Compositions and methods for expanding tregs and methods of use thereof

Abstract

This invention relates to methods of autologously expanding regulatory T-cells from biological samples with culture medium, T cell growth factors, and / or regulatory cytokines and without the addition of exogenous mitogens and / or antigens. In addition, this invention relates to autologously expanded regulatory T-cells and their use in methods of treating immune disorders, inflammatory diseases, pathogen infections, allergic diseases, transplantation rejection diseases, neurodegenerative diseases, metabolic diseases, aging-related diseases, and / or cardiovascular diseases.

Description

COMPOSITIONS AND METHODS FOR EXPANDING TREGS AND METHODS OFUSE THEREOFRelated Application Information

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 626,557, filed January 30, 2024, the disclosure of which is incorporated herein by reference in its entirety.Government Support Clause

[0002] This invention was made with government support under R01AI126398 awarded by the National Institutes of Health. The government has certain rights in the invention.Field of the Invention

[0003] The present invention relates to compositions and methods for expanding Tregs and methods of use thereof.Background of the Invention

[0004] CD25hlghFOXP3+regulatory T cells (Tregs) play a central role in suppressing pathogenic inflammation and represent the only regulatory T cell subset indispensable for self- tolerance. Deficiency of Foxp3 abrogates Treg development and causes the fatal systemic autoimmune disease IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked). The indispensable tolerogenic functions of FOXP3 are intrinsic to Tregs rather than other cell types because Treg-specific deletion of Foxp3 in adult mice leads to systemic lethal autoimmune disease. Circulating FOXP3+Tregs are depleted during autoimmune attacks, which supports the concept that Treg deficiencies may unleash autoimmune disease. Given that Tregs are necessary for self-tolerance, adoptive Treg-based immunotherapies may alleviate chronic inflammatory disease, including autoimmune disease, allergic disease, transplant rejection responses, graft versus host disease (GVHD), or any disease class where chronic inflammation drives pathogenesis. Treg-based immunotherapy is based on the concept that Tregs from a patient could be expanded in culture and infused at high cell doses into the same patient to resolve the inflammatory affliction. Due to the clinical potential, over 50 clinical trials have focused on Treg adoptive immunotherapy for broad categories of chronic inflammatory disease. However, due to problems in ‘Treg manufacture’, contemporary Treg expansion protocols produce Treg preparations that lack clinical efficacy. Technical problemsthat hamper Treg manufacture include phenotypic and functional instability, Treg exhaustion, Treg cell death, and effector T cell contamination. Thus, improved methods of Treg manufacture are desirable.Summary

[0005] The present invention is based on the finding that certain growing conditions can lead to autologous T cell expansion. Thus, a first aspect of the invention is related to a method of expanding regulatory T-cells (Tregs), the method comprising: a) providing a biological sample comprising Tregs; b) culturing the biological sample in vitro with a culture medium comprising a basal cell culture medium, a T cell growth factor, and a regulatory cytokine, to provide cultured Tregs; and d) passaging the cultured Treg population one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50 or more times) with a xeno-free culture medium comprising a basal cell culture medium, a T cell growth factor, and a regulatory cytokine to provide expanded Tregs.

[0006] Another aspect of the invention is related to a population of Tregs produced by the methods described herein.

[0007] Another aspect of the invention is related to methods of treating a disease, disorder, and / or pathogen infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs produced by the methods described herein to the subject, thereby treating the disease, disorder, and / or pathogen infection. In some embodiments, the disease, disorder, and / or pathogen infection is an immune disorder, an inflammatory disease, a viral infection, an allergic disease, a transplantation rejection disease, a neurodegenerative disease, a metabolic disease, an aging-related disease, and / or a cardiovascular disease.Brief Description of the Drawings

[0008] Fig. 1, panel A is a series of histograms showing the flow cytometric analyses of CD4+T cells, activated for 3 days with phytohemagglutinin (PHA), interleukin 2 (IL-2), and with or without transforming growth factor-P (TGF-P); and then cultured for 38 days with or without TGF-P according to some embodiments of the invention (n = 2 technical replicates) after staining with anti-CD3 PerCP-Cy5.5 (HIT3a), anti-CD4 BV421 (OKT4), anti-CD8 AF700 (SKI), anti-CD25 PE (M-A251), and anti-HLA-DR BUV395 (G46-6) antibodies to measure expression of the indicated gene. Forward scatter area (FSC-A) was used to measure cell size. Fig. 1, panel B is a series of graphs showing the flow cytometric analyses of CD4+T cells,activated for 3 days with PHA, IL-2, and with or without TGF-P; and then cultured for 38 days with or without TGF-P according to some embodiments of the invention (n = 2 technical replicates). Staining for CD3, CD4, CD8, CD25, and HLA-DR was used as described previously to measure expression of the indicated gene. FSC-A was used to measure cell size. Differences in mean values were analyzed using two-tailed unpaired T-tests (*p < 0.05, and ** p < 0.01). Fig. 1, panel C is a series of graphs showing uptake and incorporation of [3H]thymidine into the DNA of proliferating cells in counts per million (CPM) of purified CD4+PBMC T cells (right) and nonfractionated PBMCs (left), activated for 3 days with PHA, IL-2, and with or without TGF-P; and then cultured for 20 days with IL-2 and with or without TGF- P according to some embodiments of the invention (n = 3 technical replicates).

[0009] Fig. 2, panel A is a series of histograms showing the flow cytometric analyses of CD4+T cells, activated for 3 days with PHA, IL-2, and with or without TGF-P; and then cultured for 7 days with or without TGF-P according to some embodiments of the invention (n = 2). Staining for CD3, CD4, CD8, CD25, and HLA-DR was used as described previously to measure expression of the indicated gene. FSC-A was used to measure cell size. Unstained cell cultures were used as a control. Fig. 2, panel B is a series of histograms showing the flow cytometric analyses of purified CD4+PBMC T cells (right) and nonfractionated PBMCs (left) activated for 3 days with PHA, IL-2, and with or without TGF-P; and then cultured for 11 days with or without TGF-P according to some embodiments of the invention. Staining for CD3, CD4, CD8, CD25, and HLA-DR was used as described previously to measure expression of the indicated gene. FSC-A was used to measure cell size and side scatter area (SSC-A) was used to measure cellular granularity. Fig. 2, panel C is a series of histograms showing the flow cytometric analyses of nonfractionated PBMCs cultured for 63 days with or without TGF-P according to some embodiments of the invention. Staining for CD3, CD4, CD8, CD25, and HLA-DR was used as described previously to measure expression of the indicated gene. Fig. 2, panel D is a series of graphs showing the pairwise comparisons of mean values shown in Fig. 2, panel B as analyzed by use of two-tailed unpaired T-tests (**p < 0.01, ***p < 0.001, and **** p < 0.0001).

[0010] Fig. 3, panel A is a series of graphs showing the gating strategy for the flow cytometry analyses of CD4+and CD8+cell subsets after staining for CD4 and CD8. FSC-A was used to measure cell size, forward scatter height (FSC-H) compared to side scattering height (SSC-H) was used to measure cell complexity (i.e., cell size vs. cell granularity). Fig. 3, panel B is a series of histograms showing the flow cytometric analyses of CD4-purified T cells or nonfractionated PBMCs, activated for 3 days with PHA (top three rows of graphs) or withoutmitogenic activation (row four of graphs) and then cultured for the indicated number of days with or without TGF-P according to some embodiments of the invention. Side-by-side staining on the same day with anti-CD4 BV421 (0KT4), anti-CD8 AF700 (SKI), and anti-FOXP3 AF488 (PCH101) FOXP3 was used to measure expression of the indicated gene. A fluorescence-minus-one staining cocktail (FMO), and unstained cells were used as controls. The labels on the right of the graph indicate the selected cell type as well as the culture conditions. Fig. 3, panel C is a histogram showing the flow cytometric analyses of nonfractionated PBMCs, activated for 3 days with PHA and then cultured for 19 days with or without TGF-P according to some embodiments of the invention. Staining for FOXP3 was used as described previously. FMO staining and unstained cells were used as controls. Fig. 3, panel D is a series of histograms showing the flow cytometric analyses of murine SJL-P lymphoblasts (left) and human primary CD8+T cells (right) after 45 days of culture with or without TGF-P according to some embodiments of the invention. The cells were stained with varying concentrations of FOXP3 PCH101. Unstained cells were used as a control.

[0011] Fig. 4, panel A is a graph showing the expansion of PBMCs from three donors activated for 3 days with PHA, IL-2, and TGF-P; and then cultured with or without TGF-P according to some embodiments of the invention. The expansion curves are calculated from a theoretical single cell starting point. Fig. 4, panel B is a series of histograms showing the flow cytometric analyses of PBMCs from three donors, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 11 or 18 days as indicated with or without TGF-P according to some embodiments of the invention. FMO staining was used as controls. Fig. 4, panel C is a series of histograms showing the flow cytometric analyses of PBMCs from three donors, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 65 days with or without TGF-P according to some embodiments of the invention. Staining with anti-CD3 NovaFluor Blue 610- 70S, anti-CD4 BV421 (OKT4), anti-CD8 AF700 (SKI), anti-CD25 APC-Fire750, anti-CD69 PE (FN50), CD80 BV711, anti-CD86 BV650, anti-CTLA4 AF647, anti-PDL2 PE-Cy7, anti- PD1 PE-Cy5 (EH12.2H7), anti-HLA-DR BUV395 (G46-6), and anti-FOXP3 (PCH101) AF488 was used to measure expression of the indicated genes. FMO staining and unstained cells were used as controls.

[0012] Fig. 5, panel A is a graph showing the expansion of nonfractionated PBMCs from a healthy volunteer cultured with or without TGF-P according to some embodiments of the invention (n = 4). Fig. 5, panel B is a graph showing the expansion of the one surviving cell line from Fig. 5, panel A kept in culture for up to 120 days. Fig. 5, panel C is a series of graphs showing the phenotypes of a control CD4-purified Treg line cultured for 33 days and thenstained with anti-CD3-NovaFluor Blue 610-70S, anti-CD4-BV421 (0KT4), anti-CD8-AF700, anti-CD25-APC-Fire750, anti-HLA-A2-NovaFluor Yellow 730, anti-FOXP3(150D)-AF488, anti-BCL6-AF647, anti-FASL-BUV615, anti-FAS-BUV737, anti-CD86-BV650, anti- CXCR5-BV605, anti-CD69-PE, and anti-PDl-PE-Cy5 to measure expression of the indicated genes. Fig. 5, panel D is a graph showing the phenotypes of the CD8+Treg line shown in Fig. 5, panel B cultured for 83 days and then stained for expression of CD3, CD4, CD8, CD25, HLA, FOXP3, BCL6, FASL, FAS, CD86, CXCR5, CD69, and PD1 as described previously.

[0013] Fig. 6, panel A is a graph showing uptake and incorporation of [3H]thymidine into the DNA of proliferating cells in CPM of purified CD4+PBMC T cells, activated for 3 days with PHA, IL-2, and / or TGF-P; and then cultured for 28 days with IL-2 and with or without TGF-P according to some embodiments of the invention (n = 3 technical replicates). Two-way ANOVA with a Tukey multiple comparisons test was used to assess significance (* p < 0 05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001). Fig. 6, panel B is a graph showing purified CD4+PBMC T cells and IL-2 indicator T cells (e.g., SJL-P T cells), activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 28 days with or without TGF-P according to some embodiments of the invention. Cell cultures initiated without IL-2 (1stand 3rdbars of each group) were used as a means to measure IL-2 production by Tregs during the initial 24-hour culture. Cultures initiated with IL-2 (2ndand 4thbars of each group) were used a means to measure IL-2 consumption by Tregs during the initial 24-hour culture. Two-way ANOVA with a Tukey multiple comparisons test was used to assess significance (* p < 0.05, p < 0.01, *** p < 0.001, and **** p < 0.0001). Fig. 6, panel C is graph showing uptake and incorporation of [3H]thymidine into the DNA of proliferating cells in CPM of responder nonfractionated PBMCs cultured stimulated with PHA with or without FOXP3+Tregs, and then cultured with or without IL-2 according to some embodiments of the invention. ACPM was defined as mean CPM from combined Tregs / responder cultures minus mean CPM from Treg cultures alone. Two-tailed unpaired T-tests were used to compare mean values (* p < 0.05, p < 0.01, *** p < 0.001, and **** p < 0.0001).

[0014] Fig. 7, panels A-C are a series of graphs showing uptake and incorporation of [3H]thymidine into the DNA of proliferating cells in CPM of CD4+purified Treg cells, CD8+purified Treg cells, or SJL mouse T cells cultured at varying concentrations of IL-2 stimulation and with or without TGF-P according to some embodiments of the invention. The CD4+and CD8+cells were activated for 3 days with PHA, IL-2, and TGF-P; the cells were then further cultured for 22 days with IL-2 and TGF-P according to some embodiments of the invention. The cells were further treated with anti-human CD4 mAb RPA-T4 (mouse IgGl -kappa,BioXCell BE0288) or OKT4 anti-CD4 mAb (mouse IgG2b-kappa). Fig. 7, panel D is a graph showing uptake and incorporation of [3H]thymidine into the DNA of proliferating cells in CPM of CD4+purified Treg cells, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 15 days at varying concentrations of IL-2 stimulation and with TGF-P according to some embodiments of the invention (n = 3 technical replicates). The cells were further treated with varying concentrations of anti-human CD4 mAb RPA-T4 (mouse IgGl -kappa, BioXCell BE0288), 0KT4 anti-CD4 mAb (mouse IgG2b-kappa), or combinations thereof. Fig. 7, panel E is a graph showing uptake and incorporation of [3H]thymidine into the DNA of proliferating cells in CPM of CD4+purified Treg cells, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 15 days at varying concentrations of IL-2 stimulation and with TGF-P according to some embodiments of the invention. The cells were further treated with antihuman CD4 mAb RPA-T4 (mouse IgGl -kappa, BioXCell BE0288). Fig. 7, panel F is a graph showing uptake and incorporation of [3H]thymidine into the DNA of proliferating cells in CPM of CD4+purified Treg cells, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 78 days at varying seeding densities according to some embodiments of the invention. The cells were further treated with anti-human CD4 mAb RPA-T4 (mouse IgGl -kappa, BioXCell BE0288. Fig. 7, panel G is a series of graphs showing uptake and incorporation of [3H]thymidine into the DNA of proliferating cells in CPM of CD4+purified Treg cells, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 23 days (left graphs) or 36 days (right graphs) at varying concentrations of IL-2 stimulation and with or without TGF- P according to some embodiments of the invention. The cells were further treated with antihuman CD4 mAb RPA-T4 (mouse IgGl -kappa, BioXCell BE0288). For all panels, a two-way ANOVA with a Tukey multiple comparisons test was used to assess significance (*** p < 0.001, and **** p < 0.0001 for comparisons of the designated mean value(s) at the same IL-2 concentration).

[0015] Fig. 8, panels A-F show the flow cytometric analyses CD4+purified T cells, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 53 days with IL-2 and TGF-P according to some embodiments of the invention; these cells were then cultured with RPA-T4 mAb or with MOPC-21 mouse IgGl-k isotype as a control for the terminal two weeks. Fig. 8, panel A is a series of graphs showing RPA-T4 mAb (right) or MOPC-21 mouse IgGl-k isotype (left) treated CD4+purified T cells stained with CD25 PE (M-A251) and FOXP3 AF488 (150D) used to measure expression of the indicated genes. Fig. 8, panel B is a histogram showing the CD4+purified T cells stained with CD25 PE (M-A251) used to measure gene expression. Fig. 8, panel C is a series of graphs showing the FSC mean florescence intensity(MFI) values (left) and CD25+percentages (right) in the CD4+purified T cells. Two-tailed unpaired T-tests were used to compare mean values. (* p < 0.05). Fig. 8, panel D is a histogram showing the CD4+purified T cells stained with FOXP3 AF488 (150D) used to measure gene expression. Unstained cells (grey) were used as a control. Fig. 8, panel E is a series of histograms showing the CD4+purified T cells stained with FOXP3 AF488 (150D) and CD25 PE (M-A251) used to measure expression of the indicated genes. Unstained cells (grey) were used as a control. Fig. 8, panel F is a graph showing the FOXP3 MFI values in the CD4+purified T cells stained with FOXP3 AF488 (150D) and CD25 PE (M-A251) used to measure expression of the indicated genes. Two-tailed unpaired T-tests were used to compare mean values. (** p < 0.01, *** p < 0.001, and p < 0.0001). Fig. 8, panel G is a series of graphs showing the flow cytometric analyses of MFI values in CD4+purified T cells cultured for 100 days with IL-2 and TGF-P, and with RPA-T4 mAh or with MOPC-21 mouse IgGl-k isotype as a control for the terminal two weeks, according to some embodiments of the invention (n = 3 technical replicates). Staining with CD3 PerCP-Cy5.5 (HIT3a); CD4 BV421 (OKT4); CD25 PE (M-A251); HLA-DR DP, DQ SparkBlue574 (Tu39); CD86 BV650 (FUN-1); FAS BUV737 (DX2); CTLA4 AF647 (BN13); GITR BV711 (108-17); CXCR5 BV605 (MU5UBEE); PD1 PE-Cy5 (EH12.2H7); CD69 PE (FN50); CD25 APC-Fire750 (BC96); and LAG3 BUV563 (3DS223H) was used to measure expression of the indicated genes. Two-tailed unpaired T-tests were used to compare mean values (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

[0016] Fig. 9, panels A-E show the flow cytometric analyses of CD4+T cells purified from PBMCs, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 73 days with IL-2 and TGF-P and then cultured for 7 days with 100 pM, 1 nM, 10 nM, or 100 nM of IL-2 with or without TGF-P according to some embodiments of the invention; these cells were then stained for specific mAbs with CD3 Novafluor Blue-610 (SK7), CD4 BV421 (OKT4), CD8 AF700 (SKI), CD25 APC-Fire750 (BC96), CD69-PE (FN50), PD1-PE-Cy5 (EH12.2H7), and FOXP3-AF488 (150D). Fig. 9, panel A is a series of graphs showing the expression of CD25 and CD69 in the CD4+purified T cells. Fig. 9, panel B is a graph showing the percent of CD25lowCD69hlghT cells in the CD4+purified T cells cultured at varying concentrations of IL- 2 with or without TGF-p. Fig. 9, panel C is a series of graphs showing the expression of PD1 and CD69 in the CD4+purified T cells. Fig. 9, panel D is a graph showing the percent of PDlhlghCD69hlghT cells in the CD4+purified T cells cultured at varying concentrations of IL- 2 with or without TGF-p. Fig. 9, panel E is a series of histograms showing the expression of CD25 (left) and PD1 (right) in the CD69low(bottom quadrants from Fig. 9, panels A and C)or CD69hlgh(top quadrants from Fig. 9, panels A and C) CD4+purified T cells at the varying concentration of IL-2 and with or without TGF-p. Fig. 9, panel F is a series of graphs showing the percentages of CD25lowCD69highversus CD25highCD69lowT cell subsets in the CD4+purified T cells after 7 days of including 100 nM of IL-2 in the culture (left) and after 7 days of culture at the varying concentrations of IL-2 (right). A two-way ANOVA with a Tukey multiple comparisons test was used to assess differences between groups as indicated (**** p < 0.0001). Fig. 9, panel G is a series of graphs showing the percentages of PDlhlghT cell subsets in the CD4+purified T cells after 7 days of including 100 nM of IL-2 in the culture (left) and at the varying concentrations of IL-2 (right) A two-way ANOVA with a Tukey multiple comparisons test was used to assess differences between groups as indicated (**** p < 0.0001).

[0017] Fig. 10, panels A-T are graphs showing the flow cytometric analyses of CD4+T cells purified from PBMCs, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 53 days with IL-2 and TGF-P and then cultured for 7 days with 10 pM, 100 pM, 1 nM, or 10 nM of IL-2 with or without TGF-P according to some embodiments of the invention; these cells were then stained for specific mAbs with CD3 Novafluor Blue-610 (SK7); CD4 BV421 (OKT4); CD8 AF700 (SKI); CD25 APC-Fire750 (BC96); CD28 BV510 (CD28.2); CD69-PE (FN50); CD80 BV711 (2D10); CD86 BV650 (FUN-1); CD127-PE-Cy5 (A019D5); PD1-PE- Cy5 (EH12.2H7); LAG3 BUV563 (3DS223H); HLA-DR BUV395 G46-6 mAb); PDL2-PE- Cy7 (MIH18); GARP-PE-Cy7 (7B11); FAS BUV737 (DX2); FASL BUV615 (NOK-1); GITR BV711 (108-17); CTLA4 AF647 (BN13); and FOXP3-AF488 (150D). Fig. 10, panel A is a graph showing the percent of CD25hlghand CD25lowsubsets of T cells in the CD4+purified T cells. Fig. 10, panel C is a graph showing the cell size (FSC-A) in the CD4+purified T cells. Fig. 10, panels B and D-T are graphs showing the expression of the indicated gene in MFI. Fig. 10, panel U is a series of graphs showing the flow cytometric analyses of CD3+CD4+gated T cells analyzed for CD25 expression and cell size (FSC-A). For all panels, two-way ANOVA with a Tukey multiple comparisons test was used to assess differences between groups as indicated (* p < 0.05, ** p < 0.01, *** p < 0.001 , and **** p < 0.0001).

[0018] Fig. 11, panels A-G show the flow cytometric analyses of CD4+T cells purified from PBMCs, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 53 days with IL-2 and TGF-P and then cultured for 7 days with 100 pM, 10 pM, 1 nM, or 10 nM of IL-2 with or without TGF-P according to some embodiments of the invention; these cells were then stained for specific mAbs with CD3 Novafluor Blue-610 (SK7), CD4 BV421 (OKT4), CD8 AF700 (SKI), CD25 APC-Fire750 (BC96), CD152 / CTLA4 AF647 (BN13), CD223 / LAG3BUV563 (3DS223H), and CD80 BV711 (2D 10). Fig. 11, panel A is a series of graphs showing the expression of CD25 vs. LAG3, CLTA4, and CD80 as well as cell size (FSC-H) in CD3+CD4+gated T cells. Fig. 11, panel B is a graph showing the CD25 MFI values in the CD25lowand CD25hlghT cell subsets of the CD4+purified T cells cultured at varying concentrations of IL-2 and with or without TGF-p. Fig. 11, panel C is a series of graphs showing the percent of CD25lowand CD25hlghT cell subsets in the CD4+purified T cells at varying concentrations of IL-2 and with or without TGF-p. Fig. 11, panel D is a graph showing the CLTA4 MFI values in the CD25lowand CD25hlghT cell subsets of the CD4+purified T cells cultured at varying concentrations of IL-2 and with or without TGF-p. Fig. 11, panel E is a graph showing the LAG3 MFI values in the CD25lowand CD25hlghT cell subsets of the CD4+purified T cells cultured at varying concentrations of IL-2 and with or without TGF-p. Fig. 11, panel F is a graph showing the CD80 MFI values in the CD25lowand CD25hlghT cell subsets of the CD4+purified T cells cultured at varying concentrations of IL-2 and with or without TGF-p. Fig. 11, panel G is a graph showing the cell size (FSC-H) in the CD25lowand CD25hlghT cell subsets of the CD4+purified T cells cultured at varying concentrations of IL-2 and with or without TGF-p. For all panels, two-way ANOVA with a Tukey multiple comparisons test was used to assess differences between groups as indicated (* p < 0.05, ** p < 0.01, *** p < 0 001 , and **»* p < 0.0001).

[0019] Fig. 12, panels A and B show the flow cytometric analyses of CD4+T cells purified from PBMCs, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 73 days with IL-2 and TGF-P and then cultured for 7 days with 10 pM, 1 nM, 10 nM, or 100 nM of IL- 2 with or without TGF-P according to some embodiments of the invention; these cells were then stained for specific mAbs with CD3 Novafluor Blue-610 (SK7), CD4 BV421 (OKT4), CD25 APC-Fire750 (BC96), FOXP3(150D)-AF488, GITR / CD357 (108-17)-BV711, and CD152 / CTLA4 AF647 (BN13). Fig. 12, panel A is a series of graphs showing the expression of CD25 vs. CD3, CD4, FOXP3, GITR, and CLTA4 in CD4+gated T cells. Fig. 12, panel B is a series of graphs showing the indicated MFI values in the CD25lowand CD25hlghT cell subsets of the CD4+purified T cells cultured at varying concentrations of IL-2 and with or without TGF-p.

[0020] Fig. 13, panels A and B show the flow cytometric analyses of CD4+T cells purified from PBMCs, activated for 3 days with PHA, IL-2, and TGF-P; and then cultured for 73 days with IL-2 and TGF-P and then cultured for 7 days with 10 pM, 100 pM, 1 nM, or 10 nM of IL- 2 with or without TGF-P according to some embodiments of the invention; these cells were then stained for specific mAbs with CD25 APC-Fire750 (BC96); CD86 BV650 (FUN-1);CD127-PE-Cy5 (A019D5); HLA-DR BUV395 G46-6 mAb; FAS / CD95 BUV737 (DX2); and FOXP3-AF488 (150D). Fig. 13, panel A is a series of graphs showing the expression of CD25 vs. FOXP3, HLA-DR, CD86, FAS / CD95, and CD127 as well as cell size (FSC-H) in CD4+gated T cells. Fig. 13, panel B is a series of graphs showing the indicated MFI values in the CD25lowand CD25hlghT cell subsets of the CD4+purified T cells cultured at varying concentrations of IL-2 and with or without TGF-p.

[0021] Fig. 14, panels A and B show the flow cytometric analyses of CD3+and CD4+gated T cells purified from PBMCs, activated for 3 days with PHA, IL-2, and with or without TGF- P; and then cultured for 86 days with IL-2 and TGF-P according to some embodiments of the invention; these cells were then selected for CD25hlghand CD69hlghCD4+Tregs using magnetic bead purification. The selected CD25hlghand CD69hlghCD4+Tregs were further cultured for 18 days with IL-2 and TGF-p. The control CD4+T cells were activated for 3 days with PHA and IL-2; and then cultured for 42 days with IL-2. All cells were stained for specific mAbs with CD3 Novafluor Blue-610 (SK7), CD25 APC-Fire750 (BC96), CD69 PE (FN50), inducible T cell costimulatory (ICOS) / CD278 PE-Cy7 (ISA-3), PD1 / CD279 PE-Cy5 (EH12.2H7), GITR / CD357 (108-17)-BV711, BCL6 AF647 (KI 12-91), FOXP3 AF488 (PCH101), and HLA-DR BUV395 G46-6 mAb. Fig. 14, panel A is a series of graphs showing the expression of PD1 vs. BCL6, FOXP3, CD69, CD25, ICOS, HLA-DR, GITR, and CD3 as well as cell size (FSC-H) in CD3+and CD4+gated T cell subsets selected for CD69 and CD25 expression. Fig. 14, panel B is a series of graphs showing the indicated MFI values in the PDllowand PDlhlghT cell subsets of the CD3+and CD4+purified T cell subsets selected for CD25+and CD69+expression.Detailed Description of the Invention

[0022] The present invention is described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art.

[0023] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an"and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0024] It will be understood that, although the terms "first," "second," etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a "first" element discussed below could also be termed a "second" element without departing from the teachings of the present invention. The sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

[0025] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0026] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0027] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0028] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.

[0029] As used herein, the transitional phrase "consisting essentially of' (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term "consisting essentially of' as used herein should not be interpreted as equivalent to "comprising."

[0030] It will also be understood that, as used herein, the terms "example," "exemplary," and grammatical variations thereof are intended to refer to non-limiting examples and / or variantembodiments discussed herein, and are not intended to indicate preference for one or more embodiments discussed herein compared to one or more other embodiments.

[0031] The term "about," as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value may include any other range and / or individual value therein.

[0032] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0033] The term “comprise,” “comprises” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0034] As used herein, the terms “increase,” “increased,” and “increasing” (and grammatical variations thereof) describe an elevation of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more such as compared to another measurable property or quantity (e.g., a control value).

[0035] As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “decrease” (and grammatical variations thereof), describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% such as compared to another measurable property or quantity (e.g., a control value). In some embodiments, the reduction can result in no or essentially no (z.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

[0036] As used herein, the term "polypeptide" encompasses both peptides and proteins (including fusion proteins), unless indicated otherwise.

[0037] An "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly foundassociated with the polypeptide. Optionally, but not necessarily, the "isolated" polypeptide is present at a greater concentration (i.e., is enriched) as compared with the starting material (e.g., at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred- fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration). In representative embodiments, the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.

[0038] The term “fragment,” as applied to a peptide, will be understood to mean an amino acid sequence of reduced length relative to a reference peptide or amino acid sequence and comprising, consisting essentially of, and / or consisting of an amino acid sequence of contiguous amino acids identical to the reference peptide or amino acid sequence. Such a peptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of peptides having a length of at least about 5, 10, 15, 20, 25, 30, 35, 46. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more consecutive amino acids of a peptide or amino acid sequence according to the invention. In some embodiments, a polypeptide fragment is one that is expected to have at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or more) of the biological activity of the full- length polypeptide.

[0039] As used herein, the term "modified" and "variant," as applied to a polynucleotide or polypeptide sequence, refers to a sequence that differs from a wild-type sequence due to one or more deletions, additions, substitutions, or any combination thereof. Modified sequences may also be referred to as "modified variant(s)." In some embodiments, a modified or variant polypeptide sequence is one that is expected to have at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, or more) of the biological activity of the wildtype sequence.

[0040] In some embodiments, the term "biological activity" when referred to a polypeptide is intended to comprise the enzymatic activity, ligand binding activity, receptor binding activity, and the like of said polypeptide.

[0041] Furthermore, an "isolated" cell is a cell that has been partially or completely separated from other components with which it is normally associated in nature. For example, an isolated cell can be a cell in culture medium and / or a cell in a pharmaceutically acceptable carrier.

[0042] As used herein, the term "antigen" refers to a molecule capable of inducing the production of immunoglobulins (e.g., antibodies). As used herein, the term "immunogen" refers to when a molecule is capable of inducing a multi-faceted humoral and / or cellular-mediated immune response. In some embodiments, an antigen may be referred to as an immunogen, e.g., under conditions when the antigen is capable of inducing a multi-faceted humoral and / or cellular-mediated immune response. A molecule and / or composition (e.g., including but not limited to a nucleic acid, protein, polysaccharide, ribonucleoprotein (RNP), whole bacterium, and / or composition comprising the same) that is capable of antibody may be referred to as "antigenic" and / or that is capable of immune response stimulation may be referred to as "immunogenic," and can be said to have the ability of antigenicity and / or immunogenicity, respectively. The binding site for an antibody within an antigen and / or immunogen may be referred to as an epitope (e.g., an antigenic epitope).

[0043] "Effective amount" as used herein refers to an amount of a vector, nucleic acid molecule, epitope, polypeptide, cell, composition or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and / or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an "effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and / or by using routine experimentation.

[0044] “Pharmaceutically acceptable,” as used herein, means a material that is not biologically or otherwise undesirable, z.e., the material can be administered to an individual along with the compositions of this invention, without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art (see, e.g., Remington's Pharmaceutical Science,' 21sted. 2005). In some embodiments, the pharmaceutically acceptable carrier is a physiological buffer such that it maintains the composition in a physiological pH range (e.g., a pH range between about 7.00 and about 7.50, (e.g., about 7.00, 7.05, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.40, 7.45, or 7.50)). Exemplary physiological buffers for pharmaceutically acceptable carriers for the compositions of this invention include, but are not limited to, phosphate buffered saline (PBS), HEPES, sterile pyrogen-free water, and other sterile pyrogen-free physiological saline solutions.

[0045] The term “administering” or “administration” of a composition of the present invention to a subject includes any route of introducing or delivering to a subject a compound to performits intended function. In some embodiments, the administration comprises intravenous administration, mucosal administration, intradermal administration, intramuscular administration, vaginal administration, rectal administration, subcutaneous administration, transdermal administration, oral administration, sublingual administration, intranasal administration, and / or intratumoral administration

[0046] The amount of the disclosed compositions administered to a subject will vary from subject to subject, depending on the nature of the disclosed compositions and / or formulations, the species, gender, age, weight and general condition of the subject, the mode of administration, and the like. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the disclosed compositions are those large enough to produce the desired effect. The dosage should not be so large as to outweigh benefits by causing extensive or severe adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like, although some adverse side effects may be expected. The dosage can be adjusted by the individual clinician in the event of any counterindications.

[0047] A “subject” of the invention may include any animal in need thereof. In some embodiments, a subject may be, for example, a livestock animal and / or a domesticated pet. In some embodiments, a subject may be, for example, a mammal, a reptile (e.g., a snake, iguana, chameleon, gecko, bearded dragon, monitor lizard, skink, etc.), a bird (e.g., a chicken, turkey, goose, duck, parakeet, parrot, canary, cockatiel, budgerigar etc.), an amphibian (e.g., a frog, toad, salamander, etc.), or a fish (e.g., a koi fish, goldfish, betta fish, etc.). A mammalian subject may include, but is not limited to, a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, primate, etc.), a farm or commercial animal (e.g., cattle, pig, horse, goat, donkey, sheep, llama, etc.), or a domestic animal (e.g., cat, dog, ferret, gerbil, hamster, etc.). In some embodiments, a mammalian subject may be a primate, or a non-human primate (e.g., a chimpanzee, baboon, macaque (e.g., rhesus macaque, crab-eating macaque, stump-tailed macaque, pig-tailed macaque), monkey (e.g., squirrel monkey, owl monkey, etc.), marmoset, gorilla, etc.). In some embodiments, a mammalian subject may be a human.

[0048] A “subject in need” of the methods of the invention can be any subject known or suspected of having increased risk of developing a disease and / or disorder (e.g., an immune disease or an inflammatory disease), a cancer, and / or a pathogen infection (e.g., a viral, a bacterial, a prion, a parasitic, and / or a fungal infection) as described herein to which administering Tregs as described herein may provide beneficial health effects.

[0049] A “sample”, “biological sample”, and / or “ex vivo sample” of this invention can be any biological material, such as a biological fluid, an extract from a cell or tissue, an extracellular matrix isolated from a tissue, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art.

[0050] By the terms "treat," "treating" or "treatment of (and grammatical variations thereof) it is meant that the severity of the subject’s condition is reduced, at least partially improved or ameliorated and / or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and / or there is a delay in the progression of the disease or disorder. In representative embodiments, the terms "treat," "treating" or "treatment of' (and grammatical variations thereof) refer to a reduction in the severity of a disease and / or disorder; and / or a delay in the progression of a disease and / or disorder.

[0051] A "treatment effective" or “therapeutically effective” amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0052] The terms "prevent," "preventing" or "prevention of (and grammatical variations thereof) refer to prevention and / or delay of the onset and / or progression of a disease, disorder and / or a clinical symptom(s) in a subject and / or a reduction in the severity of the onset and / or progression of the disease, disorder and / or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. In representative embodiments, the terms "prevent," "preventing" or "prevention of (and grammatical variations thereof) refer to prevention and / or delay of the onset and / or progression of a disease and / or disorder in the subject. The prevention can be complete, e.g., the total absence of the disease, disorder and / or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and / or clinical symptom(s) in the subject and / or the severity of onset and / or the progression is less than what would occur in the absence of the present invention.

[0053] A "prevention effective" amount as used herein is an amount that is sufficient to prevent (as defined herein) the disease, disorder and / or clinical symptom in the subject. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

[0054] The efficacy of treating and / or preventing a disorder by the methods of the present invention can be determined by detecting a clinical improvement as indicated by a change in the subject’s symptoms and / or clinical parameters, as would be well known to one of skill in the art.

[0055] Unless indicated otherwise, the terms "protect," "protecting," "protection" and "protective" (and grammatical variations thereof) encompass both methods of preventing and treating a disorder in a subject.

[0056] The terms "protective" immune response or "protective" immunity as used herein indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence and / or severity and / or duration of disease or any other manifestation of infection. Alternatively, a protective immune response or protective immunity may be useful in the therapeutic treatment of existing disease.

[0057] An "active immune response" or "active immunity" is characterized by "participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both." Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to immunogens by infection or by vaccination. Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a non-immune host." Id.

[0058] Provided according to embodiments of the present invention are compositions and methods for expanding Tregs and methods of use thereof. In some embodiments, it was determined that autologous TCR engagement in synergy with TGF-P signaling drives stable exponential expansion of primary human CD4+ and CD8+ FOXP3+ Tregs in long-term culture. The autologous nature of the culture (i.e., without added mitogens or antigens) allows for continuous passaging (i.e., continuous exponential growth) of Tregs without producing T cell exhaustion for at least several months. Moreover, this invention allows for the culture and / or expansion of Tregs from a biological sample without needing to isolate T cells from said sample (i.e., wherein the method is devoid of a step involving isolating said T cells) and without needing to sort the expanded cell population to isolate desired cells. In some embodiments, the method produces Tregs that have a clonotypic specificity which is distinct from those produced by other methods (e.g., those that incorporate exogenous mitogens and / or antigens in the culture medium) currently used in the art. In some embodiments, the clonotypic specificity of the Tregs produced by the methods as described herein may be a result of the autologous T cell reaction provided by said methods. In some embodiments, the method comprises a) providing a biological sample comprising Tregs; b) culturing the biologicalsample in vitro with a culture medium comprising a basal cell culture medium, a T cell growth factor, and a regulatory cytokine, to provide cultured Tregs; and c) passaging the cultured Treg population one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50 or more times) with a xeno-free culture medium comprising a basal cell culture medium, a T cell growth factor, and a regulatory cytokine, to provide expanded Tregs. In some embodiments, the T cell growth factor and / or regulatory cytokine are produced by the Tregs in the culture medium.

[0059] As used herein, "autologous expansion" and "autologous cell growth" are intended to mean cell growth (e.g., expansion) which occurs without exogenous mitogens and / or antigens added to the cell culture medium. As used herein, "autologously expanded Tregs" is intended to mean Tregs which are expanded without the addition of exogenous mitogens and / or antigens to the cell culture medium during passaging. In some embodiments, the expanded Tregs are homogeneous Tregs. In some embodiments, the expanded Tregs are autologously expanded Tregs. In some embodiments, the Tregs produce autologous T-cell peptides during the culturing and / or passaging steps (i.e., step b) and / or step c)). In some embodiments, step b) and / or c) produce an autologous T cell reaction (e.g., an autologous T cell response) in the cultured and / or expanded Tregs. In some embodiments, the autologous T cell reaction is a continuous autologous T cell reaction.

[0060] A xeno-free culture medium, as used herein, is intended to mean a cell culture medium which is devoid of exogenous mitogens (e.g., added mitogens) and / or exogenous antigens (e.g., added antigens) such as fetal bovine serum (FBS), bovine serum albumin (BSA), anti T cell receptor (TCR) antibodies, anti-CD28 antibodies, and the like. In some embodiments, the culture medium and / or xeno-free culture medium is devoid of rapamycin, retinoic acid, and / or dendritic cells. In some embodiments, the culture medium and / or xeno-free culture medium consists of a basal cell culture medium, a T cell growth factor, and a regulatory cytokine.

[0061] A T cell growth factor, as used herein, is intended to mean a signaling molecule (e.g., a ligand peptide or fragment or variant thereof) which stimulate the growth and / or development of T cells in vitro and / or in vivo either directly (e.g., by binding directly to T cells and / or T cell receptors) or indirectly (e.g., by increasing the number of MHC peptides processed and presented by antigen-presenting cells (APCs)). In some embodiments, the T cell growth factor is interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 15 (IL- 15), interleukin 21 (IL-21), or any combination thereof.

[0062] A regulatory cytokine, as used herein, is intended to mean a signaling molecule (e.g., a ligand peptide or fragment or variant thereof) produced by regulatory B cells and / or regulatoryT cells which may or may not be used to modulate the immune response of a subject. In some embodiments, the regulatory cytokine is transforming growth factor P 1 (TFG-pi), transforming growth factor P 2 (TFG-P2), transforming growth factor P 3 (TFG-P3), interleukin 10 (IL-10), an interferon (e.g., a Type I interferon such as interferon beta (IFN-P) and / or interferon alpha (IFN-a)), or any combination thereof.

[0063] In some embodiments, the T cell growth factor is present in the culture medium and / or xeno-free culture medium in a concentration from about 0.001 nM to about 100 nM (e.g., about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nM) and the regulatory cytokine is present in the culture medium and / or xeno-free culture medium in a concentration from about 0.1 nM to about 100 nM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nM).

[0064] In some embodiments, the T cell growth factor is present in the culture medium and / or xeno-free culture medium in a low level, i.e., in a concentration from about 0.001 nM to about 1 nM (e.g., about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 nM). In some embodiments, a low level of T cell growth factor may lead to high levels of CD25 expression in the Tregs. In some embodiments, the T cell growth factor is present in the culture medium and / or xeno-free culture medium in a high level, i.e., in a concentration from about 10 nM to about 100 nM (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nM). In some embodiments, a high level of T cell growth factor may lead to low levels of CD25 expression in the Tregs.

[0065] In some embodiments, the regulatory cytokine is present in the culture medium and / or xeno-free culture medium in a low level, i.e., in a concentration from about 0.1 nM to about 1 nM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 nM). In some embodiments, the regulatory cytokine is present in the culture medium and / or xeno-free culture medium in a high level, i.e., in a concentration from about 10 nM to about 100 nM (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nM).

[0066] The terms "base medium," "basal medium," and "basal cell culture medium," are used herein to refer to a basal salt nutrient or an aqueous solution of salts, amino acids, vitamins, buffers and other elements that provides cells with water and inorganic ions that are needed for normal cell metabolism and maintaining intracellular and / or extracellular osmotic balance. In some embodiments, a base media may include a buffering system to maintain the mediumwithin a physiological pH range of about 7 to about 8, or a pH range of about 7.2 to about 7.8, or a pH of about 7.3 to about 7.5. In some embodiments, the basal cell culture medium comprises LymphoOne™ T-Cell Expansion Medium, X-Vivo 15™ Serum-free Hematopoietic Cell Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CelThera™ GMP T Cell Expansion Medium, ImmunoCult™-XF T Cell Expansion Medium, ExCellerate Human T Cell Expansion Media, CellGenix® GMP T Cell Medium, 4Cell® Nutri-T GMP Media, and / or ArtMedia® Human T Cell Serum-Free Medium.

[0067] In some embodiments, the culture medium used in the culturing step (i.e., step b)) further comprises a mitogen (e.g., phytohaemagglutinin (PHA)). In some embodiments, the culture medium used in step b) and / or the xeno-free culture medium used in step c) further comprises granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the culture medium used in step b) and / or the xeno-free culture medium used in step c) further comprises GM-CSF and IL-4.

[0068] In some embodiments, the expanded Tregs express forkhead box P3 protein (FOXP3), cluster of differentiation 4 protein (CD4), cluster of differentiation 8 protein (CD8), cluster of differentiation 69 protein (CD69), programmed cell death protein 1 (PD1), IL-2 receptor a protein (CD25), cytotoxic T-lymphocyte associated protein 4 (CTLA4; i.e., CD152), glucocorticoid-induced TNFR-related protein (GITR), B-cell lymphoma 6 (BCL6), cluster of differentiation 3 protein (CD3), cluster of differentiation 28 protein (CD28), cluster of differentiation 80 protein (CD80), cluster of differentiation 86 protein (CD86), FAS (i.e., CD95), FAS ligand (i.e., CD178), interleukin-7 receptor subunit alpha (IL7R-a; i.e., CD127), C-X-C chemokine receptor type 5 (CXCR5; i.e., CD185), lymphocyte-activation gene 3 (LAG3), programmed death-ligand 1 (PDL1), programmed death-ligand 2 (PDL2), glycoprotein A repetitions predominant (GARP; i.e., LRRC32), human leukocyte antigen isotypes (HLA-DR, HLA-DP, HLA-DQ, and / or HLA-A2), zinc-finger protein Helios (e g., Ikaros (Ikzfl), Helios (Ikzf2), Aiolos (Ikzf3), Eos (Ikzf4), and / or Pegasus (Ikzf5)), or any combination thereof.

[0069] In some embodiments, the expanded Tregs express FOXP3 and CD25, but do not express CD127. In some embodiments, the expanded Tregs express CD4 and do not express CD8. In some embodiments, the expanded Tregs express CD8 and do not express CD4. In some embodiments, the duration over which the expanded Tregs are expanded alters the level of one or more proteins expressed by the expanded Tregs. For example, in some embodiments, the expanded Tregs are passaged for between about 1 and 14 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) and express CD4 and CD8. In some embodiments, theexpanded Tregs are passaged for about 2 weeks or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more) and express CD8 but do not express CD4.

[0070] In some embodiments, one or more subsets of the expanded Tregs may be isolated from the expanded Tregs culture based on, for example, expression of one or more proteins in said expanded Tregs. The one or more subsets of the expanded Tregs may be isolated using any method known to those in the art, including, but not limited to, purification by magnetic beads (e.g., anti-CD4 beads, anti-CD8 beads, streptavidin beads, and the like) and / or fluorescence- activated cell sorting (FACS). In some embodiments, one or more subsets of the expanded Tregs are isolated based on the expression of FOXP3, CD4, CD8, CD69, PD1, CD25, CTLA4 (CD152), GITR, BCL6, CD3, CD28, CD80, CD86, FAS (CD95), FAS ligand (CD178), IL7R- a (CD127), CXCR5, LAG3, PDL1, PDL2, GARP (LRRC32), HLA-DR, HLA-DP, HLA-DQ, HLA-A2, zinc-finger protein Helios (e.g., Ikaros (Ikzfl), Helios (Ikzf2), Aiolos (Ikzf3), Eos (Ikzf4), and / or Pegasus (Ikzf5)), or any combination thereof. In some embodiments, a subset of the expanded Tregs may be isolated that express CD4 but do not express CD8. In some embodiments, a subset of the expanded Tregs may be isolated that express CD8 but do not express CD4. In some embodiments, an isolated subset of the expanded Tregs may then be further passaged as described herein to provide isolated Tregs (e.g., isolated CD8+Tregs and / or isolated CD4+Tregs).

[0071] In some embodiments, the biological sample is a blood sample or a tissue sample, e.g., umbilical blood, lymph nodes, and / or thymic tissue. In some embodiments, the biological sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments, the biological sample is not processed to isolate T cells.

[0072] In some embodiments, the expanded Tregs express major histocompatibility complex class I (MHC1) molecules (e.g, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, and / or HLA-L) and major histocompatibility complex class II (MHC2) molecules (e.g., HLA- DP, HLA-DQ, and / or HLA-DR). In some embodiments, the expanded Tregs do not become exhausted Tregs during step c). In some embodiments, the presence of exhausted Tregs is indicated by the expanded Tregs comprising a percentage of Tregs expressing high levels of PD1 of 10% or more (e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the presence of exhausted Tregs is indicated when the expanded Tregs do not exhibit exponential growth between the one or more passages of step c). In some embodiments, the expanded Tregs do not contain a population of senescent Tregs that make up more than 5% (e.g, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%) of the total number of expanded Tregs. In someembodiments, the expanded Tregs are passaged for about 1 week or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more). In some embodiments, the expanded Tregs are expanded 2-fold or more (e.g., 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 30- , 40-, 50-, 100-, 200-, 300-, 400-, 500-, 1000-, 10000-, 50000-fold or more) compared to the Tregs in the biological sample. In some embodiments, the method does not include genetic engineering of the Tregs.

[0073] According to some embodiments, provided herein is a population of Tregs produced by the methods described herein. In some embodiments, the population of Tregs are autologous Tregs. In some embodiments, the population of Tregs may be used in a method of treatment for any disease, disorder, and / or pathogen infection in which it is known, or believed to be known, that Tregs may produce a beneficial effect.

[0074] According to some embodiments, provided herein is a method of treating an immune disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of Tregs as produced by methods described herein to the subject, thereby treating the immune disorder. One of ordinary skill in the art would understand that the Tregs as described herein would be useful for treating any immune disorder for which administering Tregs is known, or believed to be known, to be beneficial. Examples of an immune disorder include, but are not limited to, type I diabetes, arthritis (e.g., rheumatoid arthritis, juvenile chronic arthritis, erosive osteoarthritis, or psoriatic arthritis), Celiac disease, scleroderma, psoriasis, autoimmune myopathy, psoriasis, CREST syndrome, inflammatory myositis, mixed connective tissue disease, palindromic rheumatism, eosinophilic fasciitis, dermatomyositis, calcium pyrophosphate crystal deposition disease, acute respiratory distress syndrome, osteoporosis, delayed hypersensitivity, autoimmune thyroiditis, Hashimoto's disease, Sjogren's disease, Grave's disease, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, leucopenia, thrombus formation, or lupus (e.g., systemic lupus erythematosus or lupus nephritis).

[0075] According to some embodiments, provided herein is a method of treating an inflammatory disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of Tregs as produced by methods described herein to the subject, thereby treating the inflammatory disease. One of ordinary skill in the art would understand that the Tregs as described herein would be useful for treating any inflammatory disease for which administering Tregs is known, or believed to be known, to be beneficial. Examples of an inflammatory disease include, but are not limited to, ulcerative colitis, Crohn's disease, pulmonary inflammation, idiopathic pulmonary fibrosis, chronic obstructivepulmonary disease (COPD), retinitis, uveitis, kidney disease, obesity, spondyloarthritis, or vasculitis..

[0076] According to some embodiments, provided herein is a method of treating a pathogen infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of Tregs as produced by methods described herein to the subject, thereby treating the pathogen infection. One of ordinary skill in the art would understand that the Tregs as described herein would be useful for treating any pathogen infection for which administering Tregs is known, or believed to be known, to be beneficial. In some embodiments, the pathogen is a virus, a bacterium, a prion, a parasite, and / or a fungus. In some embodiments, the pathogen infection comprises influenza virus, herpes simplex virus, Epstein-Barr virus, cytomegalovirus, adenovirus, respiratory syncytial virus, coronavirus (e.g., CoVID- 19 and variants thereof), BK virus, JC virus, streptococcal pharyngitis, tuberculosis, bacterial gastroenteritis, cellulitis, tetanus, pneumonia, Clostridioides difficile, Lyme disease, Rocky Mountain spotted fever (RMSF), meningitis, conjunctivitis, Methicillin-resistant Staphylococcus aureus, gonorrhea, bacterial sinusitis, bacterial vaginosis, giardia, Chagas disease, malaria, toxoplasmosis, cryptosporidiosis, cyclosporiasis, cysticercosis, hookworm, trypanosomiasis, echinococcosis, or any combination thereof.

[0077] In some embodiments, the pathogen is a bacterium, and the bacterium is a Bacillus bacterium, a Bordatella bacterium, a Borrelia bacterium, a Brucella bacterium, a Burkholderia bacterium, a Campylobacter bacterium, a Chlamydia bacterium, a Coxiella bacterium, a Ehrlichia bacterium, a Escherichia bacterium, a Francisella bacterium, a Leptospira bacterium, a Listeria bacterium, a Rickettsia bacterium, a Salmonella bacterium, a Shigella bacterium, a Tuberculosis bacterium, and the like.

[0078] In some embodiments, the pathogen is a parasite, and the parasite is a protozoan (e.g., Balamuthia mandrillaris, Cryptosporidium parvum, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Naegleria fowleri, Toxoplasma gondii and the like) and / or a helminth (e.g., a Taenia, an Echinococcus, a Schistosoma, a Fasciola, an Ascaris, an Enterobius, a Rhabditis, a Trichuris, a Necator, an Ancylostoma, and the like).

[0079] In some embodiments, the pathogen is a fungus, and the fungus is Coccidioides, Microsporidia, Mucorales, and the like.

[0080] According to some embodiments, provided herein is a method of treating an allergic disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs as produced by methods described herein to the subject, thereby treating the allergic disease. One of ordinary skill in the art would understand that the Tregs asdescribed herein would be useful for treating any allergic disease for which administering Tregs is known, or believed to be known, to be beneficial. Examples of an allergic disease include, but are not limited to, allergic rhinitis, allergic asthma, atopic dermatitis, eczema, or any combination thereof.

[0081] According to some embodiments, provided herein is a method of treating a transplantation rejection disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs as produced by methods described herein to the subject, thereby treating the transplantation rejection disease. One of ordinary skill in the art would understand that the Tregs as described herein would be useful for treating any transplantation rejection disease for which administering Tregs is known, or believed to be known, to be beneficial. Examples of a transplantation rejection disease include, but are not limited to, graft-versus host disease, solid organ rejection, or any combination thereof.

[0082] According to some embodiments, provided herein is a method of treating a neurodegenerative disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs as produced by methods described herein to the subject, thereby treating the neurodegenerative disease. One of ordinary skill in the art would understand that the Tregs as described herein would be useful for treating any neurodegenerative disease for which administering Tregs is known, or believed to be known, to be beneficial. Examples of a neurodegenerative disease include, but are not limited to, Alzheimer’s disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, frontotemporal dementia, Lewy body dementia, Huntington's disease, Pick’s disease, multisystem atrophy, progressive supranuclear palsy, inclusion body myositis, prion protein cerebral amyloid angiopathy, argyrophilic grain disease, tangle predominant dementia, chronic traumatic encephalopathy, traumatic brain injury, or any combination thereof.

[0083] According to some embodiments, provided herein is a method of treating a metabolic disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs as produced by methods described herein to the subject, thereby treating the metabolic disease. One of ordinary skill in the art would understand that the Tregs as described herein would be useful for treating any metabolic disease for which administering Tregs is known, or believed to be known, to be beneficial. Examples of a metabolic disease include, but are not limited to, type II diabetes, obesity, metabolic syndrome, or any combination thereof.

[0084] According to some embodiments, provided herein is a method of treating an aging- related disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs as produced by methods described herein to the subject, thereby treating the aging-related disease. One of ordinary skill in the art would understand that the Tregs as described herein would be useful for treating any aging-related disease for which administering Tregs is known, or believed to be known, to be beneficial. Examples of an aging-related disease include, but are not limited to, inflammaging, hypertension, atherosclerosis, nonalcoholic fatty liver disease, macular degeneration, or any combination thereof.

[0085] According to some embodiments, provided herein is a method of treating a cardiovascular disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs as produced by methods described herein to the subject, thereby treating the cardiovascular disease. One of ordinary skill in the art would understand that the Tregs as described herein would be useful for treating any cardiovascular disease for which administering Tregs is known, or believed to be known, to be beneficial. Examples of a cardiovascular disease include, but are not limited to, myocardial infarction, stroke, chronic heart disease, arteriosclerosis, coronary artery disease, heart failure, abnormal heart rhythm or arrhythmia, aorta disease, Marfan syndrome, cardiomyopathy, carotid artery disease, peripheral artery disease, or any combination thereof.

[0086] In some embodiments, administering the Tregs as described herein to treat a disease, disorder, and / or infection as described herein comprises administering 109Tregs or more (e.g., 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, IO20, or more Tregs) to the subject. In some embodiments, administering the Tregs comprises administering one or more doses of Tregs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses). In some embodiments, the Tregs that are administered to the subject are autologous Tregs. In some embodiments, the Tregs that are administered to the subject are exponentially expanding Tregs.

[0087] According to some embodiments, a Treg expansion strategy includes continuous autologous TCR-dependent antigen recognition and continuous TGF-P signaling. The combination of these two synergistic strategies can enable the derivation of continuous FOXP+regulatory T cell lines. This novel strategy offers several advantages compared to other contemporary methods of Treg expansion, as noted in Table 1.

[0088] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.ExamplesContinuous exponential expansion of human FOXP+Tregs

[0089] Improved strategies are needed to advance FOXP3+regulatory T cell (Treg)-based adoptive immunotherapy. To address this unmet clinical need, we focused on xeno-free human T cell culture systems that support autologous expansion of primary human T cells that constitutively expressed FOXP3+in long term cultures. Autologous T cell expansion was supported by continuous TGF-P exposure, which augmented HLA-DR and CD25 expressionwith consequent enhancements of antigen presentation, T cell antigen recognition, and IL-2 responsiveness. These Tregs homogeneously expressed FOXP3, exhibited TCR responsiveness against culture-intrinsic peptides, expressed signature Treg markers (e.g., CD25, CTLA-4, GITR), and maintained a rapid cell division rate over months of culture without exhaustion. Autologous expansion yielded several FOXP3+lineages, including a MHCII-restricted CD4+single-positive subset and a MHCI-restricted CD8+single-positive subset together with distinct subsets defined by qualitative variation in CD25, CD69, and PD1 expression. Low IL-2 concentrations (e.g., less than 1 nM IL-2) favored CD25hlghCTLA4hlghCD69lowPDllowsubsets, whereas high IL-2 concentrations (e.g., more than 10 nM IL-2) favored CD25lowCTLA4lowCD69hlghPDlhlghsubsets. In addition to stable homogeneous expression of FOXP3, these T cells lacked IL-2 production capacity, exhibited in vitro suppressive activity, and responded to TGF-P via enhanced IL-2 consumption. TCR engagement was required for autologous IL-2-driven expansion of CD4+Tregs because growth was blocked by neutralizing mAbs against CD4. Autologous FOXP+Treg expansion had distinct advantages compared to mitogen-driven Treg expansion protocols, given that autologous Treg expansion required only a single small PBMC sample as the Treg source material but yielded over 1021-fold expansions. Autologous expansion neither required nor included Treg purification, rapamycin, all-trans retinoic acid, genetic manipulation, supplementation with dendritic cells (DCs), or repeated donations from PBMC donors. Although mitogens accelerated initiation of autologous Treg cultures, use of mitogens was also unnecessary. Without wishing to be bound by any particular theory, the interpretation is that continuous homeostatic expansion of Tregs in vitro mimics a primary mechanism underlying homeostatic expansion of the Treg repertoire in vivo. That is, in xeno-free culture media, MHCIL Tregs exclusively express intrinsic MHCI / II peptide (MHCI / IIp) complexes that agonistically engage the native self-reactive TCR repertoire of Tregs via cognate autologous antigen recognition. Autologous self-presentation of MHCIIp and MHCIp complexes coupled with continuous TGF-P signaling drives autologous Treg expansion with a stable FOXP3+IL- 2-reactive phenotype, which enables stable clonal expansion of FOXP3+Tregs in the presence of exogenous IL-2.Introduction

[0090] FOXP3+regulatory T cells (Tregs) are required for immune homeostasis and selftolerance (1-8). Treg biology is defined by two primary subsets, including the natural Treg / thymic Treg (nTreg) subset and the induced / peripheral Treg (pTreg) subset that are based onnascent differentiation of the Treg lineage in the thymus versus peripheral tissues, respectively (9-11). Aside from distinctions in origin and TCR specificity, nTregs and pTregs exhibit overlapping phenotypic and functional attributes. For example, both F0XP3+Treg subsets are maintained by chronic agonistic recognition of MHC-restricted peptide antigens in homeostatic quiescent tissues and are sustained by IL-2 in inflamed tissues. The nTreg repertoire comprises a diverse TCR repertoire that exhibits self-reactive specificity and agonistic reactivity to clonotypic self-MHC / self-peptide complexes (12-15). Treg recognition of a specific selfpeptides preempts pathogenic inflammation by targeting autoimmune effector T cells that agonistically recognize the same self-antigens, cross-reactive mimicry antigens, or proximal tissue-resident self-antigens that exist within the same inflammatory sites. By these mechanisms, nTregs induce tolerance to their cognate self-antigens and clonotypically distinct self-peptides presented on the same APCs (e.g., dendritic cells). Conversely, the pTreg repertoire exhibits reactivity, at least in part, to innocuous foreign antigens to enforce tolerance to foreign environmental antigens that constantly permeate self-tissues during life but do no harm. These pTregs preempt allergic reactions to environmental foreign antigens, including gut microbiota, food antigens, and airborne antigens. The pTreg subset may also differentiate upon recognition of tissue-specific self-antigens to thereby enforce self-tolerance throughout the body (9). These pTregs, like nTregs, may enforce tolerance in specialized endocrine organs, immune-privileged tissues, pregnancy-associated tissues, and / or nervous tissue. Thus, these pTregs, like nTregs, may enforce self-tolerance in specialized endocrine organs, immune- privileged tissues, pregnancy-associated tissues, nervous tissue, among other specialized tissues. Thus, the modus operand! of both nTreg and pTreg subsets includes the following precepts: TCR specificity for MHC-restricted agonistic peptide antigens, chronic activation in response to ever-present peptide antigens, resistance to exhaustion-induced cell death or anergy despite continual presence of antigen, and immune suppression of effector responses that target these peptide antigens.

[0091] Given the dominant agonistic self-reactivity of Tregs, the main prediction of this study is that FOXP3+Tregs will exhibit dominant autologous TCR autoreactivity in vitro when exposed to self-peptides in MHCII+ / MHCI+culture systems that lack foreign (xenogeneic) antigens. That is, Treg expansion should outpace conventional T cell expansion when autologous self-antigens rather than foreign antigens represent the predominant TCR ligands in a culture system. Furthermore, autologous Treg expansion may be advantageous for ‘Treg manufacture’ in Treg-based adoptive immunotherapy protocols because Treg TCR interactions with autologous MHCIIp and MHCIp complexes may exhibit a homeostatic bandwidth ofsignal strength intensities conducive for continuous Treg expansion without exhaustion. In contrast, mitogenic expansion strategies (i.e., expansion with anti-CD3 and anti-CD28 mAbs) or expansion via mixed lymphocyte reactions (MLR) are the basis for many contemporary Treg manufacture protocols. High-intensity stimulation however may exceed optimal signal strength and instead may cause T cell exhaustion and the collapse of functionally intact Treg expansion cultures. Mitogen-driven and alloantigen driven Treg expansion protocols are the basis for many early-stage Treg-based clinical trials, which focus on chronic inflammatory diseases including autoimmune disease, allergic disease, transplant rejection, and GVHD (16- 25). These clinical trials met safety criteria, but contemporary Treg products did not reach clinically-relevant endpoints and were associated with poor Treg survivability in adoptive hosts. Given the cost and logistics of Treg adoptive immunotherapy, robust clinical responses are needed for economic viability. Curative responses represent the goal. To close this gap, qualitative advances are needed to optimize quality and quantity of the derivative Tregs.

[0092] This study offers potential solutions for Treg manufacture by providing evidence that the inherent autologous TCR reactivity of Tregs can be used to exponentially expand stable FOXP3+Tregs in TGF-P-supplemented culture systems with minimal T cell exhaustion. Autologous expansion strategies can deliver large numbers of functional CD4+and CD8+Tregs bearing stable FOXP3+phenotypes marked by qualitative variation in several Treg-associated markers (e.g., CD25, CD69, PD1, and GITR). Compared to mitogenic driven expansion protocols, autologous Treg expansion platforms for ‘Treg manufacture’ may provide a strategic alternative for adoptive Treg-based immunotherapy.Materials and Methods:

[0093] Human PBMC isolation, T cell culture, and derivation of T cell lines

[0094] PBMCs were obtained from healthy human volunteers who provided informed consent as approved by the University and Medical Center Institutional Review Board (UMCIRB) of East Carolina University (UMCIRB 23-000339). PBMCs were isolated by density gradient centrifugation. After washing in HBSS, PBMCs were cultured in LymphoOne Media and were passaged every 3-7 days. During the first passage, PBMCs were cultured with IL-2 in the presence or absence of recombinant rat TGF-P with or without 5 pg / ml PHA as designated. GM-CSF and IL-4 (1 nM) were added to the initial 1-3 passages but were not added thereafter. T cells were passaged every 3-7 days in 1 nM recombinant human IL-2 in the presence and / or absence of 10 nM TGF-P (unless designated otherwise) without other exogenous antigenic or mitogenic stimuli. The entire culture duration spanned several weeks to several months.Several distinct human FOXP3+Treg lines from multiple human PBMC donors were used in this study, including the CL1, CL2, CL3, CL4, CL5, CL6, CL7, NL1, NL2, Pl, P2, P3, VI, and V3 lines. The CL4, VI, and V3 lines were derived without use of any mitogen exposure whereas the other lines were derived after a single initial culture with PHA. For all cultures, mitogenic stimulation was never used after the first passage.

[0095] Cell purification

[0096] Human CD4+and CD8+T cell subsets were magnetically sorted by use of the REAlease CD4 or CD8 Microbead Kits (Miltenyi Biotec, catalog numbers # 130-117-037 and #130-117- 036). CD25hlghand CD69hlghT cells were isolated by labeling T cells with PE-conjugated anti- CD25 or PE-conjugated anti-CD69 mAbs followed by purification on anti-PE Microbeads (Miltenyi Biotec, catalog # 130-048-801).

[0097] Recombinant proteins

[0098] Recombinant rat Transforming Growth Factor-pi (TGF-P) was expressed and purified from transfected HEK293F cultures as described in previous studies (21). Purified TGF-P was activated by 10 min of exposure to 70°C, and each TGF-P preparation was verified for bioactivity by inhibition of GM-CSF-stimulated TF-1 cell growth (i.e., TF-1 erythroleukemia cells, ATCC, CRL-2003) (22). Recombinant human IL-2 was purified from a transfected stable HEK293F cell line, and bioactivity was assessed by proliferation of the IL-2-dependent SJL-PLP.l T cell line (23). Recombinant human IL-2 was also obtained from a NIH bioresource program (Teceleukin, Hoffman LaRoche) and from Peprotech (#200-02). Recombinant human IL-4 and GM-CSF cytokines were obtained from transfected stable HEK293F cell lines, and bioactivity was assessed by proliferation of the TF-1 cell line. Phytohemagglutinin lectin from Phaseolus vulgaris (PHA or PHA-L) was purchased from Sigma Aldrich (St. Louis, MO).

[0099] Cell proliferation assays

[0100] To measure IL-2 dependent proliferation, human T cells were cultured in LymphoOne media in 96 well plates with IL-2 in the presence of designated mAbs (BioXcell). These mAbs included the anti-CD4 mAbs OKT4 (mouse IgG2b-K) or RPA-T4 (mouse IgGl-K) or the respective isotype controls (MPC-11 mouse IgG2b-K or MOPC-21 mouse IgGl-K). Cultures were maintained for designated durations and were pulsed with 1 pCi [3H]thymidine (6.7 Ci / mmol, New England Nuclear, Perkin Elmer, Waltham, MA) during the last 24 hours ofculture. Cultures were harvested onto filters by use of a Tomtec Mach III harvester (Hamden, CT). [3H]thymidine incorporation into DNA was measured by use of a Perkin Elmer MicroBeta2 liquid scintillation counter.

[0101] Flow cytometric analysis

[0102] Cultured T cells were washed in PBS and were stained with Live / Dead Fixable Blue Dead Cell Stain Kit (ThermoFisher L23105) for 1 hour at room temperature in the dark. Cells were extensively washed and then incubated in Cell Staining Buffer (PBS + 2% BSA + 1% Human Serum) for 30 minutes at 4°C in the dark to remove unincorporated dye and to block Fc receptors / nonspecific binding sites. For surface staining, cells were stained with mAb cocktails in the dark at 4°C for 4 hours with rocking (200 pl volume). Intracellular staining was performed with the FOXP3 Intracellular Staining Kit (Invitrogen, Catalog # 00-5523-00). More specifically, cells were resuspended in Fixation / Permeabilization solution (3: 1 parts dilutant to concentrate) and incubated in the dark for 30 min at room temperature. Cells were washed twice in Permeabilization buffer and were incubated with designated mAbs in Permeabilization buffer in the dark at 4°C overnight with rocking (200pl / tube). Cells were washed 3 times in Permeabilization buffer and then with Diluted Flow Staining Buffer (PBS + 0.2% BSA + 0.1% Human Serum). Cells were analyzed on a Cytek Aurora Spectral Cytometer (Fremont, CA) with unmixing via unstained and single-stained control samples followed by analysis with De Novo Software FCS Express 7 (Glendale, CA). The following mAbs were used for staining (Table 2).Table 2. MAb reagents used for flow cytometric analyses.

[0103] Statistical analyses, data presentation, and experimental reproducibility

[0104] Comparisons among three groups or more were assessed via two-way analysis of variance (ANOVA) with the Tukey multiple comparisons test. Comparisons between two groups were analyzed by Student’s t-test. A p-value < 0.05 was considered significant. Significance was indicated as follows: (* p < 0.05, ** p < 0.01 , *** p < 0.001 , and p < 0.0001). Each data point represents the mean value, and error bars represent the standard deviation (SD). Experiments shown in the figures are representative of three independent experiments (i.e., biological replicates).Results

[0105] Continuous TGF-fl exposure enabled higher CD25 expression and superior IL-2-driven growth in long-term T cell cultures.

[0106] As shown in Fig. 1, panels A-B, continuous TGF-P exposure of purified human CD4+T cells in 38-day IL-2 expansion cultures enhanced cell enlargement compared to control T cells that were cultured without TGF-p. TGF-P-cultured T cells also exhibited higher expression levels of HLA-DR and CD25, lower expression levels of CD3, and steady expansion during the 38-day duration. In support, a previous study focusing on mitogen- stimulated human PBMC cultures also showed that TGF-P elicited higher HLA-DR expression as measured by increased percentages of HLA-DR+T cells and higher HLA-DR MFI values on a per cell basis in association with higher FOXP+Treg accumulation (31). Overall, the data in Fig. 1 provided evidence that continuous TGF-P exposure supported maintenance of T cells in an activated dividing state. Without wishing to be bound by any particular theory, one interpretation is that TGF-P caused higher levels of MHCII expression, which in turn caused higher levels of autologous TCR engagement, which in turn caused CD3 downregulation, heightened CD25hlghexpression, and greater IL-2 responsiveness. Consistent with this possibility (Fig. 1, panel C), a continuous 20-day exposure to TGF-P (i.e., since the initial derivation of the line) enabled high levels of IL-2-driven growth in proliferative bioassays, whereas control T cells (derived in the absence of TGF-P) lacked IL-2 reactivity at these cell densities. The main conclusion was that the prior continuous exposure to TGF-P augmented subsequent IL-2 reactivity. In contrast, TGF-P added directly to the assay lacked stimulatory activity and instead had a slight inhibitory activity at low IL-2 concentrations. These data provided evidence that TGF-P was not directly mitogenic, but rather, TGF-P primed IL-2 responsiveness and enhanced growth in subsequent IL-2 supplemented cultures. Similar results were obtained with both purified CD4+T cells and nonfractionated T cells (Fig. 1, panel C left and right panels, respectively).

[0107] Continuous culture with TGF-fl elicited higher CD25 expression during autologous reactions initiated with or without mitogen.

[0108] In cultures of CD4+T cells that were purified directly from PBMCs and then activated with PHA, continuous TGF-P exposure augmented CD25 expression by day 7 of culture (Fig. 2, panel A). The same effect was noted for the CD8+lineage (Fig. 2, panel B and Fig. 2, panel D) in that continuous TGF-P enhanced CD25 expression by day 11 of culture in both CD4+and CD8+subsets (Fig. 2, panel B, left and right panels respectively). In nonfractionated cultures, CD8+T cells were dominant and spontaneously overgrew CD4+T cells. For example, approximately 87% (Fig. 2, panel B, right column) and 99% (Fig. 2, panel C) of nonfractionated PBMC-derived T cells were of the CD8+lineage, respectively. The use ofPHA as a mitogen in the initial culture accelerated stable growth and the establishment of the continuous T cell line. However, the use of mitogen was not necessary. In cultures initiated without mitogen (Fig. 2, panel C), exponential T cell growth of the CD8+lineage was evident in 1-2 weeks, and these CD8+T cells exhibited high CD25 expression and stable continuous expansion in the presence of TGF-P and IL-2 in cultures that were maintained for over 58 days. Conversely, T cells cultured long-term in the absence of TGF-P did not survive. Overall, these data provide evidence that continuous TGF-P exposure sustains continuous high-level expression of CD25 and long-term IL-2 dependent growth in both CD4+and CD8+lineages.

[0109] CD4+and CD8+subsets from both TGF-fl-supplemented and control cultures expressed F0XP3.

[0110] These T cell lines expressed stable FOXP3 levels over prolonged culture durations in contrast to human conventional T cells which exhibit transitory FOXP3 expression during acute stimulation (32-35). The gating strategy is shown in Fig. 3, panel A. Both CD4+and CD8+subsets constitutively expressed FOXP3 and thereby represented a homogeneous source of human Tregs (Fig. 3, panel B). These analyses (Fig. 3, panel B and Fig. 3, panel D) used the anti-FOXP3 PCH101 clone which binds exon 1 and therefore may bind full-length FOXP3, the exon 2-spliced isoform (FOXP3 A2), or the exon 2 - exon 7 spliced isoform (FOXP3 A2A7) (36). These analyses (Fig. 3, panel C) also included the anti-FOXP3 150D clone that binds exon 2 and thereby solely recognizes full-length FOXP3, which is known to be important for optimal suppressive regulatory activities of Tregs (37-40). Specificity of FOXP3 staining was assessed in comparison to FMO (fluorescence-minus-one staining cocktails that lacked anti- FOXP3 mAb in the full panel) and unstained controls (Fig. 3, panels B-D). Anti-FOXP3 staining was uniform across 1-10 pl of anti-FOXP3 mAb reagent stain (Fig. 3, panel D). Hence, FOXP3 staining exhibited high sensitivity because staining was evident at low anti- FOXP3 concentrations. In contrast, the lymphoblastic CD25hlghmurine SJL-P T cell line did not show specific anti-human FOXP3 binding (Fig. 3, panel D). Overall, these data provide evidence that these long-term FOXP+Treg lines express the full-length form of FOXP3. However, these data did not exclude co-expression of other spliced isoforms of FOXP3.[OHl] Continual TGFfr exposure drove stable IL-2-dependent expansion ofFOXP3+Tregs.

[0112] To better document the role of TGF-P in stable continuous T cell expansion, three PHA- stimulated PBMC cultures were propagated in 1 nM IL-2 in the presence or absence of 10 nM TGF-P for 80 days of continuous culture (Fig. 4). T cells were passaged at approximately105 / ml, and cell yields were tallied for each passage. Starting at day 0 with a theoretical value of a single T cell, over trillion-fold expansions were noted for TGF-P cultures over 80 days of culture, whereas T cell lines without TGF-P lost IL-2 responsiveness and viability after approximately 30-50 days of continuous culture (Fig. 4, panel A). The human Tregs exhibited high levels of FOXP3 expression in both the presence and absence of TGF-P (Fig. 4, panel B). Thus, T cell exposure to TGF-P was not required for FOXP3 expression, although by day 18, all TGF-P-cultures exhibited higher levels of FOXP3 compared to control cultures without TGF-P (p = 0.0190, unpaired parametric two-tailed t test of median values). At day 65, TGF- P expansion cultures were comprised of primary CD8 FOXP3+Tregs that expressed MHCII (HLA-DR), CD80, CD86, CD69, CD25, PD-L2 (CD273), PD1 (CD279), and CTLA4 (Fig. 4, panel C). These data indicate that TGF-P facilitates the long-term exponential growth of human Tregs, with over a trillion to quadrillion-fold expansion potential. No special culture facilities were used in that Treg cultures were initiated and maintained in vented-cap tissueculture flasks in standard CO2 incubators. The day 80 timepoint was a chosen end of the experiment rather than a limitation of expansion potential. We conclude that this simple technology has the potential to provide large doses of human FOXP3+Tregs from small blood donations (20-40 ml) without Treg purification.

[0113] Limited numbers of nonfractionated PBMCs seeded long-term Treg cultures.

[0114] The expansion potential of autologous Treg cultures was evident from limited density cultures in which 50,000 nonfractionated PBMCs were seeded in individual wells of a 96 well flat-bottomed plate in the presence of IL-2 with or without TGF-P (Fig. 5, panel A). Cultures without exogenous TGF-P exhibited superior growth during the first several weeks of culture but these lines did not exhibit sustained long-term growth or survival. Conversely, all four of the TGF-P-supplemented cultures exhibited delayed growth. Although three of the TGF-P supplemented lines did not sustain long-term expansion, one TGF-P-supplemented culture exhibited an approximate 1022-fold expansion during three months of culture (Fig. 5, panel B). Because long-term nonfractionated lines spontaneously exhibit dominant outgrowth of CD8+Tregs, we used a CD4+Treg line (purified with anti-CD4 magnetic beads) (Fig. 5, panel C) as a comparison for the CD8+Treg line (Fig. 5, panel B and Fig. 5, panel D). The CD4+Treg line expressed bimodal CD25lowand CD25hlghpopulations along with high levels of CD3, CD4, class I MHC (HLA-A2), FOXP3, BCL6, CD86, CD69, and PD1 (Fig. 5, panel C). Although this CD4+Treg line expressed high levels of PD1, other CD4+and CD8+Treg lines exhibited low PD1 expression. For example, this CD8+Treg line expressed a dominant CD25hlghsubsetalong with high levels of CD3, CD8, MHC class I, FOXP3, BCL6, CD86, CD69, but lacked PD1 expression. Both lines exhibited high levels of CD95 (FAS) but lacked expression of CD 178 (FASL). Overall, these data indicated that autologous expansion represents a feasible approach to generate ample FOXP+Treg cell doses from small nonfractionated PBMC samples.

[0115] F0XP+T cells exhibited canonical activities of Tregs, including enhanced consumption of IL-2, lack of IL-2 production, and suppression of responder T cells.

[0116] Additional experiments confirmed that TGF-P-cultured Tregs exhibited superior IL-2 dependent growth compared to the control Tregs (Fig. 6, panel A, compare bars 1-4 vs bars 5- 8). Although TGF-P facilitated heightened IL-2 responsiveness in long-term cultures, TGF-P added directly to the culture had an acute inhibitory effect (Fig. 6, panel A, compare bar 2 vs bar 4). Thus, TGF-P promoted future propensity for IL-2 dependent growth, while TGF-P also had an acute inhibitory activity. The addition of PHA to established Treg lines inhibited growth of TGF-P Tregs (Fig. 6, panel A, compare bars 2 and 4 to bars 10 and 12, respectively). Conversely, PHA augmented growth of control Tregs (Fig. 6, panel A, compare bars 5-8 vs bars 13-16). Thus, mitogenic stimulation was contraindicated in the maintenance of these established Treg lines. These data indicated that homeostatic growth conditions facilitated better survivability and expansion compared to overt mitogenic stimulation for human Tregs. As shown in Fig. 6, panel B, neither TGF-P Tregs nor control Tregs produced detectable IL-2 bioactivity, either in the presence or absence of mitogen (Fig. 6, panel B, 1stand 3rdbar of each group). Cultures initiated with exogenous IL-2 (2ndand 4thbars of each group) provided a measure of IL-2 consumption during the first 24 hrs of culture, as represented by responses of IL-2 indicator cells in Fig. 6, panel B. Addition of TGF-P to the bioassay facilitated IL-2 consumption (Fig. 6, panel B, compare bars 2 and 4; 10 and 12; and 14 and 16). Tregs also mediated suppressive activity in that addition of Tregs to responder cultures dampened mitogenic proliferative responses attributed to PHA-stimulated PBMC responder T cells (Fig. 6, panel C). Thus, these FOXP+T cell lines exhibited core features of the Treg lineage.

[0117] A neutralizing anti-CD4 mAb inhibited autologous MHCII-restricted autoantigen recognition and IL-2 -dependent growthof CD4+Tregs.

[0118] A well-established hallmark of T cell biology is that productive T cell activation elicits a temporary CD25hlgh, IL-2 responsive, blastogenic phenotype. After clearance of antigen or removal of the TCR stimuli, blastogenic T cells revert to a quiescent CD25lowphenotype andshow limited IL-2 responsiveness (even in the presence of IL-2) coupled with growth cessation and eventual cell death. The notable observation here was that small PBMC-derived T cells transitioned to blastogenic clusters of rapidly growing FOXP+Tregs in xeno-free culture systems in the absence of mitogen or known exogenous antigens. Blastogenic Treg growth and positive cell yields occurred spontaneously and were sustained in long-term culture. Sustained blastogenic growth in the absence of identifiable xeno-TCR stimuli suggested that MHCI / IL Tregs responded to self-peptides endogenous within the culture system via selfrecognition by the Treg TCR repertoire, which is a known defining characteristic of the FOXP+Treg subset.

[0119] To assess whether TCR engagement may underlie IL-2 dependent blastogenic growth, we tested whether CD4 was needed for IL-2 driven-growth of CD4+Tregs given that CD4 is required for full TCR-response potency, but lacks known direct effects on the IL-2 receptor complex. We focused on the CD4 lineage due to the commercial availability of defined neutralizing anti-human CD4 mAb reagents. Notably, the neutralizing anti-human CD4 mAb RPA-T4 (mouse IgGl -kappa, BioXCell BE0288) but not by the non-neutralizing OKT4 anti- CD4 mAb (mouse IgG2b-kappa) inhibited IL-2-stimulated growth of CD4+Tregs (Fig. 7, panel A). This inhibitory activity was specific for CD4+Tregs, because neither RPA-T4 nor OKT4 mAbs affected IL-2 dependent growth of CD8+Tregs (Fig. 7, panel B). Also, the RPA- T4 IgGl -kappa isotype control (MOPC-21, BioXCell BE0083) and the OKT4 isotype control (mouse IgG2b-kappa, BioXCell MPC-11) lacked modulatory activity for both CD4+and CD8+Tregs (Fig. 7, panels A and B). None of these mAbs affected the IL-2 dependent growth of the mouse SJL-P T cell line (Fig. 7, panel C). The RPA-T4 and OKT4 mAbs bind distinct epitopes in the D 1 and D3 domains of CD4, respectively, but the two sites may exhibit allosteric interactions because the OKT4 mAb reversed the inhibitory activity of the RPA-T4 mAb (Fig. 7, panel A and Fig. 7, panel D). The competitive interactions of RPA-T4 and OKT4 were also noted at both 10 and 20 pg / ml concentrations (Fig. 7, panel D). Given that anti-CD4 mAb are established inhibitors of MHCII-restricted antigen recognition but not IL-2 signaling, these data provide evidence that TCR recognition events were needed for continuous IL-2 dependent Treg expansion. The RPA-T4 mAb reliably inhibited growth in a bioassay of 5 days or longer but was not inhibitory in shorter 3-day assays (Fig. 7, panel E). These data indicate that inhibition of TCR signaling requires time to impact IL-2 growth responses. Also, the RPA-T4 mAb exhibited optimal inhibition at higher cell densities (50,000 or IO47cells / well), whereas the mAb lacked detectable inhibitory activity at lower cell densities (e.g., 12,500 or 104 1cells / well) (Fig. 7, panel F). These data indicated that RPA-T4-mediated inhibition was optimalwhen cell contact was optimal. Overall, these data are consistent with a model whereby culture-intrinsic MHCIIp / TCR interactions in pure Treg cultures elicit CD25hlghexpression, enabling sustained growth in IL-2-supplemented cultures.

[0120] The FOXP+Treg lines used in these assays were derived via continuous expansion in IL-2 and TGF-P, and the bioassay was performed in the presence (Fig. 7, panels A-C top graph in each panel) or absence of TGF-P (Fig. 7, panels A-C bottom graph in each panel). Thus, cultures lacking TGF-P are Tregs withdrawn from chronic TGF-P exposure. For CD4+Tregs, TGF-P was inhibitory in the bioassay (e.g., compare Fig. 7, panel A top and bottom graphs). These data indicated that TGF-P enabled long-term growth competence while exerting shortterm acute inhibition. The presence or absence of TGF-P did not alter RPA-T4 mAb-mediated patterns of inhibition. Similar findings were noted in Fig. 7, panel G.

[0121] The RPA-T4 mAb inhibited Treg growth by limiting IL-2 responsiveness rather by limiting free IL-2 concentrations because IL-2 supplementation of cultures at subsequent phases of the bioassay did not reverse mAb-mediated inhibition (Fig. 7, panel G). That is, the RPA-T4 mAb had similar inhibitory activity when IL-2 was added on days 0 and 2 (Fig. 7, panel G, left two graphs) or on days 0, 2, and 4 (Fig. 7, panel G, right two graphs) compared to a single IL-2 addition on day 0. These data were not consistent with the possibility that mAb-mediated inhibition was due to degradation or neutralization of a limited IL-2 pool. Rather, without wishing to be bound by any particular theory, these data were consistent with the possibility that anti-CD4 mAbs blocked IL-2 responsiveness, perhaps by controlling CD25 expression.

[0122] The anti-CD4 mAb RPA-T4 promotes a distinct CD25lowTreg subset that had diminished T-APC (T cell APC) and Treg-signatures.

[0123] Given the possibility that the RPA-T4 mAb may inhibit IL-2 responsiveness, we hypothesized that the RPA-T4 mAb would inhibit CD25 expression (Fig. 8). Accordingly, long-term CD4+Treg cultures (53 days in culture) exhibited bimodal expression of CD25 (Fig. 8, panels A and B), and a 15-day exposure to the RPA mAb decreased the CD4+CD25hlghTreg percentages from 72% to 34% (Fig. 8, panel C, right graph). The CD25hlghsubset was also comprised of larger blast cells compared to the CD25lowsubset (higher FSC values, Fig. 8, panel C, left graph). Both CD4+CD25hlghand CD4+CD25lowsubsets expressed FOXP3, although the CD4+CD25hlghsubset expressed relatively higher levels of FOXP3 compared to the CD25lowTreg subset (Fig. 8, panel D). Notably, the RPA-T4 mAb primarily affected FOXP3 MFI values among CD25hlghTregs (Fig. 8, panels E and F). Overall, without wishingto be bound by any particular theory, these data support the hypothesis that CD4-dependent TCR engagement of MHCII-restricted culture-intrinsic peptides was needed for autologous responsiveness, which was required downstream for maintenance of a CD25hlghphenotype and competitive IL-2-dependent growth of the CD4+CD25hlghF0XP3+Tregs.

[0124] The anti-CD4 RPA-T4 mAb also inhibited surface markers involved in antigenic responsiveness (i.e., CD3 and CD4), T cell-mediated antigen presentation (HLA-DR), and costimulation (CD86) (Fig. 8, panel G). The observation that the RPA-T4 anti-CD4 mAb caused downregulation of CD3 provided evidence that continued culture-intrinsic antigen recognition is needed for maintenance of antigenic responsiveness. Likewise, the observation that the RPA-T4 anti-CD4 mAb caused downregulation of CD86 and HLA-DR provided evidence that continued culture-intrinsic antigen recognition is needed to maintain efficient T cell-mediated antigen presentation. The RPA-T4 mAb also inhibited expression of several Treg markers, including FAS, CTLA4, GITR, CXCR5, PD1, CD69, and LAG3. Overall, these data provide evidence that ongoing TCR engagement maintains an autologous self-reinforcing cycle of T cell-mediated antigen presentation, TCR responsiveness, IL-2 responsiveness, and Treg phenotypic identity (Fig. 8, panel G).

[0125] CD4+Tregs encompass distinct IL-2-stratefied subsets marked by differential expression of CD25, CD69, and PD1.

[0126] A central finding (Fig. 8) was that the anti-CD4 RPA-T4 mAb shifted the CD25low / CD25hlghratio of CD4+FOXP3+Tregs to favor the CD25lowsubset. However, because RPA- T4 mAb inhibited IL-2 responsiveness (Fig. 7), this mAb may consequently result in less IL-2 consumption and thereby may sustain higher IL-2 concentrations over time. Thus, the RPA- T4 mAb may perturb CD25low / CD25hlghratios indirectly, at least in part, by facilitating increased IL-2 concentrations, which would favor the CD25lowsubset because the CD25 does not confer a competitive advantage at high IL-2 concentrations. To assess this issue, we performed experiments to assess whether higher IL-2 concentration competitively favored the CD251OWFOXP+subset (Figs. 9 and 10).

[0127] To assess IL-2 and TGF-P response profiles, a long-term TGF-P-conditioned CD4+Treg line (73 days in culture) was cultured in triplicate for 7 days in the presence of designated IL-2 concentrations (100 pM vs 1, 10, or 100 nM) in the presence or absence of 10 nM TGF-P (Fig. 9). Although higher IL-2 concentrations are classically associated with higher CD25 expression, these experiments revealed a paradox in that higher IL-2 concentrations, in synergy with TGF-P, facilitated emergence of a CD25lowsubset that was enriched with a CD69hlghsubset (Fig. 9, panels A and B) and a PDlhlghsubset (Fig. 9, panels C and D). In contrast, Tregs maintained in low IL-2 concentrations were dominated by the canonical CD25hlghCD69lowPDllowphenotype, even though low IL-2 concentrations also sustained limited percentages of Tregs expressing either or both CD69 and PD1 (Fig. 9, panel A and Fig. 9, panel C). The ‘IL-2 + TGF-P’ dependent transition of cultures wherein a dominant CD25hlghCD69lowPDllowTreg subset was replaced by CD25lowCD69+and the CD69+PDlhlghsubsets are shown graphically as a function of IL-2 concentration (x-axis) (Fig. 9, panel B and Fig. 9, panel D).

[0128] As shown by histograms (Fig. 9, panel E), high concentrations of IL-2 and TGF-P promoted bimodality of CD25lowversus CD25hlghTreg subsets in both CD69hlghand CD69lowgates (compare 1stand 2ndcolumns of histograms). Conversely, high concentrations of IL-2 and TGF-P did not fundamentally alter the bimodality of the PDlhlgh / PDllowphenotypes within each respective CD69hlghand CD69lowsubset, although the distribution of the PDlhlgh / PDllowphenotypes differed fundamentally between the CD69hlghand CD69lowsubsets (compare 3rdand 4thcolumn of histograms). These findings are portrayed graphically in Fig. 9, panel F, wherein TGF-P and IL-2 caused increased frequencies of CD69hlghCD25lowTregs and a reciprocal loss of the opposite subset. The PDlhlghphenotype was enriched in the CD69hlghsubset across a wide range of IL-2 and TGF-P concentrations (Fig. 9, panel G). These data reveal two Treg subsets, including classical CD25hlghTregs as well as a distinct CD25lowCD69hlghsubset enriched for a PD1+subpopulation. These analyses did not distinguish whether high IL-2 / TGF-P concentrations elicited Treg differentiation or outgrowth of pre-existing subsets. One possibility is that high IL-2 concentrations saturated both low-affinity IL2RPy and high-affinity IL2RaPy receptors, thereby erasing the competitive advantage conferred by CD25 in CD25hlghTregs. An associated possibility is that CD25lowTregs may have competitive advantages that mediate reciprocal inhibition of CD25hlghTregs at high IL-2 concentrations. These data showed that the CD25hlghFOXP3+Treg population, which was dominant at low IL- 2 concentrations, bifurcated into qualitatively distinct CD25lowand CD25hlghpopulations when the culture was transitioned to high TGF-p / IL-2 concentrations.

[0129] High concentrations of IL-2 and TGF-fl caused dominance of CD25lowTregs even though CD25hlghTregs maintained an enhanced IL-2 signaling signature .

[0130] The CD25lowand CD25hlghpopulations were correlated with altered expression of several other Treg markers (Fig. 10), which were analyzed at designated IL-2 concentrations (10 pM to 10 nM) in the presence or absence of continued culture in 10 nM TGF-p. That is,these Treg lines were cultured long-term with TGF-P since day 0 (initiation of the line) and then were cultured for 7 days with or without TGF-P in the assay. Thus, experimental groups set up without TGF-P nonetheless may contain some TGF-P, because some exogenous TGF-P may carry over from the previous expansion culture into subsequent assay culture. Tregs may also synthesize bioactive TGF-P during culture. Thus, experimental groups set up with or without exogenous TGF-P (10 nM) likely compare high versus low levels of TGF-p. Importantly, Treg lines derived continuously in the presence of TGF-P may maintain a TGF- P-signature for prolonged durations well after TGF-P is withdrawn from the culture as part of a terminally differentiated phenotype.

[0131] Furthermore, high TGF-P concentrations may elicit differentiation. To assess the role of exogenously added TGF-P, we compared a control autologous Treg line derived in the absence of TGF-P to CD25-sorted and CD69-sorted Tregs derived in the presence of TGF-P (Fig. 14) Because TGF-P is known to elicitPDl expression, we analyzed several Treg markers (y-axis) versus PD1 expression (x-axis). These Treg lines expressed distinct subsets of pjjjnegative, pj-j j intermediate,an(j pp | bngin phenotypes. CD69-sorted Tregs exhibited a pronounced bias toward high PD1 expression including distinct poiintermediateand PDlbrightphenotypes. Conversely, CD25-sorted Tregs a strong bias toward a pDllow'negatlvephenotype, in that only a minority of CD25-sort Tregs expressed a PDlbrightphenotype. Control Tregs were predominantly PDlnegatlveand only 1-2% exhibited a PDlhlghphenotype (Fig. 14, panel B). Overall, these data reveal an associative linkage of TGF-P signaling, CD69 expression, and a PDlhlghphenotype. Derivation of Treg lines with high TGF-P-signaling also was needed for GITR expression and high levels of HLA-DR expression. As is evident from the dotplots (Fig. 14, panel A), the CD69-sorted poiintermediatesubset and the CD25 PDllowsubset expressed the BCL6 transcription factor, whereas in all three Treg lines, the PDlbrightphenotype was correlated with the lack of BCL6 expression. These findings provide suggestive evidence that the PDlbrightphenotype may demarcate a unique BCL6negatlveTreg subset. In CD69-sorted and CD25-sorted Tregs, the PDlbrightsubset was associated with the lack of BCL6, reduced levels of FOXP3, ICOS, and HLA-DR, a reduced cell size (lower FSC-H), together with elevated levels of CD69, GITR, and CD3. In control Tregs, the caveat was that the PDlbrightsubset only represented a small minority of cells, but these few Tregs expressed higher levels of FOXP3, CD69, CD25, ICOS, and CD3. However, the very low frequency of PDlhlghTregs in the control line may confer a lack intra-subset competition, which may engender a unique phenotype. In summary, the autologous TCR reactivity inherent to the human FOXP3+Treg lineage, coupled with continuous TGF-P signaling, enables continuous blastogenic growthwithout limitation in vitro. This strategy enables derivation of continuous FOXP3+Treg lines representing multiple Treg subsets, including CD4+and CD8+lineages, the canonical CD25hlghFOXP3+lineage, and additional Treg lineages bearing differential expression of CD25 and CD69 and the evolution of a unique PDlbrightphenotype.

[0132] These data presentations (Fig. 10, panels A-T) required different y-axis scales to appropriately show relative differences. One should note that many markers exhibited high fluorescence (MFI values > 104), including CD25, FOXP3, HLA-DR, CD86, CD3, CD4, FAS, and CD69. Other marker exhibited lower levels of fluorescence (MFI values < 104) including CTLA4, CD80, CD28, CD 127, PDL2, FASL, LAG3, GARP, and GITR. PD1 and CD69 were differentially expressed at high levels in the some but not other Treg lines and / or Treg subsets (Figs. 4, 5, 9, and 10), based on subset-specific differential pathways that are incompletely understood.

[0133] The qualitative distinction of CD25lowversus CD25hlghsubsets reflected large differences in CD25 expression (Fig. 10, panel B). Paradoxically, the CD25lowsubset had a competitive growth advantage compared to CD25hlghTregs at high concentrations of IL-2 and TGF-P as shown by increased CD25lowTreg percentages (Fig. 9 and Fig. 10, panel A). The paradox was that the CD25lowTregs had competitive growth advantage in high-zone IL-2 / TGF-P concentrations even though the CD25hlghsubset had a superior IL-2 signature. For example, CD25hlghTregs had larger cell sizes (FSC-A, Fig. 10, panel C) and higher expression of CTLA4 and FOXP3 (Fig. 10, panels D and E). The CD25hlghsubset also expressed higher levels of APC markers (HLA-DR, CD86, CD80) (Fig. 10, panels F-H), and T cell antigen recognition proteins (CD3, CD4) (Fig. 10, panels I and J). These data are consistent with the possibility that the CD25lowsubset might have slower growth rates coupled with lower levels of activation-induced cell death compared to the CD25hlghsubset. Hence, the relative growth rates versus death rates may balance to favor dominance of the CD25lowsubset at high TGF-p / IL-2 concentrations. In addition to IL-2 dependent upregulation of multiple markers, IL-2 also caused the downregulation of other markers. Both CD25hlghand CD25lowsubsets exhibited IL- 2 dependent downregulation of CD28, and this inhibitory mechanism was potentiated by TGF- P (Fig. 10, panel K). Both subsets also showed an IL-2 dependent inhibition of CD127 (IL7Ra) expression (Fig. 10, panel L), which was expected given that IL-2-stimulated FOXP3+Tregs exhibit a CD127lowphenotype. Overall, these data reveal the relative IL-2 dependent regulatory patterns among multiple canonical markers in the CD25hlghand CD25lowhuman Treg subsets. We also consider the possibility that the two Treg subsets may compete,such that purified subsets may exhibit altered properties in the presence or absence of the other subset.

[0134] A PD1 / PDL2 regulatory system was evident in these cultures. In both CD25lowand CD25hlghsubsets, TGF-P facilitated higher PD1 expression, which was pronounced in the CD25lowsubset (Fig. 10, panel M). These data support previous studies showing that TGF-P elicits PD1 expression (41, 42). In comparing two of our lab-derived Treg lines, PD1 expression in the CL6 Treg line (Fig. 9, panel E) was substantially higher than PD1 expression levels in the CL7 line (Fig. 10, panel M) (PD1 MFI values > 105compared to PD1 MFI values < 104, respectively). These data provided evidence that PD1 -mediated regulation may vary substantially among different Treg culture systems. Compared to CD25lowTregs, CD25hlghTregs expressed higher level of PDL2 by an IL-2 driven mechanism, although expression levels were relatively low (< 104MFI values). The differential MFI values obtained for PD1 versus PDL2 detection likely represented different expression levels, because both PD1 and PDL2 mAbs were coupled with bright fluorochromes (PE-Cy5 and PE-Cy7, respectively). PDL1 was not detected on these Treg subsets (APC-eFluor780, MIH1). Thus, TGF-P-dependent PD1 / PDL2 signaling pathways were present in these Treg cultures and are well-positioned as pivotal regulators of these Treg subsets.

[0135] FAS / FASL was another prominent regulatory system in that both CD25lowand CD25hlghsubsets expressed both FAS and FASL by IL-2 dependent mechanisms. The CD25hlghsubset expressed higher levels of FAS and FASL compared to the CD25lowsubset (Fig. 10, panels O and P). FAS was expressed at levels substantially higher than FASL, which was expressed at or slightly above detection levels (> 104MFI values for FAS versus < 2.5 x 103MFI values for FASL). Although high concentrations of IL-2 may facilitate FAS-induced death among CD25hlghTregs, at relatively low IL-2 concentrations (1 nM), these Tregs exhibited consistent blastogenic growth without evident cell death. Thus, we surmise that FAS was not generally engaged at lower IL-2 concentrations, probably because FASL levels were below the threshold needed to elicit FAS signaling. Nonetheless, FAS may represent a ‘kill’ switch that limits Treg survival in environments replete in FASL.

[0136] Several other Treg-associated markers were expressed by these Tregs, including LAG3, GARP, GITR, and CD69 (Fig. 10, panels Q-T). CD25hlghTregs expressed higher levels of LAG3 and GITR compared to the CD25lowsubset, and expression was largely driven by IL-2 rather than TGF-p. IL-2 also augmented GARP expression, particularly in the CD25lowsubset, whereas TGF-P either inhibited expression or blocked detection of GARP. CD69 also exhibited IL-2 driven expression in several subsets, including the TGF-P-cultured CD25lowsubset. Notably, the CL6 (Fig. 9) and CL7 (Fig. 10) lines had differences in precursor frequencies of the CD25lowCD69hlghPDlhlghsubset, such that high IL-2 / TGF-P concentrations enabled the emergence of this subset in the CL6 line during the 7-day assay culture in contrast to the CL7 line, where this CD25lowCD69hlghPDlhlghsubset was just beginning to emerge at the end of the 7-day assay culture. The implication is that the CD25lowsubset may encompass multiple phenotypes including distinct CD69hlgh / CD69lowand PDlhlgh / PDllowsubsets. Whether these phenotypes represent different lineages or distinct stages of differentiation is currently unknown.Discussion

[0137] This study revealed a novel in vitro Treg expansion strategy. TCR antigen recognition in the presence of TGF-P is well known to elicit the differentiation of FOXP3+Tregs, and this novel Treg expansion strategy couples autologous TCR recognition of culture-intrinsic autoantigens with continuous TGF-P signaling in long-term cultures. This strategy circumvented T cell exhaustion to sustain long-term proliferation (Figs. 4 and 5). These T cell lines had functional attributes of FOXP3+Tregs, including lack of IL-2 production, heightened consumption of IL-2, and in vitro suppressive activity (Fig. 6). Autologous TCR stimulation and TGF-P signaling were intertwined mechanistically because TGF-P sustained expression of MHCII on Tregs which enabled autologous CD4-dependent T cell-mediated antigen presentation in xeno / allo-free culture systems (Figs. 7 and 8). CD8-dependent MHCI / selfantigen recognition was implicated as a possible mechanism in the continuous exponential expansion of CD8+Tregs. Overall, this study supports the hypothesis that continuous interactions among Treg TCRs and MHCI and MHCII-restricted self-peptides on the same or neighboring Tregs sustained blastogenic Treg growth. This novel strategy offers advantages compared to established contemporary methods of Treg expansion, as noted in Table 3. (16- 25)

[0138] Starting Treg numbers and cell-based purification'.

[0139] A major advantage of autologous Treg expansion strategies is that these cultures were initiated with small samples of nonfractionated human PBMCs. Autologous Treg expansion strategies neither involved nor required Treg-based purification of CD127lowCD25hlghCD4+T cells. Thus, autologous Treg expansion methods will be feasible in a wide array of laboratory settings. Conversely, contemporary Treg expansion strategies require high starting Treg numbers to ensure that Treg purification strategies provide a sufficient starting cell yield to enable final protocol success. Treg purification is challenging because Tregs cannot be distinguished from activated effector T cells via surface markers, which results in contamination of Treg preparations with non-Tregs (24). Cell purification strategies involve flow cytometric or magnetic bead-based strategies that necessitate specialized facilities including FDA-approved instrumentation, procedures, and reagents (25). Thus, autologous Treg expansion strategies have significant feasibility advantages compared to other contemporary methods.

[0140] Genetic engineering'.

[0141] Technical limitations have impaired clinical development of Treg-based products, and the impetus for the CAR Treg field, at least in part, is to overcome these technical barriers via engineering solutions. However, without wishing to be bound by any particular theory, autologous Treg expansion strategies may bypass many of these technical challenges and thereby provide simpler alternative to CAR Treg / engineered Treg products. Alternatively,without wishing to be bound by any particular theory, autologous Treg expansion strategies may interface with CAR Treg applications as potential substrates for cell-based engineering.

[0142] Mitogenic expansion versus autologous expansion:

[0143] Many Treg expansion protocols use repeated mitogenic stimulation (anti-CD3 / anti- CD28 mAbs) to mimic native TCR / costimulation signals. However, without wishing to be bound by any particular theory, these activation signals likely exceed signaling intensities that physiologically delimit agonistic Treg thresholds, given that Tregs are selected in vivo within a bandwidth of TCR-signaling efficiencies defined by limited-efficiency agonistic recognition of self. Excessive TCR stimulation of Tregs in the presence of high IL-2 concentrations drive T cell exhaustion, emergence of ex-Tregs, and eventual overgrowth by non-Treg effector T cells. Conversely, autologous Treg expansion in vitro is physiologically matched to the same self-MHC peptide ligands and tuned to the same signaling intensities operative in vivo, thereby providing optimal TCR signaling with minimal T cell exhaustion.

[0144] Drugs that enhance Treg stability :

[0145] Many Treg manufacture strategies include agents such as rapamycin or all-trans retinoic acid that stabilize FOXP3 expression. However, we did not test whether these or similar agents would augment the efficiency of autologous Treg expansion. Only three ingredients were needed for autologous Treg expansion (xeno-free media, a T cell growth factor such as IL-2, and a regulatory cytokine such as TGF-P). In clinical development programs, simplicity is a virtue.

[0146] Autologous recognition of self MHC peptides:

[0147] Mitogens were used in initial experiments to jump-start Treg lines per the prevailing methodology of the field, but mitogens were not used thereafter during long-term Treg expansion. We subsequently showed mitogens were not needed at any phase during development of autologous Treg lines. Regarding use of mitogen, the main advantage was that mitogen accelerated establishment of Treg cultures. The main disadvantage was the concept that the initial mitogenic stimulation may contaminate the Treg pool with induced Treg clonotypes (not native to the existing Treg repertoire). Rather, the autologous Treg expansion strategy leverages intrinsic self-antigens within xeno-free culture systems to drive continual exponential expansion of pure primary FOXP3+Tregs. Theoretically, the self-MHC ligands driving Treg differentiation and survival in vivo (both before PBMC donation and after Tregadoptive immunotherapy) should extensively overlap the self-MHC ligands driving ex vivo Treg expansion because the Treg cells are the source of many self-peptides. Because these FOXP3+Tregs are expanded in vitro based on the same MHC ligands that drive in vivo survival and growth (6), we predict that Tregs derived by autologous expansion will exhibit long-term persistence in vivo because these Tregs will colonize their native Treg niche based on homeostatic recognition of a common set of self-peptides. In contrast, strategies based on repeated or continuous mitogenic stimulation or on artificial CAR Treg specificities cannot match the endogenous antigen specificities or the TCR signal strength needed to establish a self-preserving Treg niche in vivo. Thus, without wishing to be bound by any particular theory, autologous expansion protocols may have qualitative advantages in Treg survival after transfer into the recipient.

[0148] Dendritic cell as supplements for Treg expansion protocols '.

[0149] In autologous Treg expansion protocols, syngeneic DCs are not needed because Tregs express high levels of both MHCII, MHCI, CD80, and CD86 (along with various levels of PD- L1 and PD-L2) and thereby superimpose Treg and DC phenotypes. These TGF-P-induced Tregs can be considered professional APC because MHCII expression is constitutive and because the MFI values are exceedingly high even though the respective anti-HLA-DR antibody is coupled to a relatively dim fluorochrome (BUV395). Because FOXP3+Tregs appear fundamentally important for self-tolerance, one would presume that the dedicated APC activity of Tregs would also subserve a tolerogenic purpose. For example, as noted in this study but without wishing to be bound by any particular theory, the professional APC activity of Tregs appears critical for clonal expansion and maintenance of the Treg repertoire, at least as defined in these defined culture conditions. Thus, one contribution of this study is the novel concept that TGF-P-induced Tregs are tolerogenic DCs bearing a TCR.

[0150] The APC activity of Tregs has interesting implications. First, the dedicated APC activity of FOXP3+Tregs appears to license Tregs to survive and grow in environments deficient in myeloid DCs. Without wishing to be bound by any particular theory, a positive amplification mechanism based on autocrine or paracrine Treg APC activity would enable robust Treg expansion wherein Tregs become the dominant APC subset in a local tissue. This mechanism may underpin maintenance of tissue-resident memory Tregs and the imposition of dominant regional tolerance. Second, it is interesting to speculate that the conjunction of Treg and DC phenotypes may represent a direct coupling mechanism whereby TCR signaling may control functional attributes of antigen presentation and perhaps qualitative aspects of thepeptide repertoire. Thus, an important question is whether TCR clonotypic specificity influences the peptidome presented by a given Treg clonotype. Without wishing to be bound by any particular theory, just as the BCR controls the array of presented peptides presented by B cells to mediate antigen-specific B cell / T cell collaboration, the TCR may influence capture and presentation of self-peptides arrays by Tregs. Third, generation of tolerogenic DCs has long been sought as a means of adoptive cell-based therapy. However, this field has been hampered by the inability to generate sufficient cell doses, because DC maturation results in a cessation of growth. This study provides a potential solution in that TGF-P-cultivated Tregs are essentially lymphoid tolerogenic DCs that can be loaded with any peptide of choice and grown to high cell densities. Overall, MHC class II expression and T cell-mediated antigen presentation by F0XP3+Tregs may represent an important aspect of human Treg biology.

[0151] Expansion potential ofFOXP3+Tregs'.

[0152] A unique observation of this study was that TGF-P enabled continual IL-2-dependent growth without limitation by T cell exhaustion and death. IL-2 is well-known to drive blastogenic expansion, but eventually effector T cells exhibit exhaustion and death, and memory T cells revert to small CD25lowquiescent cells. This study provided evidence that TGF-P augmented MHCII expression to sustain autologous TCR stimulation, but this activity probably does not solely account for the continual exponential expansion. This study provided evidence that TGF-P was necessary for long-term expansion. Based on this observation, we postulate that TGF-P inhibited one or more mechanisms underlying activation-induced cell death because. Without wishing to be bound by any particular theory, one possibility is that TGF-P augmented FOXP3 expression levels, and FOXP3 is known to promote sustained protection from activation-induced death. TGF-P may also have inductive activities on other transcription factors such a BCL6 to promote sustained Treg growth.

[0153] F0XP3+purity.

[0154] Once established, these Treg lines expressed FOXP3 at nearly 100% frequencies without indication of instability. Without wishing to be bound by any particular theory, this observation provides evidence that culture conditions were compatible with FOXP3+Tregs but no other T cell lineages. The combination of xeno-free media, IL-2, and TGF-P appeared to be a selective media enabling Treg dominant outgrowth. This supposition fits well with the observation that T cell cultures were entirely FOXP3 Tregs without any initiating Treg purification. This study showed differential FOXP3 expression consistent with markers of theTreg differentiation pathways. CD25hlghTregs expressed the highest levels of F0XP3 whereas the CD69 PDlhigh Treg subset expressed lower levels of FOXP3. In fact, IL-2 concentration appeared to be a major controlling factor of F0XP3 expression.

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Claims

THAT WHICH IS CLAIMED IS:

1. A method of expanding regulatory T-cells (Tregs), the method comprising: a) providing a biological sample comprising Tregs; b) culturing the biological sample in vitro in a culture medium comprising a basal cell culture medium, a T cell growth factor, and a regulatory cytokine, to provide cultured Tregs; and c) passaging the cultured Treg population one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50 or more times) in a xeno-free culture medium comprising a basal cell culture medium, a T cell growth factor, and a regulatory cytokine, to provide expanded Tregs.

2. The method of claim 1, wherein the expanded Tregs are homogeneous Tregs.

3. The method of claim 1 or 2, wherein step b) and / or c) produce an autologous T cell reaction in the cultured and / or expanded Tregs.

4. The method of claim 3, wherein the autologous T cell reaction is a continuous autologous T cell reaction.

5. The method of any one of claims 1-4, wherein the biological sample is a blood sample or a tissue sample, e.g., umbilical blood, lymph nodes, thymic tissue.

6. The method of any one of claims 1-5, wherein the biological sample comprises peripheral blood mononuclear cells (PBMCs).

7. The method of any one of claims 1-6, wherein the cultured and / or expanded Tregs produce autologous T-cell peptides during step b) and / or c).

8. The method of any one of claims 1-7, wherein the xeno-free culture medium is devoid of added mitogens (e.g., exogenous mitogens) and / or antigens.

9. The method of any one of claims 1-8, wherein the culture medium in step b) and / or xeno-free culture medium in step c) is devoid of rapamycin, retinoic acid, and / or dendritic cells.

10. The method of any one of claims 1-9, wherein the culture medium and / or xeno-free culture medium consists of a basal cell culture medium, a T cell growth factor, and a regulatory cytokine.

11. The method of any one of claims 1-10, wherein the T cell growth factor is interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 15 (IL- 15), interleukin 21 (IL-21), or any combination thereof.

12. The method of any one of claims 1-11, wherein the regulatory cytokine is transforming growth factor P 1 (TFG-pi), transforming growth factor P 2 (TFG-P2), transforming growth factor P 3 (TFG-P3), interleukin 10 (IL- 10), or any combination thereof.

13. The method of any one of claims 1-12, wherein the T cell growth factor is present in the culture medium in a concentration from about 0.001 nM to about 100 nM (e.g., about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nM) and the regulatory cytokine is present in the culture medium in a concentration from about 1 nM to about 100 nM (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nM).

14. The method of any one of claims 1-13, wherein the basal cell culture medium comprises LymphoOne™ T-Cell Expansion Medium, X-Vivo 15™ Serum -free Hematopoietic Cell Medium, CTS™ OpTmizer™ T Cell Expansion SFM, CelThera™ GMP T Cell Expansion Medium, ImmunoCult™-XF T Cell Expansion Medium, ExCellerate Human T Cell Expansion Media, CellGenix® GMP T Cell Medium, 4Cell® Nutri-T GMP Media, and / or ArtMedia® Human T Cell Serum -Free Medium.

15. The method of any one of claims 1-14, wherein the culture medium used in step b) further comprises a mitogen (e.g., phytohaemagglutinin (PHA)).

16. The method of any one of claims 1-15, wherein the culture medium used in step b) and / or the xeno-free culture medium used in step c) further comprises granulocyte-macrophage colony-stimulating factor (GM-CSF).

17. The method of claim 16, wherein the culture medium used in step b) and / or the xeno- free culture medium used in step c) further comprises IL-4.

18. The method of any one of claims 1-17, wherein the expanded Tregs express forkhead box P3 protein (FOXP3), cluster of differentiation 4 protein (CD4), cluster of differentiation 8 protein (CD8), cluster of differentiation 69 protein (CD69), programmed cell death protein 1 (PD1), IL-2 receptor a protein (CD25), cytotoxic T-lymphocyte associated protein 4 (CTLA4), glucocorticoid-induced TNFR-related protein (GITR), or any combination thereof.

19. The method of any one of claims 1-18, wherein the T cell growth factor is present in the culture medium in a concentration from about 0.001 nM to about 1 nM (e.g., about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 nM).

20. The method of any one of claims 1-18, wherein the T cell growth factor is present in the culture medium in a concentration from about 10 nM to about 100 nM (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nM).

21. The method of any one of claims 1-20, wherein the expanded Tregs express major histocompatibility complex class I (MHC1) molecules (e.g., HLA-A, HLA-B, HLA-C, HLA- E, HLA-F, HLA-G, HLA-K, and / or HLA-L) and major histocompatibility complex class II (MHC2) molecules (e g., HLA-DP, HLA-DQ, and / or HL A-DR).

22. The method of any one of claims 1-21, wherein the expanded Tregs do not become exhausted Tregs during step c).

23. The method of claim 22, wherein the presence of exhausted Tregs is indicated when 10% or more (e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the Tregs in the expanded Tregs express high levels of PD1.

24. The method of claim 22, wherein the presence of exhausted Tregs is indicated when the expanded Tregs do not exhibit exponential growth between the one or more passages of step c).

25. The method of any one of claims 1-24, wherein the expanded Tregs are passaged for about 1 week or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more).

26. The method of any one of claims 1-25, wherein the expanded Tregs are expanded 2- fold or more (e.g., 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 30-, 40-, 50-, 100-, 200-, 300-, 400-, 500-, 1000-fold or more) compared to the isolated Tregs.

27. The method of any one of claims 1-26, wherein the method does not include genetic engineering of the Tregs.

28. A population of Tregs produced by the method of any one of claims 1-27.

29. The population of Tregs of claim 28, wherein the Tregs are autologous Tregs.

30. A method of treating an immune disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs of claim 28 or 29 to the subject, thereby treating the immune disorder.

31. The method of claim 30, wherein the immune disorder is an autoimmune disorder.

32. The method of claim 30 or 31, wherein the immune disorder comprises type I diabetes, arthritis (e.g., rheumatoid arthritis, juvenile chronic arthritis, erosive osteoarthritis, or psoriatic arthritis), Celiac disease, scleroderma, psoriasis, autoimmune myopathy, psoriasis, CREST syndrome, inflammatory myositis, mixed connective tissue disease, palindromic rheumatism, eosinophilic fasciitis, dermatomyositis, calcium pyrophosphate crystal deposition disease, acute respiratory distress syndrome, osteoporosis, delayed hypersensitivity, autoimmune thyroiditis, Hashimoto's disease, Sjogren's disease, Grave's disease, primary biliary cirrhosis, idiopathic thrombocytopenic purpura, leucopenia, thrombus formation, or lupus (e.g., systemic lupus erythematosus or lupus nephritis).

33. A method of treating an inflammatory disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs of claim 28 or 29 to the subject, thereby treating the inflammatory disease.

34. The method of claim 33, wherein the inflammatory disease is a chronic inflammatory disease.

35. The method of claim 33 or 34, wherein the inflammatory disease comprises ulcerative colitis, Crohn's disease, pulmonary inflammation, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), retinitis, uveitis, kidney disease, obesity, spondyloarthritis, or vasculitis.

36. A method of treating a pathogen infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs of claim 28 or 29 to the subject, thereby treating the pathogen infection.

37. The method of claim 36, wherein the pathogen infection comprises influenza virus, herpes simplex virus, Epstein-Barr virus, cytomegalovirus, adenovirus, respiratory syncytial virus, coronavirus, BK virus, JC virus, streptococcal pharyngitis, tuberculosis, bacterial gastroenteritis, cellulitis, tetanus, pneumonia, Clostridioides difficile, Lyme disease, Rocky Mountain spotted fever (RMSF), meningitis, conjunctivitis, Methicillin-resistant Staphylococcus aureus, gonorrhea, bacterial sinusitis, bacterial vaginosis, giardia, Chagas disease, malaria, toxoplasmosis, cryptosporidiosis, cyclosporiasis, cysticercosis, hookworm, trypanosomiasis, echinococcosis, or any combination thereof.

38. A method of treating an allergic disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs of claim 28 or 29 to the subject, thereby treating the allergic disease.

39. The method of claim 38, wherein the allergic disease comprises allergic rhinitis, allergic asthma, atopic dermatitis, eczema, or any combination thereof.

40. A method of treating a transplantation rejection disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs of claim 28 or 29 to the subject, thereby treating the transplantation rejection disease.

41. The method of claim 40, wherein the transplantation rejection disease comprises graft- versus host disease, solid organ rejection, or any combination thereof.

42. A method of treating a neurodegenerative disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs of claim 28 or 29 to the subject, thereby treating the neurodegenerative disease.

43. The method of claim 42, wherein the neurodegenerative disease comprises Alzheimer’ s disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, frontotemporal dementia, Lewy body dementia, Huntington's disease, Pick’s disease, multisystem atrophy, progressive supranuclear palsy, inclusion body myositis, prion protein cerebral amyloid angiopathy, argyrophilic grain disease, tangle predominant dementia, chronic traumatic encephalopathy, traumatic brain injury, or any combination thereof.

44. A method of treating a metabolic disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs of claim 28 or 29 to the subject, thereby treating the metabolic disease.

45. The method of claim 44, wherein the metabolic disease comprises type II diabetes, obesity, metabolic syndrome, or any combination thereof.

46. A method of treating an aging-related disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs of claim 28 or 29 to the subject, thereby treating the aging-related disease.

47. The method of claim 46, wherein the aging-related disease comprises inflammaging, hypertension, atherosclerosis, nonalcoholic fatty liver disease, macular degeneration, or any combination thereof.

48. A method of treating a cardiovascular disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the Tregs of claim 28 or 29 to the subject, thereby treating the cardiovascular disease.

49. The method of claim 48, wherein the cardiovascular disease comprises myocardial infarction, stroke, chronic heart disease, arteriosclerosis, coronary artery disease, heart failure, abnormal heart rhythm or arrhythmia, aorta disease, Marfan syndrome, cardiomyopathy, carotid artery disease, peripheral artery disease, or any combination thereof.

50. The method of any one of claims 30-49, wherein administering the Tregs comprises administering 109Tregs or more (e.g., 109, IO10, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, IO20, or more Tregs).

51. The method of any one of claims 30-50, wherein administering the Tregs comprises administering one or more doses of Tregs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses).

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