Treg, and natural killer (NK) cell counts in PBMCs from healthy donors and T1D patients. Figure 31C shows histograms (left) and mean fluorescence intensity (MFI) quantification (right) of Primeflow®, a flow cytometry-based assay for measuring IL37 mRNA expression in CD3+, CD4+, CD8+, Treg cells, and NK cells in PBMCs from healthy donors and T1D patients. IgG was used as a negative control staining (black-gray histogram). Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 5 human donors per group). *p < 0.05, **p < 0.01, ***p < 0.001. (Student's t-test). Data are representative of two independent experiments. [Figure 31B]Figure 31A shows a series of graphs depicting Treg and IL37-expressing cells in PBMCs from healthy donors and type 1 diabetes (T1D) patients using the flow cytometry-based PrimeFlow® RNA assay. Figure 31A shows contour plots of CD25+CD127lo cell gating on human CD3+CD4+ T cells from healthy donors (left) and T1D patients (center), as well as quantification of the percentage of CD3+CD4+CD25hiCD127lo cells (right). Figure 31B shows bar graphs of CD3+, CD4+, CD8+, Treg, and natural killer (NK) cell counts in PBMCs from healthy donors and T1D patients. Figure 31C shows histograms (left) and mean fluorescence intensity (MFI) quantification (right) of Primeflow®, a flow cytometry-based assay for measuring IL37 mRNA expression in CD3+, CD4+, CD8+, Treg cells, and NK cells in PBMCs from healthy donors and T1D patients. IgG was used as a negative control staining (black-gray histogram). Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 5 human donors per group). *p < 0.05, **p < 0.01, ***p < 0.001. (Student's t-test). Data are representative of two independent experiments. [Figure 31C]Figure 31A shows a series of graphs depicting Treg and IL37-expressing cells in PBMCs from healthy donors and type 1 diabetes (T1D) patients using the flow cytometry-based PrimeFlow® RNA assay. Figure 31A shows contour plots of CD25+CD127lo cell gating on human CD3+CD4+ T cells from healthy donors (left) and T1D patients (center), as well as quantification of the percentage of CD3+CD4+CD25hiCD127lo cells (right). Figure 31B shows bar graphs of CD3+, CD4+, CD8+, Treg, and natural killer (NK) cell counts in PBMCs from healthy donors and T1D patients. Figure 31C shows histograms (left) and mean fluorescence intensity (MFI) quantification (right) of Primeflow®, a flow cytometry-based assay for measuring IL37 mRNA expression in CD3+, CD4+, CD8+, Treg cells, and NK cells in PBMCs from healthy donors and T1D patients. IgG was used as a negative control staining (black-gray histogram). Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem. (n = 5 human donors per group). *p < 0.05, **p < 0.01, ***p < 0.001. (Student's t-test). Data are representative of two independent experiments. [Figure 32] A series of bar graphs depicting gene expression analysis of ex vivo expanded human Treg cells. qRT-PCR analysis of IL37, FOXP3, IL17, STAT3, and GATA3 expression in T conventional (Tconv) cells, unexpanded Treg cells (Treg Ctl), and expanded human Treg cells (Treg Blast). GAPDH served as an internal control. Each symbol represents an individual mouse. Small horizontal lines indicate the mean ± sem. (n = 3 human donors per group). Data represent the mean ± sem. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (Student's t-test). Data are representative of three independent experiments. [Figure 33] The vector map of IL37-BFP_Lenti is shown. [Figure 34A] Figure 34A shows a series of graphs depicting gene expression changes in ex vivo expanded human Treg cells after IL37 transduction. Figure 34A shows a density plot of IL-37 expression from purified human Treg cells transduced with a control vector or an IL37 overexpression (OE) vector (left) and quantification of the percentage of IL-37+ Treg cells (far right). No staining was used as a control. Right. Figure 34B shows a bar graph of IL37, FOXP3, and Helios gene expression in human Treg cells transduced with a control vector or an IL37 OE vector. GAPDH served as an internal control. Each symbol represents an individual healthy donor. Small horizontal lines indicate the mean ± sem. (n = 3 donors per group). Data represent the mean ± sem. ***p < 0.001, ****p < 0.0001. (Student's t-test). Data are representative of three independent experiments. [Figure 34B] Figure 34A shows a series of graphs depicting gene expression changes in ex vivo expanded human Treg cells after IL37 transduction. Figure 34A shows a density plot of IL-37 expression from purified human Treg cells transduced with a control vector or an IL37 overexpression (OE) vector (left) and quantification of the percentage of IL-37+ Treg cells (far right). No staining was used as a control. Right. Figure 34B shows a bar graph of IL37, FOXP3, and Helios gene expression in human Treg cells transduced with a control vector or an IL37 OE vector. GAPDH served as an internal control. Each symbol represents an individual healthy donor. Small horizontal lines indicate the mean ± sem. (n = 3 donors per group). Data represent the mean ± sem. ***p < 0.001, ****p < 0.0001. (Student's t-test). Data are representative of three independent experiments. [Figure 35A]Figure 35A shows a series of graphs demonstrating that IL-37 overexpression in expanded human Treg cells maintains their suppressive function in a mouse model of graft-versus-host disease (GVHD). Figure 35A shows a schematic diagram illustrating a xenogeneic GVHD experiment using adoptive cell transfer of human Treg cells expanded ex vivo using control or IL37 OE vectors to control xenogeneic GVHD in immunocompromised BRGS mice (recipients). Figure 35B shows a line graph depicting the clinical scores of BRGS mice over 34 days after injection of PBS, PBMCs only, PBMCs + control Treg cells (pbmc ctl), or PBMCs + IL37 OE Treg cells (pbmc IL37). Figure 35C shows a bar graph of CD45+ cell counts in the spleen on day 34. Figure 35D shows dot blots of splenic CD4 and CD8 cells in mice receiving PBMCs and treated with control or IL37 OE Treg cells. Figure 35E shows a dot blot (left) and bar graph (right) of splenic Treg cells in mice receiving PBMCs and treated with control or IL37 OE Treg cells. Data represent mean ± sem. *p<0.05, **p<0.01 (Student's t-test). Data are representative of two independent experiments. [Figure 35B]Figure 35A shows a series of graphs demonstrating that IL-37 overexpression in expanded human Treg cells maintains their suppressive function in a mouse model of graft-versus-host disease (GVHD). Figure 35A shows a schematic diagram illustrating a xenogeneic GVHD experiment using adoptive cell transfer of human Treg cells expanded ex vivo using control or IL37 OE vectors to control xenogeneic GVHD in immunocompromised BRGS mice (recipients). Figure 35B shows a line graph depicting the clinical scores of BRGS mice over 34 days after injection of PBS, PBMCs only, PBMCs + control Treg cells (pbmc ctl), or PBMCs + IL37 OE Treg cells (pbmc IL37). Figure 35C shows a bar graph of CD45+ cell counts in the spleen on day 34. Figure 35D shows dot blots of splenic CD4 and CD8 cells in mice receiving PBMCs and treated with control or IL37 OE Treg cells. Figure 35E shows a dot blot (left) and bar graph (right) of splenic Treg cells in mice receiving PBMCs and treated with control Treg cells or IL37 OE Treg cells. Data represent mean ± sem. *p<0.05, **p<0.01 (Student's t-test). Data are representative of two independent experiments. [Figure 35C]Figure 35A shows a series of graphs demonstrating that IL-37 overexpression in expanded human Treg cells maintains their suppressive function in a mouse model of graft-versus-host disease (GVHD). Figure 35A shows a schematic diagram illustrating a xenogeneic GVHD experiment using adoptive cell transfer of human Treg cells expanded ex vivo using control or IL37 OE vectors to control xenogeneic GVHD in immunocompromised BRGS mice (recipients). Figure 35B shows a line graph depicting the clinical scores of BRGS mice over 34 days after injection of PBS, PBMCs only, PBMCs + control Treg cells (pbmc ctl), or PBMCs + IL37 OE Treg cells (pbmc IL37). Figure 35C shows a bar graph of CD45+ cell counts in the spleen on day 34. Figure 35D shows dot blots of splenic CD4 and CD8 cells in mice receiving PBMCs and treated with control or IL37 OE Treg cells. Figure 35E shows a dot blot (left) and bar graph (right) of splenic Treg cells in mice receiving PBMCs and treated with control Treg cells or IL37 OE Treg cells. Data represent mean ± sem. *p<0.05, **p<0.01 (Student's t-test). Data are representative of two independent experiments. [Figure 35D]Figure 35A shows a series of graphs demonstrating that IL-37 overexpression in expanded human Treg cells maintains their suppressive function in a mouse model of graft-versus-host disease (GVHD). Figure 35A shows a schematic diagram illustrating a xenogeneic GVHD experiment using adoptive cell transfer of human Treg cells expanded ex vivo using control or IL37 OE vectors to control xenogeneic GVHD in immunocompromised BRGS mice (recipients). Figure 35B shows a line graph depicting the clinical scores of BRGS mice over 34 days after injection of PBS, PBMCs only, PBMCs + control Treg cells (pbmc ctl), or PBMCs + IL37 OE Treg cells (pbmc IL37). Figure 35C shows a bar graph of CD45+ cell counts in the spleen on day 34. Figure 35D shows dot blots of splenic CD4 and CD8 cells in mice receiving PBMCs and treated with control or IL37 OE Treg cells. Figure 35E shows a dot blot (left) and bar graph (right) of splenic Treg cells in mice receiving PBMCs and treated with control Treg cells or IL37 OE Treg cells. Data represent mean ± sem. *p<0.05, **p<0.01 (Student's t-test). Data are representative of two independent experiments. [Figure 35E]Figure 35A shows a series of graphs demonstrating that IL-37 overexpression in expanded human Treg cells maintains their suppressive function in a mouse model of graft-versus-host disease (GVHD). Figure 35A shows a schematic diagram illustrating a xenogeneic GVHD experiment using adoptive cell transfer of human Treg cells expanded ex vivo using control or IL37 OE vectors to control xenogeneic GVHD in immunocompromised BRGS mice (recipients). Figure 35B shows a line graph depicting the clinical scores of BRGS mice over 34 days after injection of PBS, PBMCs only, PBMCs + control Treg cells (pbmc ctl), or PBMCs + IL37 OE Treg cells (pbmc IL37). Figure 35C shows a bar graph of CD45+ cell counts in the spleen on day 34. Figure 35D shows dot blots of splenic CD4 and CD8 cells in mice receiving PBMCs and treated with control or IL37 OE Treg cells. Figure 35E shows a dot blot (left) and bar graph (right) of splenic Treg cells in mice receiving PBMCs and treated with control Treg cells or IL37 OE Treg cells. Data represent mean ± sem. *p<0.05, **p<0.01 (Student's t-test). Data are representative of two independent experiments. [Figure 36A]Figure 36A shows a series of graphs demonstrating that IL-37 overexpression in Jurkat cells induces a Treg-like phenotype and function in a mouse model of xenogeneic graft-versus-host disease (GVHD). Figure 36A shows a schematic diagram illustrating a GVHD experiment using adoptive cell transfer of human PBMCs with control or IL37 OE Jurkat cells (donor) into immunocompromised BRGS mice (recipients). Figure 36B shows a line graph (top) depicting the clinical scores of BRGS mice over 12 days after injection of PBS, PBMCs only, PBMCs + control Jurkat cells, or PBMCs + IL37 OE Jurkat cells, as well as the clinical scoring criteria for GVHD used in the experiment (bottom). Figure 36C shows images of the colon (left) and spleen (right) from BRGS mice injected with PBMCs only, PBMCs + IL37 OE Jurkat cells, or PBMCs + control Jurkat cells. Figure 36D shows a bar graph depicting splenocyte counts from BRGS mice injected with PBS, PBMCs only, PBMCs plus control Jurkat cells, or PBMCs plus IL37 OE Jurkat cells. Data represent mean ± sem. *p<0.05, **p<0.01 (Student's t-test). Data are representative of three independent experiments. [Figure 36B]Figure 36A shows a series of graphs demonstrating that IL-37 overexpression in Jurkat cells induces a Treg-like phenotype and function in a mouse model of xenogeneic graft-versus-host disease (GVHD). Figure 36A shows a schematic diagram illustrating a GVHD experiment using adoptive cell transfer of human PBMCs with control or IL37 OE Jurkat cells (donor) into immunocompromised BRGS mice (recipients). Figure 36B shows a line graph (top) depicting the clinical scores of BRGS mice over 12 days after injection of PBS, PBMCs only, PBMCs + control Jurkat cells, or PBMCs + IL37 OE Jurkat cells, as well as the clinical scoring criteria for GVHD used in the experiment (bottom). Figure 36C shows images of the colon (left) and spleen (right) from BRGS mice injected with PBMCs only, PBMCs + IL37 OE Jurkat cells, or PBMCs + control Jurkat cells. Figure 36D shows a bar graph depicting splenocyte counts from BRGS mice injected with PBS, PBMCs only, PBMCs plus control Jurkat cells, or PBMCs plus IL37 OE Jurkat cells. Data represent mean ± sem. *p<0.05, **p<0.01 (Student's t-test). Data are representative of three independent experiments. [Figure 36C]Figure 36A shows a series of graphs demonstrating that IL-37 overexpression in Jurkat cells induces a Treg-like phenotype and function in a mouse model of xenogeneic graft-versus-host disease (GVHD). Figure 36A shows a schematic diagram illustrating a GVHD experiment using adoptive cell transfer of human PBMCs with control or IL37 OE Jurkat cells (donor) into immunocompromised BRGS mice (recipients). Figure 36B shows a line graph (top) depicting the clinical scores of BRGS mice over 12 days after injection of PBS, PBMCs only, PBMCs + control Jurkat cells, or PBMCs + IL37 OE Jurkat cells, as well as the clinical scoring criteria for GVHD used in the experiment (bottom). Figure 36C shows images of the colon (left) and spleen (right) from BRGS mice injected with PBMCs only, PBMCs + IL37 OE Jurkat cells, or PBMCs + control Jurkat cells. Figure 36D shows a bar graph depicting splenocyte counts from BRGS mice injected with PBS, PBMCs only, PBMCs plus control Jurkat cells, or PBMCs plus IL37 OE Jurkat cells. Data represent mean ± sem. *p<0.05, **p<0.01 (Student's t-test). Data are representative of three independent experiments. [Figure 36D]Figure 36A shows a series of graphs demonstrating that IL-37 overexpression in Jurkat cells induces a Treg-like phenotype and function in a mouse model of xenogeneic graft-versus-host disease (GVHD). Figure 36A shows a schematic diagram illustrating a GVHD experiment using adoptive cell transfer of human PBMCs with control or IL37 OE Jurkat cells (donor) into immunocompromised BRGS mice (recipients). Figure 36B shows a line graph (top) depicting the clinical scores of BRGS mice over 12 days after injection of PBS, PBMCs only, PBMCs + control Jurkat cells, or PBMCs + IL37 OE Jurkat cells, as well as the clinical scoring criteria for GVHD used in the experiment (bottom). Figure 36C shows images of the colon (left) and spleen (right) from BRGS mice injected with PBMCs only, PBMCs + IL37 OE Jurkat cells, or PBMCs + control Jurkat cells. Figure 36D shows a bar graph depicting splenocyte counts from BRGS mice injected with PBS, PBMCs only, PBMCs plus control Jurkat cells, or PBMCs plus IL37 OE Jurkat cells. Data represent mean ± sem. *p<0.05, **p<0.01 (Student's t-test). Data are representative of three independent experiments. [Figure 37A]Figure 37A shows a series of graphs illustrating that binding partner screening of FOXP3-regulating transcription factors identifies IL-37 as a key molecule for maintaining FOXP3 in human Treg cells. Figure 37A shows qRT-PCR analysis of proteins associated with transcription factor (STAT5, NFAT, and Smad3) expression in purified human Tconv and Treg cells. GAPDH served as an internal control. Figure 37B shows the dot plot (left) and frequency (right) of FOXP3 expression between Tconv (CD25-) and Treg (CD25+) cells purified from human donors using the human CD4+CD25+CD127dim kit. Numbers in the dot plots indicate the percentage of FOXP3+ cells in Tconv or Treg cells. Figure 37C shows a contour plot of FOXP3+ cell gating in human CD4+ T cells transfected 48 hours prior with scrambled siRNA (Ctrl), siCHK1, siCREB1, siIL-37, siRPTOR, and siRUNX1 in the upper panel, and quantification of the percentage of FOXP3+ T cells in the lower panel. *p-values were measured using Scrambled Ctrl. Figure 37D shows Western blot analysis of protein expression levels in purified human CD4+ T cells 24 hours after transfection with scrambled siRNA (Ctrl) or siRNAs against CREB1, CHEK1 (pooled), IL37 (pooled), RPTOR (pooled), and RUNX1. Actin served as a loading control. Figure 37E shows qRT-PCR analysis of FOXP3 gene expression after siRNA knockdown of IL37 in purified Treg cells. IL37 expression is shown on the top, and FOXP3 expression is shown on the bottom. GAPDH served as an internal control. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem (n = 4 donors per group) (A), (n = 8 donors per group) (Figure 37B), (n = 5 donors per group) (Figure 37C), (n = 3 donors per group) (Figure 37D, Figure 37E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t-test).Data are representative of two (Figure 37E), three (Figures 37C, 37D), four (Figure 37A), or seven (Figure 37B) independent experiments. [Figure 37B]Figure 37A shows a series of graphs illustrating that binding partner screening of FOXP3-regulating transcription factors identifies IL-37 as a key molecule for maintaining FOXP3 in human Treg cells. Figure 37A shows qRT-PCR analysis of proteins associated with transcription factor (STAT5, NFAT, and Smad3) expression in purified human Tconv and Treg cells. GAPDH served as an internal control. Figure 37B shows the dot plot (left) and frequency (right) of FOXP3 expression between Tconv (CD25-) and Treg (CD25+) cells purified from human donors using the human CD4+CD25+CD127dim kit. Numbers in the dot plots indicate the percentage of FOXP3+ cells in Tconv or Treg cells. Figure 37C shows a contour plot of FOXP3+ cell gating in human CD4+ T cells transfected 48 hours prior with scrambled siRNA (Ctrl), siCHK1, siCREB1, siIL-37, siRPTOR, and siRUNX1 in the upper panel, and quantification of the percentage of FOXP3+ T cells in the lower panel. *p-values were measured using Scrambled Ctrl. Figure 37D shows Western blot analysis of protein expression levels in purified human CD4+ T cells 24 hours after transfection with scrambled siRNA (Ctrl) or siRNAs against CREB1, CHEK1 (pooled), IL37 (pooled), RPTOR (pooled), and RUNX1. Actin served as a loading control. Figure 37E shows qRT-PCR analysis of FOXP3 gene expression after siRNA knockdown of IL37 in purified Treg cells. IL37 expression is shown on the top, and FOXP3 expression is shown on the bottom. GAPDH served as an internal control. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem (n = 4 donors per group) (A), (n = 8 donors per group) (Figure 37B), (n = 5 donors per group) (Figure 37C), (n = 3 donors per group) (Figure 37D, Figure 37E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t-test).Data are representative of two (Figure 37E), three (Figures 37C, 37D), four (Figure 37A), or seven (Figure 37B) independent experiments. [Figure 37C]Figure 37A shows a series of graphs illustrating that binding partner screening of FOXP3-regulating transcription factors identifies IL-37 as a key molecule for maintaining FOXP3 in human Treg cells. Figure 37A shows qRT-PCR analysis of proteins associated with transcription factor (STAT5, NFAT, and Smad3) expression in purified human Tconv and Treg cells. GAPDH served as an internal control. Figure 37B shows the dot plot (left) and frequency (right) of FOXP3 expression between Tconv (CD25-) and Treg (CD25+) cells purified from human donors using the human CD4+CD25+CD127dim kit. Numbers in the dot plots indicate the percentage of FOXP3+ cells in Tconv or Treg cells. Figure 37C shows a contour plot of FOXP3+ cell gating in human CD4+ T cells transfected 48 hours prior with scrambled siRNA (Ctrl), siCHK1, siCREB1, siIL-37, siRPTOR, and siRUNX1 in the upper panel, and quantification of the percentage of FOXP3+ T cells in the lower panel. *p-values were measured using Scrambled Ctrl. Figure 37D shows Western blot analysis of protein expression levels in purified human CD4+ T cells 24 hours after transfection with scrambled siRNA (Ctrl) or siRNAs against CREB1, CHEK1 (pooled), IL37 (pooled), RPTOR (pooled), and RUNX1. Actin served as a loading control. Figure 37E shows qRT-PCR analysis of FOXP3 gene expression after siRNA knockdown of IL37 in purified Treg cells. IL37 expression is shown on the top, and FOXP3 expression is shown on the bottom. GAPDH served as an internal control. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem (n = 4 donors per group) (A), (n = 8 donors per group) (Figure 37B), (n = 5 donors per group) (Figure 37C), (n = 3 donors per group) (Figure 37D, Figure 37E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t-test).Data are representative of two (Figure 37E), three (Figures 37C, 37D), four (Figure 37A), or seven (Figure 37B) independent experiments. [Figure 37D]Figure 37A shows a series of graphs illustrating that binding partner screening of FOXP3-regulating transcription factors identifies IL-37 as a key molecule for maintaining FOXP3 in human Treg cells. Figure 37A shows qRT-PCR analysis of proteins associated with transcription factor (STAT5, NFAT, and Smad3) expression in purified human Tconv and Treg cells. GAPDH served as an internal control. Figure 37B shows the dot plot (left) and frequency (right) of FOXP3 expression between Tconv (CD25-) and Treg (CD25+) cells purified from human donors using the human CD4+CD25+CD127dim kit. Numbers in the dot plots indicate the percentage of FOXP3+ cells in Tconv or Treg cells. Figure 37C shows a contour plot of FOXP3+ cell gating in human CD4+ T cells transfected 48 hours prior with scrambled siRNA (Ctrl), siCHK1, siCREB1, siIL-37, siRPTOR, and siRUNX1 in the upper panel, and quantification of the percentage of FOXP3+ T cells in the lower panel. *p-values were measured using Scrambled Ctrl. Figure 37D shows Western blot analysis of protein expression levels in purified human CD4+ T cells 24 hours after transfection with scrambled siRNA (Ctrl) or siRNAs against CREB1, CHEK1 (pooled), IL37 (pooled), RPTOR (pooled), and RUNX1. Actin served as a loading control. Figure 37E shows qRT-PCR analysis of FOXP3 gene expression after siRNA knockdown of IL37 in purified Treg cells. IL37 expression is shown on the top, and FOXP3 expression is shown on the bottom. GAPDH served as an internal control. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem (n = 4 donors per group) (A), (n = 8 donors per group) (Figure 37B), (n = 5 donors per group) (Figure 37C), (n = 3 donors per group) (Figure 37D, Figure 37E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t-test).Data are representative of two (Figure 37E), three (Figures 37C, 37D), four (Figure 37A), or seven (Figure 37B) independent experiments. [Figure 37E]Figure 37A shows a series of graphs illustrating that binding partner screening of FOXP3-regulating transcription factors identifies IL-37 as a key molecule for maintaining FOXP3 in human Treg cells. Figure 37A shows qRT-PCR analysis of proteins associated with transcription factor (STAT5, NFAT, and Smad3) expression in purified human Tconv and Treg cells. GAPDH served as an internal control. Figure 37B shows the dot plot (left) and frequency (right) of FOXP3 expression between Tconv (CD25-) and Treg (CD25+) cells purified from human donors using the human CD4+CD25+CD127dim kit. Numbers in the dot plots indicate the percentage of FOXP3+ cells in Tconv or Treg cells. Figure 37C shows a contour plot of FOXP3+ cell gating in human CD4+ T cells transfected 48 hours prior with scrambled siRNA (Ctrl), siCHK1, siCREB1, siIL-37, siRPTOR, and siRUNX1 in the upper panel, and quantification of the percentage of FOXP3+ T cells in the lower panel. *p-values were measured using Scrambled Ctrl. Figure 37D shows Western blot analysis of protein expression levels in purified human CD4+ T cells 24 hours after transfection with scrambled siRNA (Ctrl) or siRNAs against CREB1, CHEK1 (pooled), IL37 (pooled), RPTOR (pooled), and RUNX1. Actin served as a loading control. Figure 37E shows qRT-PCR analysis of FOXP3 gene expression after siRNA knockdown of IL37 in purified Treg cells. IL37 expression is shown on the top, and FOXP3 expression is shown on the bottom. GAPDH served as an internal control. Each symbol represents an individual donor. Small horizontal lines indicate the mean ± sem (n = 4 donors per group) (A), (n = 8 donors per group) (Figure 37B), (n = 5 donors per group) (Figure 37C), (n = 3 donors per group) (Figure 37D, Figure 37E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t-test).Data are representative of two (Figure 37E), three (Figures 37C, 37D), four (Figure 37A), or seven (Figure 37B) independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure is based, at least in part, on the discovery that nuclear expression of the immunosuppressive cytokine IL-37 is required for the maintenance of FOXP3 expression in human regulatory T (Treg) cells and that IL-37 plays an important role in the suppressive properties of these cells not only in steady-state environments but also in inflammatory environments. FOXP3+ Treg cells play an essential role in regulating immune responses and maintaining peripheral self-tolerance. Continuous expression of FOXP3 in Treg cells is crucial for preserving tolerance and stabilizing Treg cell populations. Elevated nuclear IL-37 expression corresponds to elevated expression of FOXP3, maintaining FOXP3+ Treg cells during inflammatory responses and resulting in increased suppression both in vitro and in vivo. The disclosure herein describes methods for generating populations of engineered regulatory T cells or Treg-like cells capable of sustained suppressive function in vitro and in vivo, even under inflammatory conditions. By avoiding conversion to conventional T (Tconv) cells (e.g., non-Treg CD4+ cells), the modified Treg cell populations of the present disclosure may provide improved clinical outcomes when used in adoptive therapy and treatment of immune diseases or disorders (e.g., autoimmune diseases and disorders).

[0024] Treg cells play a key role in peripheral tolerance by suppressing and preventing autoreactive immune responses. Understanding how Treg cells function in maintaining peripheral tolerance is important for preventing and treating autoimmunity and for the future development of immune-targeted therapies, such as adoptive Treg cell therapy. However, culturing and maintaining sufficient primary human Treg cells to conduct molecular-based experiments is time-consuming and expensive. Although novel genome editing technologies have improved the effectiveness of modifying primary Treg cells, experimental challenges remain due to many factors, including the inter- and intra-individual heterogeneity of primary human Treg cells. An alternative to using primary Treg cells is to develop Treg cell lines that are easily usable for experiments. Although several studies have attempted to identify or establish Treg-like cell lines by altering gene expression in T cell lines or inducing Treg cell differentiation, no artificial Treg cell lines have yet been generated that are stable or readily available for use.

[0025] Methods described herein utilize the suppressive properties of the modified CD4+ T cells disclosed herein, including methods for generating and using a population of modified CD4+ T cells that express nuclear IL-37 to treat immune diseases or disorders (e.g., autoimmune diseases and disorders). In some embodiments, the method of treatment involves adoptive transfer using the modified population of CD4+ T cells. One of the key challenges with adoptive transfer of regulatory T cells in autoimmunity is that regulatory T cells are unstable and can convert into inflammatory T cells, exacerbating the disease. Disclosed herein are methods for inducing nuclear IL-37 expression in CD4+ T cells (e.g., Treg cells or Treg-like cells) and using the same to treat immune diseases or disorders (e.g., autoimmune diseases and disorders). In some embodiments, the method of treatment involves adoptive transfer using a population of modified regulatory T cells. The methods disclosed herein provide a way to minimize potential adverse effects of regulatory T cell therapy, such as, but not limited to, immunosuppression.

[0026] The disclosed methods can generate antigen-specific regulatory T cells, where the T cells have increased specificity and increased potency in suppressing autoimmunity compared to unmodified regulatory T cells. Antigen-specific Treg cells are difficult to develop because antigens are not always known or uniform for each disease population. Using the methods disclosed herein, antigen-specific T effector cells can be converted from autoimmune patients to generate antigen-specific Treg cells (e.g., by modifying autologous T cells). Thus, it is not necessary to identify specific antigens from each patient for successful therapy. Antigen-specific Treg cells are less likely to suppress other immune cells or other inflammatory conditions, minimizing potential side effects such as nonspecific immunosuppression.

[0027] Disclosed herein are methods for producing a population of engineered regulatory T cells for use in treating an autoimmune disease or disorder, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a population of engineered regulatory T cells that express nuclear IL-37. In some embodiments, the methods disclosed herein utilize the patient's T effector cells. In some embodiments, the methods disclosed herein generate individualized Treg cells.

[0028] Provided herein are methods for generating a population of engineered regulatory T cells, the method comprising introducing into a plurality of human T cells a composition comprising IL-37 or a nucleic acid sequence encoding IL-37 under conditions suitable for expressing IL-37 in the nuclei of the human T cells, thereby generating a plurality of engineered regulatory T cells.

[0029] Also provided herein is a method of treating an immune disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a population of engineered CD4+ T cells that express IL-37.

[0030] CD4+ T cells and regulatory T cells "CD4+ T cells" are intended to have their standard definition as used in the art. In some embodiments, the CD4+ T cells are CD3+CD4+CD8- T cells.

[0031] As used herein, "Tconv cells" or "T conventional cells" are synonymous and are intended to have their standard definitions used in the art. In some embodiments, Tconv cells are non-Treg CD4+ T cells. Tconv cells include naive CD4+ T cells (non-pathogenic).

[0032] As used herein, "Teff cells," "CD4+ Teff cells," "effector T cells," or "T effector cells" are synonymous and are intended to have their standard definitions used in the art. These are also called T helper (Th) cells and are involved in immune responses. In some embodiments, Teff cells include Th1, Th2, Th9, Th17, Th22, Thf cells, or combinations thereof.

[0033] As used herein and in the claims, the terms "regulatory T lymphocytes," "T regulatory cells," "regulatory T cells," "Treg cells," or "Treg" are synonymous and are intended to have their standard definitions used in the art. Treg cells are a specialized subpopulation of T cells that act "regulatory" to suppress immune system activation, thereby maintaining immune system homeostasis and tolerance to self-antigens. Treg cells are sometimes called suppressor T cells. Treg cells are characterized by expression of the forkhead family transcription factor FOXP3 (forkhead box p3). Treg cells are characterized by expression of CD4. Treg cells may express CD25 (also known as interleukin 2 receptor subunit alpha or IL2RA).

[0034] Treg cells are generally identified as a CD4+CD25+ T cell population capable of suppressing immune responses. Identification of Foxp3 as a "master regulator" of Treg cells helped define Treg cells as a distinct T cell lineage. Identification of additional antigenic markers on the surface of Treg cells allowed for the identification and FACS sorting of viable Treg cells to greater purity, resulting in a more highly enriched suppressive Treg population. In addition to CD4 and CD25, both mouse and human Treg cells express GITR / AITR, CTLA-4, and low levels of CD127 (IL-7Ra). Exemplary Treg expression markers include, but are not limited to, FOXP3, CD25, CD4, CTLA4, IL-10, GITR, TGF-beta, and CD127.

[0035] Furthermore, Treg cells can exist in different states that can be identified based on the expression of surface markers. Treg cells that develop in the thymus from CD4+ thymocytes are known as "natural" Treg cells. However, Treg cells can also be induced in the periphery from naive CD4+ T cells in response to low-dose binding of TCR, TGF-beta, and IL-2. These "induced" Treg cells secrete the immunosuppressive cytokine IL-10. The phenotype of Treg cells changes again as they are activated, and markers including GARP in mice and humans, CD45RA in humans, and CD103 in mice have been shown to be useful for identifying activated Treg cells. Treg cells are important for maintaining immune cell homeostasis, as evidenced by the undesirable consequences of genetic or physical ablation of Treg populations. Treg cells generally maintain order in the immune system by enforcing dominant-negative regulation on other immune cells. Broadly classified as natural or adaptive (induced) Treg cells, natural Treg cells are CD4+CD25+ T cells that develop and emigrate from the thymus and play a role in immune homeostasis. Adaptive Treg cells are non-regulatory CD4+ T cells that acquire CD25 (IL-2R alpha) expression outside the thymus and can be induced by inflammation and disease processes such as autoimmunity and cancer. Functional Treg cells can also be forced via overexpression of either of two common human FoxP3 isoforms in CD4+CD25- cells. These are classified as "forced Treg cells." In some embodiments, "Treg-like cells" can be generated via expression of nuclear IL-37 in CD4+ T cells. Treg-like cells exhibit properties of T regulatory cells, such as tolerance and stability. Treg-like cells can play a role in regulating immune responses and maintaining self-tolerance.

[0036] In some embodiments, the modified CD4+ T cells express one or more markers selected from the group consisting of FOXP3, CD25, CD4, CTLA4, IL-10, GITR, TGF-β, and CD127. In some embodiments of the present disclosure, at least one cell surface marker is FOXP3 and CD25. In some embodiments, at least one cell surface marker is FOXP3.

[0037] In certain embodiments, Treg cells are CD4+CD25+ and FOXP3+. In certain embodiments, Treg cells are CD4+CD25+FOXP3+ and CD127low. In certain embodiments, Treg cells are CD4+CD25+CD127low. In certain embodiments, Treg cells are CD4+CD25+FoxP3+CD127low and CD45RA+.

[0038] In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the plurality of engineered CD4+ T cells express at least one marker for regulatory T cells. In some embodiments, at least about 75% of the plurality of engineered CD4+ T cells express at least one marker for regulatory T cells. In some embodiments, at least about 95% of the plurality of engineered CD4+ T cells express at least one marker for regulatory T cells.

[0039] Loss of FOXP3 under inflammatory conditions induces instability and plasticity of Treg cells, which raises considerable concerns when using Treg cells or T cells to treat immune pathologies or anti-tumor immunity, respectively.

[0040] In some embodiments of the disclosed methods, the modified population of CD4+ T cells are allogeneic regulatory T cells. In some embodiments, the modified population of CD4+ T cells are autologous regulatory T cells.

[0041] Fork Head Box P3 (FOXP3) Expression of the transcription factor forkhead box P3 (FOXP3) is required for the suppressive function of regulatory T cells. Mutation or deletion of FOXP3 leads to the loss of functional Treg cell populations and the subsequent development of severe autoimmunity and / or inflammation in both humans and mice. Studies have shown that sustained FOXP3 expression and subsequent peripheral Treg (pTreg) cell stability are maintained by transcriptional and epigenetic regulation of FOXP3 in its promoter and conserved non-coding DNA sequences.

[0042] As disclosed herein, human Treg cells express the highest IL-37 mRNA levels among blood cells isolated from healthy individuals. Knockdown of IL-37 expression in CD4+ T cells significantly affected FOXP3 expression (72% reduction in FOXP3 mRNA after IL-37 knockdown). Knockdown of FOXP3 expression did not significantly affect IL-37 expression. Thus, evidence suggests an important role for IL-37 in regulating Treg cell function. Without wishing to be bound by theory, IL-37 may promote Treg suppressive function through its effect on FOXP3, and high-level expression of IL-37 functions to promote and maintain human Treg cell stability.

[0043] In some embodiments, CD4+ T cells that are not Treg cells can be genetically engineered into Treg cells via forced expression of FOXP3. In some embodiments, FOXP3 may be encoded in a transgene with an inducible promoter, such that FOXP3 expression can be induced to generate engineered or "forced" Treg cells. In some embodiments, the FOXP3 is wild-type (WT) FOXP3.

[0044] In some embodiments of the disclosed methods, expression of FOXP3 in the plurality of engineered CD4+ T cells is greater than expression of FOXP3 in a population of wild-type human T cells, hi some embodiments, expression of FOXP3 in the plurality of engineered CD4+ T cells is about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold greater than expression of FOXP3 in a population of wild-type human T cells.

[0045] In some embodiments, expression of FOXP3 in the plurality of engineered CD4+ T cells is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% greater than expression of FOXP3 in a population of wild-type human T cells.

[0046] In some embodiments, increased expression of FOXP3 in the modified CD4+ T cells is dependent on the introduction of IL-37 or a composition comprising a nucleic acid sequence encoding IL-37. In some embodiments, increased expression of FOXP3 in a population of modified CD4+ T cells maintains the immunosuppressive function of the modified CD4+ T cells in vitro and in vivo. In some embodiments, increased expression of FOXP3 in a population of modified CD4+ T cells prevents the conversion of the modified CD4+ T cells to proinflammatory T cells in vitro and in vivo. In some embodiments, increased expression of FOXP3 in a population of modified CD4+ T cells prolongs the immunosuppressive function of the modified CD4+ T cells in vitro and in vivo.

[0047] Interleukin-37 Interleukin-37 (IL-37) is a unique dual-function cytokine that functions both intracellularly and extracellularly. IL-37 is one of 11 IL-1 family members and the only one known to be broadly anti-inflammatory. IL-37 is transcribed as five distinct splice variants (IL-37a-e) and is expressed in human cells but not in mouse cells. However, like other IL-1 family members, IL-37 does not exhibit species specificity and exhibits effects on mouse cells that are comparable to those on human cells.

[0048] IL-37 is an anti-inflammatory cytokine that is involved in immune tolerance by generating semi-mature tolerogenic dendritic cells (DCs) in antigen-specific adaptive immune responses. Human regulatory T cells express the highest IL-37 levels among all T cell subsets. Because T cells do not secrete IL-37, IL-37 may play an intracellular role.

[0049] A plurality of the modified human CD4+ T cells of the population generated by the methods of the disclosure comprise interleukin-37 (IL-37) or a nucleic acid sequence encoding IL-37 under suitable conditions to express IL-37 in the nuclei of the T cells, wherein at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, comprise nuclear IL-37 or a nucleic acid sequence encoding IL-37. In some embodiments, about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage therebetween, of a plurality of engineered T cells of the population further express one or more markers of regulatory T (Treg) cells.

[0050] In some embodiments, nuclear expression of IL-37 in the plurality of engineered CD4+ T cells is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold greater than nuclear expression of IL-37 in a population of wild-type human T cells. In some embodiments, nuclear expression of IL-37 in the plurality of engineered CD4+ T cells is at least about 5-fold greater than nuclear expression of IL-37 in a population of wild-type human T cells.

[0051] In some embodiments, the nuclear expression of IL-37 in the plurality of engineered CD4+ T cells is about 1-2 fold, about 1-3 fold, about 1-4 fold, about 1-5 fold, about 1-6 fold, about 1-7 fold, about 1-8 fold, about 1-9 fold, about 1-10 fold, about 2-3 fold, about 2-4 fold, about 2-5 fold, about 2-6 fold, about 2-7 fold, about 2-8 fold, about 2-9 fold, about 2-10 fold, about 3-4 fold, about 4-5 fold, about 4-6 fold, about 4-7 fold, about 4-8 fold, about 4-9 fold, about 2-10 fold, about 3-4 fold, about 4-5 fold, about 4-6 fold, about 4-7 fold, about 4-8 fold, about 4-9 fold, about 5-10 fold, about 5-4 fold, about 5-6 fold, about 5-7 fold, about 5-8 fold, about 5-9 fold, about 5-10 fold, about 5-4 fold, about 5-8 fold, about 5-10 fold, about 5-4 fold, about 5-10 ... 3-5 fold, about 3-6 fold, about 3-7 fold, about 3-8 fold, about 3-9 fold, about 3-10 fold, about 4-5 fold, about 4-6 fold, about 4-7 fold, about 4-8 fold, about 4-9 fold, about 4-10 fold, about 5-6 fold, about 5-7 fold, about 5-8 fold, about 5-9 fold, about 5-10 fold, about 6-7 fold, about 6-8 fold, about 6-9 fold, about 6-10 fold, about 7-8 fold, about 7-9 fold, about 7-10 fold, about 8-9 fold, about 8-10 fold, about 9-10 fold. In some embodiments, nuclear expression of IL-37 in the plurality of engineered CD4+ T cells is about 5-10 fold greater than nuclear expression of IL-37 in a population of wild-type human T cells.

[0052] In some embodiments, nuclear expression of IL-37 in the plurality of engineered CD4+ T cells is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%, or any percentage therebetween, greater than nuclear expression of IL-37 in a population of wild-type human T cells.

[0053] In some embodiments, nuclear IL-37 expression in the engineered population of CD4+ T cells is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% greater than nuclear IL-37 expression in a population of wild-type CD4+ T cells. In some embodiments, nuclear IL-37 expression in the engineered population of CD4+ T cells is at least about 50% greater than nuclear IL-37 expression in a population of wild-type CD4+ T cells. In some embodiments, nuclear IL-37 expression in the engineered population of CD4+ T cells is at least about 85% greater than nuclear IL-37 expression in a population of wild-type CD4+ T cells.

[0054] In some embodiments, the expression of nuclear IL-37 in the population of engineered CD4+ T cells is about 10% to 20%, about 10% to 30%, about 10% to 40%, about 10% to 50%, about 10% to 60%, about 10% to 70%, about 10% to 80%, about 10% to 90%, about 10% to 100%, about 20% to 30%, about 20% to 40%, about 20% to 50%, about 20% to 60%, about 20% to 70%, about 20% to 80%, about 20% to 90%, about 20% to 100%, about 30% to 40%, about 30% to 50%, about 40% to 50%, about 50% to 6 ... Approximately 30% to 60%, approximately 30% to 70%, approximately 30% to 80%, approximately 30% to 90%, approximately 30% to 100%, approximately 40% to 50%, approximately 40% to 60%, approximately 40% to 70%, approximately 40% to 80%, approximately 40% to 90%, approximately 40% to 100%, approximately 50% to 60%, approximately 50% to 70%, approximately 50% to 80%, approximately 50% to 90%, approximately 50% to 100%, approximately 60% to 70%, approximately 60% to 80%, approximately 60% to 90%, approximately 60% to 100%, approximately 70% to 80%, approximately 70% to 90%, approximately 70% to 100%, approximately 80% to 90%, approximately 80% to 100%, approximately 90% to 100% larger. In some embodiments, nuclear IL-37 expression in the population of engineered CD4+ T cells is about 50% to about 80% greater than nuclear IL-37 expression in a population of wild-type CD4+ T cells.

[0055] In some embodiments of the disclosed methods, increasing nuclear expression of IL-37 in a population of engineered CD4+ T cells causes increased expression of FOXP3 in the same population. In some embodiments, higher expression of nuclear IL-37 in a population of engineered CD4+ T cells compared to IL-37 expression in a population of wild-type human T cells causes higher expression of FOXP3 in the population of engineered CD4+ T cells compared to FOXP3 expression in a population of wild-type human T cells. In some embodiments, increasing nuclear IL-37 expression in a population of engineered CD4+ T cells maintains the immunosuppressive function of the engineered CD4+ T cells in vitro and in vivo. In some embodiments, increasing nuclear IL-37 expression in a population of engineered CD4+ T cells prevents conversion of the engineered CD4+ T cells to proinflammatory T cells in vitro and in vivo. In some embodiments, increasing nuclear IL-37 expression in a population of engineered CD4+ T cells prolongs the immunosuppressive function of the engineered CD4+ T cells in vitro and in vivo. In some embodiments, increased expression of nuclear IL-37 in a population of engineered CD4+ T cells maintains the stability of the immunosuppressive phenotype of the engineered CD4+ T cells in vitro and in vivo. In some embodiments, expression of nuclear IL-37 in engineered CD4+ T cells prevents loss of FOXP3 expression in the engineered CD4+ T cells under inflammatory or pathogenic environments, compared to loss of FOXP3 expression in wild-type human T cells under similar conditions in vitro and in vivo.

[0056] Among all IL-1 family members, IL-37 is unique due to its broad suppression of innate and adaptive immunity. Because IL-37 is such a potent anti-inflammatory / anti-immune cytokine, its synthesis, activation, and secretion are tightly regulated. In humans, IL-37 mRNA and protein levels are normally low.

[0057] The present disclosure provides methods for generating a population of engineered CD4+ T cells, the method comprising introducing into a plurality of human T cells a composition comprising IL-37 or a nucleic acid sequence encoding IL-37 under conditions suitable for expression of IL-37, thereby generating a plurality of engineered CD4+ T cells. In some embodiments of the methods of the present disclosure, the plurality of engineered CD4+ T cells express IL-37 in the nucleus and cytoplasm of the cells. In some embodiments, the engineered CD4+ T cells express IL-37 in the nucleus. In some embodiments, the level of IL-37 in the nucleus is higher than the level of IL-37 in the cytoplasm. In some embodiments, the levels of IL-37 in the nucleus and cytoplasm are the same. In some embodiments, the level of IL-37 is lower in the nucleus than in the cytoplasm. In some embodiments, introducing into a plurality of human T cells a composition comprising IL-37 or a nucleic acid sequence encoding IL-37 increases the level of IL-37 in the nucleus of the engineered T cells.

[0058] In some embodiments, the ratio of nuclear IL-37 to cytoplasmic IL-37 in the plurality of engineered CD4+ T cells is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the ratio of nuclear IL-37 to cytoplasmic IL-37 in the plurality of engineered CD4+ T cells is about 0:10, 1:9; 2:8, 3:7, 4:6, 5:5; 6:4, 7:3, 8:2, 9:1, or 10:0. In some embodiments, the ratio of nuclear IL-37 to cytoplasmic IL-37 in the plurality of engineered CD4+ T cells is about 4:6, 5:5; 6:4, 7:3, or 8:2. In some embodiments, the ratio of nuclear IL-37 to cytoplasmic IL-37 in the plurality of engineered CD4+ T cells is about 4:6.

[0059] In some embodiments, the engineered CD4+ T cell population comprises non-Treg CD4+ T cells, and the ratio of nuclear IL-37 to cytoplasmic IL-37 is about 0:100. In some embodiments, the engineered CD4+ T cell population comprises Treg cells, and the ratio of nuclear IL-37 to cytoplasmic IL-37 is about 5:5. In some embodiments, the engineered CD4+ T cell population comprises Treg cells, and the ratio of nuclear IL-37 to cytoplasmic IL-37 is about 4:6. In some embodiments, the engineered CD4+ T cell population comprises Treg cells, and the ratio of nuclear IL-37 to cytoplasmic IL-37 is about 6:4.

[0060] Similar to macrophages and monocytes, T cells have been shown to express IL-37. However, unlike macrophages and monocytes, T cells do not secrete IL-37, suggesting an intracellular role for IL-37 in T cells. Initial data were obtained from studies of human Treg cells, which showed that cytoplasmic expression of IL-37 in human Treg cells resulted in the expression of Treg signatures, such as secretion of TGF-β and IL-10 and expression of CTLA-4 and FOXP3, resulting in their immunosuppressive function. IL-37 expression was detected in T cells from IL37 transgenic (Tg) mice, and adoptive transfer of IL-37-expressing CD3+ T cells attenuated DSS-induced chronic inflammatory bowel disease in mice.

[0061] IL-37 mRNA and protein levels are normally low and are induced by stimulation with pro-inflammatory cytokines in macrophages. However, this is not the case in Treg cells. Compared to monocytes, IL-37 mRNA levels are higher (more than 10-fold) in unstimulated human Treg cells.

[0062] Recently, human Treg cells have been shown to express the highest IL-37 mRNA levels among blood cells isolated from healthy individuals, and expression is much higher in melanoma patients, suggesting that Treg cells with high IL-37 expression may be highly suppressive and therefore may weaken anti-tumor immunity. Furthermore, a novel role for IL-37 in regulating FOXP3 expression and maintaining human Treg cell stability has recently emerged. Loss of FOXP3 under inflammatory conditions induces Treg cell instability and plasticity, raising considerable concerns when using Treg cells or T cells to treat immune pathologies or anti-tumor immunity, respectively.

[0063] The present disclosure provides methods for generating a population of modified CD4+ T cells, comprising introducing into a plurality of human T cells a composition comprising IL-37 or a nucleic acid sequence encoding IL-37 under conditions suitable for expressing IL-37 in the human T cells, thereby generating a plurality of modified regulatory T cells, wherein IL-37 regulates FOXP3 expression in the modified T cells. In some embodiments, the increase in IL-37 maintains FOXP3 levels in the modified CD4+ T cells under inflammatory conditions. In some embodiments, FOXP3 levels in the modified CD4+ T cells are maintained at a higher level compared to wild-type human T cells under inflammatory conditions. In some embodiments, the increase in IL-37 in the modified CD4+ T cells maintains the stability of the immunosuppressive phenotype of the modified T cells under steady-state or inflammatory conditions.

[0064] Dysregulated IL-37 expression has been reported in autoimmune diseases (RA, SLE, Hashimoto's thyroiditis, inflammatory bowel disease, Graves' disease, multiple sclerosis), ankylosing spondylitis, asthma, cardiovascular disease, cerebral ischemia, liver injury, infection, type 2 diabetes, and cancer. Downregulated IL-37 levels in active psoriatic skin were normalized when the disease was controlled with the JAK inhibitor tofacitinib. Similarly, downregulated IL-37 levels in active atopic dermatitis skin were restored after treatment with the JAK / SYK inhibitor ASN002. These data demonstrate a clear correlation between IL-37 expression and T cell-mediated skin inflammation (Th17 in psoriasis and Th2 in atopic dermatitis), suggesting an immunosuppressive role for IL-37 in human disease.

[0065] The present disclosure provides methods of treating an autoimmune disease or disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a population of engineered CD4+ T cells that express IL-37. In some embodiments, the engineered CD4+ T cells express IL-37 in the nucleus and cytoplasm. In some embodiments, the engineered CD4+ T cells express IL-37 in the nucleus. In some embodiments, the engineered CD4+ T cells express higher levels of IL-37 in the nucleus than in the cytoplasm. In some embodiments, the engineered CD4+ T cells express similar levels of IL-37 in the nucleus as in the cytoplasm. In some embodiments, the engineered CD4+ T cells express lower levels of IL-37 in the nucleus than in the cytoplasm.

[0066] Exemplary Engineered Regulatory T Cells (Treg-Like Cell Lines) The Jurkat cell line is an immortalized T lymphocyte cell line originally derived from the peripheral blood of a 14-year-old male patient with T-cell leukemia. The Jurkat clone E6-1 is a clone of the Jurkat-FHCRC cell line, a derivative of the Jurkat cell line, which was established from the peripheral blood of the aforementioned patient. This cell line can be used in immune system disorders and immunology and immuno-oncology research. The Jurkat cell line has been most frequently used as the prototype T cell line to study multiple events in T cell biology.

[0067] The development of Treg-like cell lines requires stable expression of the transcription factor forkhead box P3 (FOXP3), a key factor used to identify Treg cells and required for their suppressive ability. In a 2007 study, Treg-like cell lines were generated by transfecting E6 Jurkat cells, a non-Treg CD4 T-cell leukemia cell line, with a FOXP3 overexpression vector (Kim et al., Functional and genomic analyses of FOXP3-transduce Jurkat-T cells as regulatory T (Treg)-like cells, in the Journal of Biochem Biophys Res. Commun). This study found that overexpression of FOXP3 in Jurkat cells resulted in increased surface expression of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD25. These cells suppressed the proliferation of conventional T (Tconv) cells, but the suppressive activity was significantly lower than that of primary Treg cells, suggesting that alternative molecules are required to generate functionally equivalent artificial Treg cells from Jurkat cells.

[0068] Recently, it has been shown that IL-37, an anti-inflammatory IL-1 cytokine family member, contributes to FOXP3 expression. Knockdown of IL-37 in primary human Treg cells resulted in a significant reduction in FOXP3 and promoted T cell proliferation and differentiation. Treatment of human Treg cells with recombinant IL-37 also upregulated FOXP3 and enhanced their suppressive activity. These studies indicate that IL-37 expression in human Treg cells contributes to FOXP3 expression and Treg cell function, prompting us to hypothesize that overexpression of IL-37 in non-Treg CD4 T cells, such as Jurkat cells, may result in the generation of human Treg-like cells.

[0069] However, it was unclear whether T cells express IL-37. It has been reported that monocytes and dendritic cells are the main producers of IL-37 in human peripheral blood mononuclear cells (PBMCs) from healthy donors, and only 0.5% of T cells express IL-37. Meanwhile, another study showed that IL-37 mRNA and IL-37 protein are highly expressed in human T cells, including Treg cells, from healthy volunteers. In fact, approximately 70% of Treg cells express IL-37 protein, indicating that Treg cells are one of the important contributors to IL-37 expression.

[0070] In some embodiments, the population of engineered CD4+ T cells is derived from human T cells. In some embodiments, the human T cells are derived from a primary human T cell population. In some embodiments, the human T cells are derived from a T cell line. In some embodiments, the T cell line is a Jurkat T cell line.

[0071] Compositions and Uses of Modified CD4+ T Cells In some embodiments, the methods of the disclosure produce compositions comprising a population of modified CD4+ T cells. In some embodiments, the methods of the disclosure produce compositions comprising a population of modified T regulatory cells or Treg-like cells.

[0072] In some embodiments of the disclosed methods, the buffer comprises a plurality of engineered regulatory T cells or precursors thereof. The buffer maintains or enhances cell viability and / or the level of Treg phenotype of immune cells, including T cells, or precursors thereof. In certain embodiments, the buffer maintains or enhances cell viability and / or the level of Treg phenotype of primary human T cells prior to nucleofection. In certain embodiments, the buffer maintains or enhances cell viability and / or the level of Treg phenotype of primary human T cells during nucleofection. In certain embodiments, the buffer maintains or enhances cell viability and / or the level of Treg phenotype of primary human T cells after nucleofection. In certain embodiments, the buffer comprises one or more of KCl, MgCl, ClNa, glucose, and Ca(NO) in any absolute or relative abundance or concentration; optionally, the buffer further comprises a supplement selected from the group consisting of HEPES, Tris / HCl, and phosphate buffer. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO). In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO), and supplements comprising 20 mM HEPES and 75 mM Tris / HCl. In certain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl, 90 mM ClNa, 10 mM glucose, and 0.4 mM Ca(NO), and supplements comprising 40 mM NaHPO / NaHPO (pH 7.2). In certain embodiments, a composition comprising engineered regulatory T cells or precursors thereof comprises 100 μl of buffer and 5×10 6 ~25×10 6 In certain embodiments, the composition comprises 250 x 10 cells per milliliter of buffer or other medium during the introducing step. 6 Contains scalable ratios of human T cells.

[0073] In some embodiments of the methods of the present disclosure, the methods comprise contacting immune cells of the present disclosure, including regulatory T cells of the present disclosure, with a T cell expansion composition. In some embodiments of the methods of the present disclosure, the step of introducing IL-37 or a nucleic acid sequence encoding IL-37 of the present disclosure into immune cells of the present disclosure can further comprise contacting the immune cells with a T cell expansion composition. In some embodiments, including embodiments in which the introducing step of the method comprises an electroporation or nucleofection step, the electroporation or nucleofection step can be performed with the immune cells contacted with the T cell expansion composition of the present disclosure.

[0074] Methods for expressing IL-37 The present disclosure provides methods for expressing IL-37 in modified CD4+ T cells, the methods including: (a) obtaining a cell population; (b) contacting the cell population with a composition comprising IL-37 or a nucleic acid sequence encoding IL-37 under conditions sufficient to translocate IL-37 or a nucleic acid encoding IL-37 across the cell membrane of at least one cell in the cell population, thereby creating a modified cell population; (c) culturing the modified cell population under conditions suitable for expression of IL-37 or a nucleic acid sequence encoding IL-37 in the nucleus of the T cells; and (d) expanding and / or selecting at least one cell from the modified cell population that expresses IL-37.

[0075] In some aspects, the cell population may comprise a plurality of engineered human CD4+ T cells that express at least one marker of regulatory T cells, wherein the at least one marker of regulatory T cells is selected from the group consisting of CD4, FOXP3, CD25, CD4, CTLA4, IL-10, GITR, TGF-β, and CD127. In some embodiments, the at least one marker is FOXP3. In some embodiments, the at least one marker is FOXP3 and CD25. The cell population may comprise an optimized ratio of FOXP3+ and FOXP3+CD25+ regulatory T cells.

[0076] In some embodiments, the conditions sufficient to translocate IL-37 or a nucleic acid sequence encoding IL-37 across the cell membrane of at least one cell in the cell population comprise at least one application of one or more electrical pulses at a specified voltage, a buffer, and one or more cofactors. In some embodiments, the conditions suitable for incorporation of the sequence encoding IL-37 comprise at least one of a buffer and one or more cofactors.

[0077] The expansion and selection steps may be performed in parallel or sequentially. Expansion may occur before selection. Expansion may occur after selection, and optionally, a further (i.e., second) selection may occur after expansion. Parallel expansion and selection may be simultaneous. The expansion and / or selection steps may proceed over a period of 1 to 7 days, inclusive.

[0078] In some aspects, when IL-37 or a nucleic acid encoding IL-37 comprises a selection gene, the selection step comprises contacting at least one cell of the modified cell population with a compound to which the selection gene confers resistance, thereby identifying cells that express the selection gene as those that survive the selection and identifying cells that are unable to express the selection gene as those that do not survive the selection step.

[0079] The present disclosure provides compositions comprising a population of multiple modified, expanded, and selected CD4+ human T cell populations according to the methods described herein.

[0080] Vector Composition The present disclosure provides compositions and methods for delivering IL-37 or a nucleic acid sequence encoding IL-37 to regulatory T cells or a population of regulatory T cells. Non-limiting examples of compositions for delivering the disclosed compositions to cells or cell populations include transposons or vectors. Thus, the present disclosure provides vectors comprising a nucleic acid sequence encoding IL-37.

[0081] The vectors comprising IL-37 of the present disclosure may further comprise a selection gene. The selection gene may encode a gene product essential for cell viability and survival. The selection gene may encode a gene product essential for cell viability and survival when challenged by selective cell culture conditions. The selective cell culture conditions may include a compound deleterious to cell viability and survival, and the gene product confers resistance to that compound. Non-limiting examples of selection genes include neo (confers neomycin resistance), DHFR (encodes dihydrofolate reductase, conferring methotrexate resistance), TYMS (encodes thymidylate synthetase), MGMT (encodes O(6)-methylguanine-DNA methyltransferase), multidrug resistance gene (MDR1), ALDH1 (encodes aldehyde dehydrogenase 1 family member A1), FRANCF, RAD51C (encodes RAD51 paralog C), GCS (encodes glucosylceramide synthase), NKX2.2 (encodes NK2 homeobox 2), or any combination thereof.

[0082] Treatment method The present disclosure provides uses of the disclosed compositions or pharmaceutical compositions for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, e.g., by administering or contacting a therapeutically effective amount of the composition or pharmaceutical composition to the cell, tissue, organ, animal, or subject. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0083] The present disclosure provides methods for treating an immune disease or disorder (e.g., an autoimmune disease or disorder) or an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a population of engineered regulatory T cells that express IL-37. Immune diseases or disorders include conditions resulting from the abnormal activity of immune cells, overreacting to or attacking the body, conditions exhibiting an extreme inflammatory response, or a loss of the ability to recognize and combat non-self cells. Exemplary immune diseases or disorders include, but are not limited to, 1) allergies, such as allergic contact hypersensitivity; 2) alloreactivity, such as graft-versus-host disease and transplant rejection; and 3) autoimmune diseases or disorders, such as type 1 diabetes, systemic lupus erythematosus, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and multiple sclerosis. In some embodiments, the immune disease or disorder is allergic contact hypersensitivity. In some embodiments, the immune disease or disorder is graft-versus-host disease. In some embodiments, the immune disease or disorder is inflammatory bowel disease. In some embodiments, the immune disease or disorder is type 1 diabetes.

[0084] Exemplary inflammatory diseases or disorders include, but are not limited to, conditions affecting the digestive system, joints, skin, respiratory system, or nervous system. In some embodiments, the inflammatory disease or disorder is selected from the group consisting of psoriasis, traumatic brain injury, bronchitis, and pneumonitis. In some embodiments, the inflammatory disease or disorder is psoriasis. In some embodiments, the inflammatory disease or disorder is traumatic brain injury. In some embodiments, the inflammatory disease or disorder is bronchitis. In some embodiments, the inflammatory disease or disorder is pneumonitis.

[0085] In some embodiments, the population of modified CD4+ T cells are allogeneic regulatory T cells. In some embodiments, the population of modified CD4+ T cells are autologous regulatory T cells.

[0086] In a preferred embodiment, the treatment of an immune disorder or disease involves adoptive cell therapy. For example, in one embodiment, the present disclosure provides modified CD4+ T cells that express IL-37 and at least one marker of a regulatory T cell, wherein the modified regulatory T cell is selected and expanded for administration to a subject in need thereof. The modified cells can be formulated for storage at any temperature, including room temperature and body temperature. The modified cells can be formulated for cryopreservation and subsequent thawing. The modified cells can be formulated in a pharmaceutically acceptable carrier for direct administration to a subject from sterile packaging. The modified cells can be formulated in a pharmaceutically acceptable carrier with an indicator of cell viability and / or IL-37 expression level to ensure a minimum level of cell function and IL-37 expression. The modified cells can be formulated in a pharmaceutically acceptable carrier at a prescribed density with one or more agents to inhibit further expansion and / or prevent cell death.

[0087] The treatment methods can include administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or both. Such methods can optionally further include co-administration or combination therapy for treating such diseases or disorders, where the administration of any of the compositions or pharmaceutical compositions disclosed herein further includes administration before, concurrently with, and / or after at least one chemotherapeutic agent (e.g., alkylating agents, antimitotic agents, and radiopharmaceuticals).

[0088] In some aspects, the subject does not develop graft versus host disease (GvHD) and / or host versus graft disease (HvGD) after administration. In one aspect, administration is systemic. Systemic administration can be by any means known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or infusion. In one aspect, administration is local. Local administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral, intrathecal, intraventricular, intraocular, or intraosseous injection or infusion.

[0089] In some embodiments, the therapeutically effective dose is a single dose, hi some embodiments, a single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or any number in between, doses produced simultaneously.

[0090] In some embodiments, when the composition is autologous or allogeneic cells, the dose is sufficient to allow the cells to engraft and / or persist for a sufficient time to treat the disease or disorder.

[0091] In some aspects of the treatment methods described herein, treatment can be modified or terminated, for example, in response to signs of recovery or a decrease in disease severity / progression, signs of disease remission / halt, and / or the occurrence of an adverse event.

[0092] Formulations, Dosages and Modes of Administration The present disclosure provides formulations, dosages and methods for administration of the modified CD4+ T cell compositions described herein.

[0093] The disclosed compositions can further comprise at least one of any suitable auxiliary agent, such as, but not limited to, a diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant, etc. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples of such sterile solutions and methods for their preparation are well known in the art, for example, but not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 and "Physician's Desk Reference," 52nd ed., Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable carriers are well known in the art or can be routinely selected to suit the mode of administration, solubility, and / or stability of the protein scaffold, fragment, or variant compositions described herein.

[0094] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars, such as alditols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which may be present alone or in combination and comprise 1 to 99.99% by weight or volume, alone or in combination. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid / protein components that may also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0095] Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myo-inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0096] The composition can also contain a buffer or pH adjuster. Typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffer. Preferred buffers are organic acid salts such as citrate.

[0097] Additionally, the disclosed compositions may include polymeric excipients / additives such as polyvinylpyrrolidone, Ficoll (polymeric sugar), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0098] Many known and developed modes can be used to administer a therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein. Non-limiting examples of modes of administration include bolus, buccal, infusion, intra-articular, intrabronchial, intraperitoneal, intravesical, intrachondral, intracavity, intracellular, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intrathecal, intrasynovial, intrathoracic, intrauterine, intratumor, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal, or vaginal means.

[0099] nucleic acid molecule The nucleic acid molecule of the present disclosure encoding IL-37 can be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA can be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or the non-coding strand, also referred to as the antisense strand.

[0100] Nucleic acid construction The isolated nucleic acids of the present disclosure can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as are well known in the art.

[0101] A nucleic acid may conveniently contain nucleotide sequences in addition to the polynucleotides of the present disclosure. For example, a multicloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of the polynucleotide. A translatable sequence can also be inserted to aid in the isolation of the translated polynucleotides of the present disclosure. For example, a hexahistidine marker sequence provides a convenient means for purifying the proteins of the present disclosure. A nucleic acid of the present disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expressing the polynucleotides of the present disclosure.

[0102] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art.

[0103] The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art. Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the present disclosure without undue experimentation, based on the teachings and guidance provided herein.

[0104] definition As used throughout this disclosure, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, and reference to "a dose" includes a reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0105] The terms "about" or "approximately" mean within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in this application and claims, unless otherwise specified, the term "about" meaning within an acceptable range of error for the particular value should be assumed.

[0106] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants or inert carriers. "Consisting of" shall mean excluding more than trace amounts of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0107] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0108] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or a protein produced by translation of mRNA. Gene products also include RNAs that have been modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that have been modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0109] "Modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.

[0110] "Operably linked" or its equivalents (e.g., "linked operatively") means that two or more molecules are positioned relative to each other so that they can interact to affect a function attributable to one or both molecules, or a combination thereof.

[0111] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked together. A depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid can also encompass the complementary strand of a depicted single strand. Nucleic acids of the present disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0112] The nucleic acids of the present disclosure may be single-stranded or double-stranded. The nucleic acids of the present disclosure may contain double-stranded sequences even when the majority of the molecule is single-stranded. The nucleic acids of the present disclosure may contain single-stranded sequences even when the majority of the molecule is double-stranded. The nucleic acids of the present disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure may contain a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure may contain a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure may be synthesized to contain non-naturally occurring amino acid modifications. The nucleic acids of the present disclosure may be obtained by chemical synthesis or recombinant methods.

[0113] The nucleic acids of the present disclosure may be non-naturally occurring, either in their entirety or in any part thereof. The nucleic acids of the present disclosure may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions that render the entire nucleic acid sequence non-naturally occurring. The nucleic acids of the present disclosure may contain one or more duplicated, inverted, or repeated sequences that render the entire nucleic acid sequence non-naturally occurring, resulting in a non-naturally occurring sequence. The nucleic acids of the present disclosure may contain non-naturally occurring modified, artificial, or synthetic nucleotides that render the entire nucleic acid sequence non-naturally occurring.

[0114] Given the redundancy in the genetic code, multiple nucleotide sequences may encode any particular protein, and all such nucleotide sequences are contemplated herein.

[0115] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene under the control of a spatially connected promoter. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Variation in the distance between the promoter and the gene can be accommodated without loss of promoter function.

[0116] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally occurring molecule that is capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences that further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, which can be located as many as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter can regulate the expression of a genetic component constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include a bacteriophage T7 promoter, a bacteriophage T3 promoter, an SP6 promoter, a lac operator promoter, a tac promoter, an SV40 late promoter, an SV40 early promoter, an RSV-LTR promoter, a CMV IE promoter, an EF-1 alpha promoter, a CAG promoter, an SV40 early promoter or an SV40 late promoter, and a CMV IE promoter.

[0117] As used throughout this disclosure, the term "substantially complementary" refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0118] As used throughout this disclosure, the term "substantially identical" refers to first and second sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or nucleic acids where the first sequence is substantially complementary to the complement of the second sequence.

[0119] As used throughout this disclosure, the term "variant," when used to describe a nucleic acid, refers to (i) a portion or fragment of a referenced nucleotide sequence, (ii) the complement of a referenced nucleotide sequence or a portion thereof, (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or its complement, or (iv) a nucleic acid that hybridizes under stringent conditions to a referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0120] As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence containing a replication origin. A vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector can contain a combination of amino acids with a DNA sequence, an RNA sequence, or both a DNA and an RNA sequence.

[0121] As used throughout this disclosure, the term "endogenous" refers to a nucleic acid or protein sequence that is naturally associated with the target gene or host cell into which it is introduced.

[0122] As used throughout this disclosure, the term "exogenous" refers to a nucleic acid or protein sequence that is not naturally associated with the target gene or the host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., a DNA sequence, or a naturally occurring nucleic acid sequence located in a non-naturally occurring genomic location.

[0123] The present disclosure provides a method for introducing a polynucleotide construct containing a DNA sequence into a host cell. The term "introducing" refers to presenting the polynucleotide construct to a plant in a manner that allows the construct to access the interior of the host cell. The method of the present disclosure does not depend on a particular method for introducing the polynucleotide construct into a host cell, but only on the polynucleotide construct accessing the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art, including, but not limited to, stable transformation, transient transformation, and virus-mediated methods. [Example]

[0124] Example 1: Expression of IL-37 induces regulatory T cell-like phenotype and function in Jurkat cells This example describes the materials and methods used in Examples 1-7.

[0125] Materials and Methods Cell preparation and culture Human leukemia cell line E6 Jurkat cells were obtained from ATCC (Manassas, VA) (clone E6-1). Cells were cultured in RPMI 1640 containing L-glutamine (Gibco, Thermo Fisher Scientific) and supplemented with 10% FBS (Gemini BioProducts), 10 nM HEPES (Life Technologies, Thermo Fisher Scientific), 1% non-essential amino acids (Life Technologies), and 1 mM sodium pyruvate (Life Technologies, Thermo Fisher Scientific) in a humidified incubator at 37°C and 5% CO2. Cells were cultured in Nunc™ EasyFlask T75 containers at 2 x 10 5 / ml, the cell count was 1 × 10 6 They were passaged when they reached 1 / ml and replated at a 1:5 dilution into new flasks.

[0126] Human peripheral blood from healthy donors was obtained from a blood cone provided by Children's Hospital Blood Donor Center (Aurora, CO), and peripheral blood mononuclear cells (PBMCs) were purified using Ficoll density gradient centrifugation. Human primary T cells were isolated from blood samples obtained from healthy human donors. After Ficoll density gradient centrifugation, the cells were purified using a Human CD4+ T Cell Isolation Kit (Miltenyi Biotech, Auburn, CA) and Human CD4+CD25+CD127. dim Cells were isolated using a regulatory T cell isolation kit (Miltenyi Biotech) according to the manufacturer's instructions, and were classified as CD4+ T cells, CD4+CD25-CD127 hi (Tconv) cells, and CD4+CD25+CD127 dimHuman Treg cells were purified to 94-96% purity by flow cytometry using CD4, CD25, and FOXP3 surface markers. Purification was repeated twice to minimize Tconv cell contamination. FOXP3 Treg cell purity check after human cell isolation is shown in Figure 37B. All use of human blood specimens at the University of Colorado AMC was approved by the Colorado Institutional Review Board.

[0127] Mouse CD4+ T cells and CD4+CD25+ Treg cells were purified from splenocytes of C57BL / 6, IL37 Tg, or D20A mice using a Mouse CD4+ T Cell Isolation Kit (Miltenyi Biotech) and a Mouse CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotech) according to the manufacturer's instructions. For the purification of either mouse or human Treg cells, MS column purification steps were performed in duplicate to ensure highly pure CD4+CD25+ T cells. Foxp3+ Treg cell purity check after mouse cell isolation. Mouse and human CD4+CD25- Tconv / Tresp cells were obtained as the dump population of CD4+ T cells remaining after CD25+ Treg cell purification.

[0128] Caspase-1 inhibitor (Ac-YVAD-CMK) was purchased from Sigma-Aldrich. PAK1 / 2 / 3 inhibitor FRAX597 was purchased from Tocris. Flagellin was purchased from Invivogen.

[0129] mouse IL37 Tg mice have been previously described (Garlanda et al., 2004; Nold et al., 2010). The Dinarello laboratory developed IL37 Tg mice, which are described herein and further described in Suzhao et al. (S. Li et al., 2019). D20ATg(D20A) mice were generated. C57BL / 6 (WT) mice were purchased from Taconic Biosciences. The presence of the transgenes encoding IL37 and D20A was confirmed by genotyping PCR (Transnetyx) using tail clips from 3- to 4-week-old mice. Only mice 6- to 12-week-old were used in the experiments. All mice were maintained in the specific pathogen-free animal facility at the University of Colorado AMC and handled in accordance with institutional guidelines. All mouse experiments were approved by the Institutional Animal Care and Use Committee of the University of Colorado Denver.

[0130] Murine CD4+ T cells and CD4+CD25+ Treg cells were purified from splenocytes of C57BL / 6, IL37 transgenic (Tg), or D20A mice using a Murine CD4+ T Cell Isolation Kit (Miltenyi Biotech) and a Murine CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotech) according to the manufacturer's instructions. For the purification of murine Treg cells, MS column purification steps were performed in duplicate to ensure high purity of CD4+CD25+ T cells. Foxp3+ Treg cell purity check was performed after murine cell isolation. Murine CD4+CD25- Tconv / T resp Cells were obtained as a dump population of CD4+ T cells remaining after CD25+ Treg cell purification.

[0131] All cells were cultured in RPMI 1640 containing L-glutamine (Gibco, Thermo Fisher) and supplemented with 10% FBS (Gemini BioProducts), 10 nM HEPES (Life Technologies, Thermo Fisher) (San Diego, CA), 1% nonessential amino acids (Life Technologies, Thermo Fisher), and 1 mM sodium pyruvate (Life Technologies, Thermo Fisher).

[0132] antibody Flow cytometry and cell stimulation were performed using the following antibodies from Thermo Fisher for mouse antigens: Foxp3 (FJK-16s), CD25 (PC61.5), CD45RB (I6A), CD4 (GK1.5), CD8 (53-6.7), CD3ε (2c11), CD28 (37.51), and CTLA-4 (UC10-4B9), and from Thermo Fisher for human antigens. The following antibodies were from Fisher Scientific: CD28 (CD28.2), CD3 (OKT3), CD4 (RPA-T4), CD8 (SK1), CTLA-4 (14D3), CD28 (CD28.2), CD45RB (PD7 / 26), IL-10 (JES3-9D7), IL-37 (37D12), CD127 (eBioRDR5), AIM2 (14-6008-93), phospho-Smad3 S208 (PA5-1042450), phospho-caspase-1 S376 (PA5-38565), phospho-NLRC4 S533 (MA5-31846), and phospho-PAK1 / 2 / 3. S144, S141, S139 (MAF-32130), and FOXP3 (3G3, SK3, and 236A / E7) were used. FOXP3 (D608C) was purchased from Cell Signaling.

[0133] Primeflow® RNA Assay Human donor PBMCs were collected and purified using Ficoll gradient centrifugation. Cells were then either left untreated, cultured with 100 ng / ml lipopolysaccharide (LPS) in complete RPMI 1640 medium, or resuspended in freezing medium (RPMI + 10% DMSO (Sigma)) and placed in a -80°C freezer for later use. After 24 hours, fluorescent mRNA in situ hybridization was performed using the PrimeFlow® RNA Assay from Thermo Fisher using a custom-designed IL-37 mRNA-specific probe according to the manufacturer's instructions. The IL37 mRNA probe was designed by Thermo Fisher based on our published IL37 qRT-PCR primer sequence (Luo et al., 2014) to obtain consistent results between the two assays. For surface staining, the manufacturer's recommended volume (5 μl / test) of fluorescently conjugated antibodies specific for surface markers was added to 1 × 10 6Cells were added to each seeded donor PMBC sample, resuspended in 100 μl of FACS buffer at 4°C for 30 minutes, and then washed twice with FACS buffer before analysis or further processing. Immune cells were stained into two groups based on the immune antibody panel provided by the manufacturer: one group for myeloid subsets and the other for lymphoid subsets (as shown by the gating strategy in Figures 23A-B). Once gated on individual subsets, cells were analyzed for expression of IL37 mRNA (AF437). PrimeFlow® specific antibodies for cell surface proteins, including CD3 (efluor450, UCHT1), CD4 (AF700, RPA-T4), CD8 (APC-efluor780, RPA-T8), CD11c (PE-efluor610, 3.9), CD14 (PECy7, 61D3), CD16 (PE, EBIOCB16), CD19 (PECy5.5), CD25 (PE-Cy7, BC96), CD45RB (AF488, PD7 / 26), CD56 (PE-Ef610, CMSSB), and HLA-DR (FITC, L243), were purchased from ThermoFisher. Fixable Viability Dye (FVD, efluor506) was used to remove dead cells. Flow cytometry of PrimeFlow® RNA samples was performed on a ZE5 Cell Analyzer (BioRad, Hercules, CA). The gating strategy for PrimeFlow® samples for specific cell analysis, such as the percentage of IL-37+ cells, is shown in Figures 1A-1B. Further analysis of flow cytometry data was performed using FlowJo_V10 software (Tree Star).

[0134] siRNA knockdown in primary human T cells Purified primary human CD4+ T cells were used for siRNA knockdown of RUNX1, RPTOR, IL37, CREB1, CHK1, NLRP2, NLRP3, NLRP4, ASC, and NLRC4, and were CD4+CD25+CD127 dimT cells were used for siRNA knockdown of FOXP3 and IL-37. Cells were purified from healthy donor PBMCs as described above and then placed in culture in complete RPMI 1640 for at least 2 hours prior to siRNA transfection by nucleofection using the Amaxa P3 Primary Cell 4D-Nucleofector X Kit L (Lonza) according to the manufacturer's instructions. For knockdown of RPTOR and IL-37, three different siRNAs (30 nM) against RPTOR and IL-37 (A, B, and C (OriGene, Rockville, MD)) were used separately or pooled, while for FOXP3 knockdown, CD4+CD25+CD127 T cells were used. dim Due to the limited number of T cells, only a pool of three different siRNAs (30 nM) against FOXP3 (A, B, and C (OriGene)) was used. For NLRP3A knockdown, three to four siRNAs were pooled together, and for NLRC4 and NLRP4, Dharmacon SMARTpool was used. For NLRP2 and ASC (Ambion), single sense and antisense siRNAs were used. Scrambled siRNAs (OriGene, Ambion, and Dharmacon) were used as controls. Knockdown was confirmed 24 hours after nucleofection by qRT-PCR or 48 hours after nucleofection by Western blot, and the relative density of the protein bands was determined using FIJI. The remaining T cells were then stimulated with anti-CD3 / CD28 Dynabeads™ and assayed for FOXP3 expression or activated caspase-1 by flow cytometry, as described above. FOXP3 knockdown was confirmed by qRT-PCR 24 hours after nucleofection, and the remaining cells were antibody stimulated using anti-CD3 / CD28 Dynabeads™ and assayed for IL-37 and CTLA-4 expression using flow cytometry. All siRNA sequences are listed in Table 1 below.

[0135] [Table 1]

[0136] In vitro T cell suppression assay For suppression assays containing Treg cells, CD4+CD25 hi T cells were purified from splenocytes of either wild-type or IL37 Tg mice using the Mouse CD4+CD25+ T cell Isolation Kit (Miltenyi). 100,000 purified CFSE-labeled wild-type CD4+CD25 T cells were collected in 50 μL of complete RPMI 1640. lo T cell responders (Tresp) were added to round-bottom 96-well plates (CellStar, Dallas, TX) containing heat-killed splenocytes (APCs), 5 μg / ml soluble anti-CD3 antibody, and the indicated dilutions of Treg cells in 150 μl of complete RPMI 1640 (Sarmento et al., 2015). After 3 days, proliferation / suppression of Tresp cells was measured using CFSE and analyzed by flow cytometry. Percent suppression was calculated using the formula: % suppression = 100 - (x / y) 100, where X is the percentage of T cells, including Treg cells, as in (McMurchy and Levings, 2012). resp represents the division index (DI) of Tresp cells, and Y is the average DI of Tresp cells only.

[0137] IL-37 ELISA IL-37 secretion into the supernatant was measured by incubating 1 × 10 cells in 1 ml of complete RPMI 1640 medium. 6 Cells were analyzed by culturing 1000 cells. Supernatants were then collected and analyzed to measure IL-37 protein abundance using the DuoSet® Human IL-37 / IL-1F7 ELISA Kit (R&D Systems) according to the manufacturer's instructions. 1 ng / ml and 2 ng / ml of recombinant human IL-37 were used as controls to determine the sensitivity of the ELISA assay.

[0138] Contact hypersensitivity assay On day 0, 6- to 8-week-old, gender-matched C57BL / 6 WT mice were sensitized by applying 25 μl of 0.5% 2,4-dinitro-fluorobenzene (DNFB) (Sigma-Aldrich) in 4:1 (vol / vol) acetone / olive oil to the shaved abdomen. As a control, 10 μl of vehicle was applied to the left ear. Ear thickness was measured using an engineer's micrometer (Mitutoyo, Takatsu-ku, Japan). Results were expressed as net ear swelling, which was calculated by subtracting the pre-treatment ear thickness from the post-treatment ear thickness.

[0139] Contact hypersensitivity assay (expanded Treg cells) On day 5, 1 hour before challenge with DNFB, 1x PBS (vehicle) or 5x10 cells from either donor WT or IL37 Tg were injected. 5 MACS purified and expanded CD4+CD25 hi T cells were injected intravenously into mice. Mice were challenged by applying 10 μl of 0.2% DNFB in 4:1 (v / v) acetone / olive oil to the right ear. Clinical measurements and analyses were as described above.

[0140] Tissue collection from CHS for purification and analysis of lymphocyte populations Forty-eight hours after DNFB challenge, draining lymph nodes (dLNs) and spleens were harvested from mice. Lymphocytes were obtained from vehicle- and hapten-sensitized ears by enzymatic digestion in room-temperature HBSS medium (Gibco) supplemented with 0.7 mg / ml collagenase D (Sigma-Aldrich), and density gradient separation was performed according to a previously described protocol (Benck et al., 2016). dLNs and spleens were macerated with 40 μM filters (Falcon, Corning, NY). RBCs were lysed from splenocytes, and single cells from both dLNs and spleens were washed with 1X PBS and prepared for antibody staining (Osborne et al., 2015). Single-cell suspensions from ears, dLNs, and spleens were Fc-blocked with purified anti-mouse CD16 / CD32 monoclonal antibody (2.4G2) and then stained for CD3, CD8, CD4, CD25, and CD45RB. The lymphocyte gating strategy is shown in Figure 23B.

[0141] Immunofluorescence (IF) and imaging / flow-based proximity ligation assay (PLA) 1×10 5Treg cells were cultured on poly-D-lysine-coated Falcon® 8-chamber polystyrene vascular tissue culture-treated glass slides (Corning, Corning, NY) for at least 30 minutes to allow for firm adhesion. Cells were fixed by adding ice-cold fixative (4% paraformaldehyde and 0.5% glutaraldehyde in 1x PBS), incubated in the dark at room temperature for 30 minutes, and then permeabilized with 0.2% Triton X-100 in PBS for 15 minutes. Cells were then cultured overnight at 4°C in a humidified chamber with 10 μg / ml mouse anti-human IL-37 (IL-1F7) monoclonal antibody (7F1A1, Proteintech) and rabbit anti-human phospho-Smad3 (Ser423, Ser425) (ThermoFisher) in IF buffer (TBS and goat serum cocktail). After washing 5–6 times in 1x PBS, cells were incubated with AF488-conjugated goat anti-mouse secondary antibody (1:500 dilution in imaging buffer) for 1 h at room temperature for IL-37 imaging only. After washing 5–6 times with PBS, ProLong™ Gold antifade reagent (Thermo Fisher) containing DAPI nuclear stain was added to each well.

[0142] For proximity ligation assay (PLA) experiments, after washing out the primary antibody, cells were incubated with anti-rabbit PLUS probe (1:5 dilution, Duo92002, Sigma-Aldrich) or anti-mouse MINUS probe (1:5 dilution, Duo92004, Sigma-Aldrich) for 1 h at 37 °C and washed twice with wash buffer A. The probes were then ligated for 30 min at 37 °C, washed twice with buffer A, and amplified with polymerase (Sigma-Aldrich) in the dark for 100 min at 37 °C. For flow-based PLA (Sigma-Aldrich), after amplification, cells were treated with 1x green detection buffer for 30 min at 37 °C, then washed twice with PLA wash buffer and prepared for flow analysis. After washing twice with 1x buffer B (Thermo Fisher), ProLong™ Gold antifade reagent (Thermo Fisher) containing DAPI nuclear stain was added to each well. Images were acquired using an Olympus FV1000 FCS / RICS system (Olympus, Tokyo, Japan). For analysis, seven different 100X / 1.4 Oil Plan-Apochromatic objective fields were imaged per well per experiment. Each field was measured for IL-37 fluorescence intensity using the FIJI Measure application. To analyze the cytoplasmic versus nuclear localization of IL-37 in T cells, overlapping images of DAPI and IL-37 were used. Along with single-slice whole-field images, 0.25 μM slice z-stack images were taken to confirm the cytoplasmic versus nuclear localization of IL-37. Colocalization of IL-37 and DAPI was assessed by Pearson's correlation coefficient using FIJI. To measure differences in fluorescence across the cell by drawing a line across the diameter of the cell, a line intensity profile was created using Plot profile in FIJI. The staining intensity measurements across the line were then imported into Prism 8 (GraphPad, Lo Jolla, CA). 3D fluorescence maps were generated using Surface plot in FIJI.

[0143] Phosphoprotein expression Purified WT and IL37 Tg CD4+CD25+CD127 dim Cell lysates from Treg cells were assayed using a Phospho Explorer antibody microarray (Full Moon, Sunnyvale, CA), which contains 1,318 antibodies. A single slide contains two replicates of each antibody and multiple positive and negative controls printed on a coated glass microscope slide. Slides were prepared according to the manufacturer's instructions, and Fullmoon scanned the slides. Fluorescence intensity for each antibody was measured using FIJI, and fold changes were calculated after background intensity was subtracted from each fluorescence measurement.

[0144] Inflammasome RT Profiler PCR Array Inflammasomes RT 2 Unstimulated purified human CD4+CD25+CD127 was isolated using a Profiler PCR Array (Qiagen, Valencia, CA). dim Treg cells and CD4+CD25-CD127 hi Gene expression of 84 inflammasome components and signaling pathways in Tconv cells was analyzed according to the manufacturer's instructions. Experiments were performed a minimum of three times for Treg and Tconv cells. PCR array data were analyzed using the GeneGlobe Data Analysis Center on the QIAGEN website at http: / / www.qiagen.com / kr / shop / genes-and-pathways / data-analysis-center-overview-page / .

[0145] Ingenuity® Pathway Analysis (IPA®) Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) to determine the association of Hsp90 with NLR family members and inflammasome-related molecules. http: / / www.ingenuity.com / . IPA® is available in Table 3.

[0146] Jurkat E6 transduction Jurkat cells were transduced with lentivirus prepared by transfecting 293T cells transfected with either the empty vector or the pLenti-IL37-C-Myc-DDK-P2A-Puro vector (IL37 OE) (Origene) together with the Lenti-V-pak packaging kit (Origene). A schematic diagram of the pLenti-IL37-C-Myc-DDK-P2A-Puro vector is shown in Figure 2. Supernatants containing lentiviral particles were collected 48 hours after transfection. The presence of lentivirus in the supernatant was confirmed using Lenti-X GoStix Plus (Takara). 1 × 10 6 Jurkat cells were transduced with 1 ml of lentivirus-containing supernatant and 8 μg / ml polybrene and incubated at 37°C for 24 hours. The medium was then replaced with RPMI 1640 supplemented with pen-strep and 10% FCS. Drug selection began 48 hours after transduction with 0.5 μg / ml puromycin. Jurkat cells were bulk-selected, and overexpression was confirmed by Western blot.

[0147] Flow cytometry: surface and intracellular antigen staining, activation, and proliferation Intracellular FOXP3 and cytokines were assayed by flow cytometry. Prior to fixation / permeabilization, cells were surface stained for 30 minutes at 4°C. Purified lymphocytes were washed with FACS buffer (5% BSA and 1x PBS) and then prepared for surface or intracellular staining. For surface staining, 1 µg or the manufacturer's recommended amount of fluorescently conjugated antibody specific for the surface marker to be visualized was added to 1x10 lymphocytes resuspended in 100 µl of FACS buffer. 6Cells were added to the medium for 30 minutes at 4°C and then washed twice with FACS buffer before analysis or further processing. For the latter, after washing twice with FACS buffer, cells were immediately fixed in 4% paraformaldehyde (PFA), permeabilized with 90% methanol, and then stained. For IL-10 intracellular cytokine staining, cells were stimulated with 1 μg / ml soluble anti-CD28 antibody or anti-CD3 / CD28-conjugated Dynabeads (Gibco, Thermo Fisher Scientific) on 1 μg / ml anti-CD3-coated plates for 24 hours, followed by treatment with BD GolgiStop™ (Franklin Lakes, NJ) in culture for 6 hours using a FOXP3 Cytoperm / Cytofix staining kit (BD Pharmingen, San Diego, CA). T cell proliferation was measured by staining cells with 5 μM CFSE (Molecular Probes, Eugene, OR) before cell culture and analyzing them using flow cytometry.

[0148] RNA extraction and quantitative RT-PCR analysis Cells were stimulated for 24 hours on anti-CD3 / CD28-coated plates as described above. Total RNA was extracted from Jurkat cells using the RNeasy Plus Mini Kit (Qiagen) and subsequently reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative RT-PCR (qRT-PCR) was performed on an AriaMx real-time PCR system (Agilent Technologies, Santa Clara, CA) using Power Up SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The primer sets used for human cells were as follows: GAPDH forward - 5'-TGC ACC ACC AAC TGC TTA GC-3' (SEQ ID NO: 178), reverse - 5'-GGC ATG GAC TGT GGT CAT GAG-3' (SEQ ID NO: 179), IL-37 forward - 5'-GCA TTC ATG ACC AGG ATC AC-3' (SEQ ID NO: 180), reverse - 5'-CAA AGA AGA TCT CTG GGC GTA-3' (SEQ ID NO: 181), FOXP3 forward - 5'-CAG AGC TCC TAC CCA CTG CT-3' (SEQ ID NO: 182), reverse - 5'-CTT CTC CTT CTC CAG CAC CA-3' (SEQ ID NO: 183), GITR forward - 5'-CAT GTG TGT CCA GCC TGA AT-3' (SEQ ID NO: 184), reverse - 5'-GGC ACA GTC GAT ACA CTG GA-3' (SEQ ID NO: 185), CTLA-4 forward - 5'-CTC TAC ATC TGC AAG GTG GAG C-3' (SEQ ID NO: 186), reverse - 5'-AGA GGA GGA AGT CAG AAT CTG GG-3' (SEQ ID NO: 187), TGF-β forward - 5'-CAC CTG GAG CTG TAC CAG AA-3' (SEQ ID NO: 188), reverse - 5'-TGC AGT GTG TTA TCC CTG CT-3' (SEQ ID NO: 189), IL17 forward - 5'-ATA TTG GGG CTT GCC TTT CT-3' (SEQ ID NO: 190), reverse - 5'-GTG TAA TTC CAG GGG GAG GT-3' (SEQ ID NO: 191), STAT3 forward - 5'-AGA GAA ATG AGT GAG TGT GGG-3' (SEQ ID NO: 192), reverse - 5'-ACA GGA GGT GTT CCC CTT TGC-3' (SEQ ID NO: 193), and GATA3 forward - 5'-AGG CAG GGA GTG TGT GAA CT-3' (SEQ ID NO: 194), reverse - 5'-GTG GTT GTG GTG GTC TGA CA-3' (SEQ ID NO: 195).

[0149] The primer sets used for mouse cells were as follows: GAPDH forward - 5'C ACC ACC AAC TGC TTA GC-3' (SEQ ID NO: 196), reverse - 5'-GGC ATG GAC TGT GGT CAT GAG-3' (SEQ ID NO: 197), IL37 forward - 5'-GCA TTC ATG ACC AGG ATC AC-3' (SEQ ID NO: 198), reverse - 5'-CAA AGA AGA TCT CTG GGC GTA-3' (SEQ ID NO: 199), FOXP3 forward - 5'-CCA GGA CAG ACC ACA TTC A-3' (SEQ ID NO: 200), reverse - 5'-CTG GAC ACC CAT TCC AGA CT-3' (SEQ ID NO: 201).

[0150] Further primer sets are listed below in Tables 2.1 and 2.2. Quantification was performed using cycling change threshold (ΔCt) and is shown relative to GAPDH mRNA expression.

[0151] [Table 2-1]

[0152] [Table 2-2]

[0153] [Table 3-1]

[0154] [Table 3-2]

[0155] Surface and intracellular flow cytometry and FAM-FLICA® caspase-1 staining Purified lymphocytes from either humans or mice were washed with FACS buffer (5% BSA (Sigma-Aldrich) and 1x PBS) and then prepared for surface or intracellular staining as described (Osborne and Wetzel, 2012). For surface staining, 1 µg or the manufacturer's recommended amount of fluorescently conjugated antibody specific for the surface marker to be visualized was added to 1 x 10 lymphocytes resuspended in 100 µl of FACS buffer. 6Cells were added to the cells for 30 minutes at 4°C and then washed twice with FACS buffer before analysis or further processing. For intracellular cytokine staining (ICS), cells were stimulated with 1 μg / ml soluble anti-CD28 antibody or anti-CD3 / CD28-conjugated Dynabeads™ (Gibco, Thermo Fisher Scientific) on 1 μg / ml anti-CD3-coated plates for 24 hours using a FOXP3 Cytoperm / Cytofix staining kit (BD Pharmingen, San Diego, CA). To measure proliferation, cells were stained with 5 μM CFSE (Molecular Probes, Eugene, OR) before cell culture and then analyzed using flow cytometry. Flow-based measurements of caspase-1 activation were performed using the FAM-FLICA® Caspase-1 Assay Kit #98 according to the manufacturer's instructions (ImmunoChemistry Tech, Bloomington, MN). For flow cytometry experiments using the caspase-1 inhibitor Ac-YVAD-CMK, cells were cultured with 50 μM Ac-YVAD-CMK for 2 hours, 24 hours (mouse), and 48 hours (human) before stimulation with anti-CD3 / CD28-conjugated Dynabeads™ and then prepared for flow cytometry analysis. For experiments using the PAK1 / 2 / 3 inhibitor FRAX597, cells were cultured with 1 μM FRAX597 for 24 hours (flow and qRT-PCR) or 48 hours (immunoblotting) and then prepared for analysis. For flow cytometry or qRT-PCR experiments using IL-6 treatment, human and mouse cells were cultured with 20 ng / ml IL-6 (Peprotech) and anti-CD3 / CD28-conjugated Dynabeads™ and then prepared for analysis. For flow cytometry and qRT-PCR experiments using flagellin, cells were cultured with 100 ng / ml flagellin for 24 hours and then prepared for analysis. During and after preparation of cells for flow cytometry analysis, cells were placed in FACS buffer.All acquisitions were performed on a Galios® or Galios® 561 cytometer with Kaluza software (Beckman-Coulter, Indianapolis, IN), and all data were analyzed with FlowJo_V10 software (Tree Star, Ashland, OR).

[0156] Western blot, immunoprecipitation (IP) and immunoblot analysis For immunoblot analysis, cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 μg / ml aprotinin, 5 μg / ml leupeptin, 1% Triton x-100, 1% sodium deoxycholate, 0.1% SDS) (Sigma-Aldrich). For Smad3 IP, cells were lysed in Pierce® IP Lysis Buffer (Thermo) and prepared using Thermo Immunoprecipitation with Dynabeads™ Protein G kit according to the manufacturer's instructions. Smad3 IP was performed using a rabbit monoclonal antibody against Smad3 (C67H9) (Thermo). Hsp90 IP was performed using a mouse monoclonal antibody against Hsp90 (610418) (BD Biosciences). 20 μg of sample was run on a 4-15% Mini-Protean TGX precast gel (Bio-Rad) and then transferred to a PVDF membrane overnight at 4°C and 30 V using a Bio-Rad Trans-Blot® transfer cell. After blocking with blocking buffer (5% milk / Tween 20 in TBS) for 1 hour, the membrane was incubated with primary antibodies overnight at 4°C. For siRNA knockdown blots, a mouse β-actin monoclonal antibody (Sigma-Aldrich) at a dilution of 1:10,000 was used as a loading control. For Smad3 IP, a rabbit anti-human IL-37 (IL-1F7) polyclonal antibody (Invitrogen, Thermo) at a dilution of 1:1000 was used to detect IL-37. HRP-conjugated anti-rabbit (1:5000) and anti-mouse IgG (1:5000) were purchased from Sigma-Aldrich. The SuperSignal West Pico Kit (Thermo Fisher) was used according to the manufacturer's instructions for chemiluminescent detection of proteins.Protein expression was further analyzed by measuring relative density using FIJI (National Institutes of Health: http: / / rsb.info.nih.gov / ij / ) or LI-COR Image Studio™ Software.

[0157] In vitro T cell suppression assay Human CD4+CD127loCD25+ T cell isolation kit (Miltenyi) was used to isolate human CD4+CD25 lo T cell responder (Tresp) and human CD4+CD25 hi Human primary Treg cells were isolated for suppression assays (Osborne et al., 2022). For mouse cells, mouse CD4+CD25+ T cells were isolated from WT or IL37 Tg mouse splenocytes using a mouse CD4+CD25+ T cell isolation kit (Miltenyi). lo T cell responder (Tresp) and mouse CD4+CD25 hi Treg cells were isolated using 100,000 purified CFSE-labeled WT CD4+CD25 in 50 μL of complete RPMI 1640. lo T cell responders (Tresp) were added to round-bottom 96-well plates (CellStar, Dallas, TX) containing heat-killed splenocytes (APCs) (as a source of antigen-presenting cells), 5 μg / ml soluble anti-CD3 antibody, and the indicated dilutions of Treg cells in 150 μl of complete RPMI 1640. Five days later, proliferation / suppression of Tresp cells was measured using CFSE and analyzed by flow cytometry. Percent suppression was calculated using the formula: % suppression = 100 - (x / y) 100, where X represents the mitotic index (DI) of Tresp cells containing Treg cells, and Y is the average DI of Tresp cells alone, as in (McMurchy and Levings, 2012).

[0158] Treg cell expansion For mouse Treg cell expansion, spleens were collected from either 6-8 week-old age- and sex-matched C57BL / 6 WT or IL37 Tg mice. Spleens in 10 ml of 1x PBS were macerated using a 10 ml plastic syringe (Fisher Scientific) on a 40 μm filter in a 6-well plate. Splenocytes were then passed through the same 40 μm filter into a 50 ml conical tube. The 6-well plate was washed with 10 ml of 1x PBS and placed into the splenocyte solution in the 50 ml conical tube. The tube was spun at 1500 rpm for 10 minutes. After the spin, the solution was aspirated, leaving only the splenocyte pellet. The splenocyte pellet was resuspended in 5 ml of RBC lysis buffer for 3 minutes, and the buffer was then neutralized by adding 15 ml of complete medium (1x PBS + 10% FBS). The tube was spun at 1500 rpm for 5 minutes. After the spin, the solution was aspirated, leaving only the splenocyte pellet. Resuspend in 10 ml of 1x PBS + 10% FBS and count using a hemocytometer. After counting, splenocytes were prepared for Treg purification using the Miltenyi Mouse CD4+CD25+ T Cell Purification Kit. Purification followed the Miltenyi Mouse CD4+CD25+ T Cell Purification Kit protocol. After purification, 5x10 cells were then cultured in complete RPMI medium supplemented with 1000 U / ml of mouse IL-2. 5 Treg cells at 1 × 10 cells / ml were plated onto 24-well plates coated with 1 μg / ml anti-CD3 (145-2C11) and 5 μg / ml anti-CD28 (37.51). Over a 10-day series of cultures, cell numbers increased to 1 × 10 cells / ml. 6 Once cells / ml was reached, each well was split and complete medium supplemented with 100 U / ml IL-2 was added. On day 10, cells were counted and Foxp3 and IL-37 expression was confirmed using qRT-PCR and flow cytometry.

[0159] For human Treg cell expansion, human peripheral blood from healthy donors was obtained from the blood cone provided by Children's Hospital Blood Donor Center (Aurora, CO), and peripheral blood mononuclear cells (PBMCs) were purified using Ficoll density gradient centrifugation. CD4+CD25+CD127 cells were isolated from PBMCs. dim Further purification of Treg cells was performed using human CD4+CD25+CD127 dim Regulatory T cell isolation kit (Miltenyi Biotech) was used according to the manufacturer's instructions. After purification, 5 × 10 cells were then cultured in complete RPMI medium supplemented with 1000 U / ml human IL-2. 6 Treg cells at 1 x 10 cells / ml were plated onto 24-well plates coated with 1 μg / ml anti-CD3 (OKT3) and 5 μg / ml anti-CD28 (CD28.6). Over a series of 10-day cultures, cell numbers increased to 1 x 10 6 Once cells / ml were reached, each well was split and complete medium supplemented with 50 U / ml IL-2 was added. On day 10, cells were counted and FOXP3 and IL37 expression was confirmed using qRT-PCR and flow cytometry.

[0160] Transduction of expanded human or NOD mouse Treg cells with IL-37 overexpression vectors After expanding either primary human or NOD mouse Treg cells for ...

Claims

1. 1. A method of generating a population of modified CD4+ T cells, the method comprising: introducing into a plurality of human T cells a composition comprising interleukin-37 (IL-37) or a nucleic acid sequence encoding IL-37 under conditions suitable for expressing IL-37 in the nuclei of said human T cells, thereby generating a plurality of modified CD4+ T cells.

2. 10. The method of claim 1, wherein the modified population of CD4+ T cells are regulatory T cells.

3. 3. The method of claim 2, wherein the modified population of CD4+ T cells are non-regulatory T cells.

4. 4. The method of any one of claims 1-3, wherein nuclear expression of IL-37 in the plurality of modified CD4+ T cells is at least 5-fold greater than nuclear expression of IL-37 in a population of wild-type human T cells.

5. 4. The method of any one of claims 1-3, wherein nuclear expression of IL-37 in the plurality of engineered CD4+ T cells is about 5-fold to about 10-fold greater than nuclear expression of IL-37 in a population of wild-type human T cells.

6. 6. The method of any one of claims 1-5, wherein at least about 75% of the plurality of modified CD4+ T cells express at least one marker of regulatory T cells.

7. 7. The method of claim 6, wherein at least about 95% of the plurality of modified CD4+ T cells express at least one marker of regulatory T cells.

8. 8. The method of claim 6 or 7, wherein the at least one marker of regulatory T cells is selected from the group consisting of FOXP3, CD25, CD4, CTLA4, IL-10, GITR, TGF-beta and CD127.

9. 9. The method of claim 8, wherein at least one marker is FOXP3.

10. 9. The method of claim 8, wherein the at least one marker is FOXP3 and CD25.

11. A composition comprising a population of modified CD4+ T cells produced by the method of any one of claims 1 to 10.

12. 12. The composition of claim 11 for use in the treatment of an immune disease or disorder selected from the group consisting of allergic contact hypersensitivity, graft versus host disease, transplant rejection, type 1 diabetes, systemic lupus erythematosus, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and multiple sclerosis.

13. 13. The composition of claim 12, wherein the immune disease or disorder is allergic contact hypersensitivity.

14. 13. The composition of claim 12, wherein the immune disease or disorder is graft-versus-host disease.

15. 13. The composition of claim 12, wherein the immune disease or disorder is type 1 diabetes.

16. 12. The composition of claim 11 for use in the treatment of an inflammatory disease or disorder selected from the group consisting of psoriasis, traumatic brain injury, bronchitis and pneumonitis.

17. 1. A method of treating an immune disease or disorder or an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising a population of engineered CD4+ T cells that express nuclear IL-37.

18. 18. The method of claim 17, wherein the modified population of CD4+ T cells are regulatory T cells.

19. 19. The method of claim 17 or 18, wherein the expression of nuclear IL-37 in the modified population of CD4+ T cells is at least 50% greater than the expression of nuclear IL-37 in a population of wild-type CD4+ T cells.

20. 20. The method of claim 19, wherein the expression of nuclear IL-37 in the modified population of CD4+ T cells is about 50% to about 80% greater than the expression of nuclear IL-37 in a population of wild-type CD4+ T cells.

21. 21. The method of any one of claims 17 to 20, wherein the modified population of CD4+ T cells is allogeneic CD4+ T cells.

22. 21. The method of any one of claims 17 to 20, wherein the modified population of CD4+ T cells is autologous CD4+ T cells.

23. 23. The method of any one of claims 17 to 22, wherein the immune disease or disorder is selected from the group consisting of allergic contact hypersensitivity, graft versus host disease, transplant rejection, type 1 diabetes, systemic lupus erythematosus, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and multiple sclerosis.

24. 24. The method of claim 23, wherein the immune disease or disorder is allergic contact hypersensitivity.

25. 24. The method of claim 23, wherein the immune disease or disorder is graft-versus-host disease.

26. 24. The method of claim 23, wherein the immune disease or disorder is type 1 diabetes.

27. 23. The method of any one of claims 17 to 22, wherein the inflammatory disease or disorder is selected from the group consisting of psoriasis, traumatic brain injury, bronchitis, and pneumonitis.

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