PREVENTION AND TREATMENT OF GRAFT-VERSUS-HOST DISEASE (GVHD)

PRIORITY CLAIM

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/119,919, filed December 1 , 2020, which is incorporated herein by reference in its entirety, including drawings.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Numbers R01 AI066008 and R01 CA228465, awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] The success of allogeneic hematopoietic cell transplantation (Allo-HCT) for treatment of hematologic malignancies depends partly on the ability of donor T cells to eliminate residual malignant cells in the recipient after the pre-transplant conditioning, but the same donor T cells also mediate graft-versus-host-disease (GVHD) [1], Prevention of GVHD in patients with immunosuppressants also suppresses graft-versus-leukem ia (GVL) activity [2-4], Development of approaches that prevent GVHD while preserving GVL activity remains a long-sought goal [5-8],

[0004] Interactions of PD-L1 with PD-1 and CD80 on activated T cells have an important role in regulating immune responses [9-11], Tumor cell PD-L1 interaction with PD-1 on activated T cells tolerizes anti-tumor T cells and prevents anti-tumor immunity [12], Similarly, recipient tissue PD-L1 interactions with PD-1 and CD80 on alloactivated donor CD8 ⁺ T cells markedly reduce GVHD severity, although this mechanism is not effective when the graft contains both CD4 ⁺ and CD8 ⁺ T cells [13], One possible reason is that IL-2 produced by CD4 ⁺ T cells could prevent tolerance induction by PD-1 signaling [14], although previous studies showed that administration of high-dose IL-2 early after Allo-HCT prevents acute GVHD (aGVHD) while preserving GVL activity [15],

[0005] Regulatory T cells including FoxP3 ⁺ Treg and FoxP3-IL-10 ⁺ Tr1 cells play important roles in ameliorating aGVHD [16-22], PD-L1 interaction with PD-1 augments conversion of activated T cells into Foxp3 ⁺ Treg cells [23], In the pathogenesis of aGVHD, most regulatory T cells are Tr1 cells that require Eomes fortheir development [17], Although PD-L1/PD-1 interaction upregulates expression of Eomes and Blimp-1 during induction of anergy and exhaustion of T effector cells, the role of PD-L1 on Tr1 cell development remains unclear. In addition, persistence of donor CD8 ⁺ T cell- induced GVHD was mediated by CD8 ⁺ T memory progenitors (Tmp) [24] that play a critical role in tumor immunity [25],

[0006] Accordingly, there is a need to develop an approach that effectively prevents graft-versus-host disease (GVHD) while preserving strong graft-versus- leukemia (GVL) activity in patients. This disclosure satisfies the need in the art.

SUMMARY

[0007] This disclosure is directed to a method of preventing or treating GVHD while preserving GVL activity in vivo in a subject receiving a hematopoietic cell transplantation (HCT). The method entails administering to the subject an effective amount of an anti-IL-2 antibody. In certain embodiments, the subject receives an allogeneic HCT. In certain embodiments, the GVHD is acute GVHD. In certain embodiments, the anti-IL-2 antibody augments IL-2 binding to IL-2Ra and blocks IL-2 binding to IL-2R|3 in vivo. In certain embodiments, the anti-IL-2 antibody is a monoclonal antibody. In certain embodiments, the anti-IL-2 antibody is a recombinant antibody. In certain embodiments, the anti-IL-2 antibody is a human antibody. In certain embodiments, the anti-IL-2 antibody is a humanized antibody. In certain embodiments, the anti-IL-2 antibody is anti-IL-2-JES6 antibody. In certain embodiments, the anti-IL-2 antibody is administered to the subject on the same day of receiving HCT. In certain embodiments, the anti-IL-2 antibody is administered to the subject after receiving HCT. In certain embodiments, multiple doses of the anti-IL-2 antibody are administered after HCT. In certain embodiments, a single dose of the anti-IL-2 antibody is administered each day. In certain embodiments, the anti-IL-2 antibody is administered every other day for a week, for two weeks, for three weeks, or for a month after HCT. In certain embodiments, the subject is human.

[0008] In a related aspect, disclosed herein is a composition for use in preventing or treating GVHD while preserving GVL activity in a subject receiving a hematopoietic cell transplantation (HCT). The composition comprises an effective amount of an anti- IL-2 antibody. In certain embodiments, the subject receives an allogeneic HCT. In certain embodiments, the GVHD is acute GVHD. In certain embodiments, the anti-IL- 2 antibody augments IL-2 binding to IL-2Ra and blocks IL-2 binding to IL-2R|3 in vivo. In certain embodiments, the anti-IL-2 antibody is a monoclonal antibody. In certain embodiments, the anti-IL-2 antibody is a recombinant antibody. In certain embodiments, the anti-IL-2 antibody is a human antibody. In certain embodiments, the anti-IL-2 antibody is a humanized antibody. In certain embodiments, the anti-IL-2 antibody is anti-IL-2-JES6 antibody. In certain embodiments, the composition comprising the anti-IL-2 antibody is administered to the subject on the same day of receiving HCT. In certain embodiments, the composition comprising the anti-IL-2 antibody is administered to the subject after receiving HCT. In certain embodiments, multiple doses of the composition comprising the anti-IL-2 antibody are administered after HCT. In certain embodiments, a single dose of the composition comprising the anti-IL-2 antibody is administered each day. In certain embodiments, the composition comprising the anti-IL-2 antibody is administered every other day for a week, for two weeks, forthree weeks, or for a month after HCT. In certain embodiments, the subject is human.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 shows that tolerogenic anti-IL-2 mAb (JES6-1 ) prevents acute GVHD while preserving strong GVL activity. After administration of tolerogenic anti- IL-2 mAb (JES6-1 ) that forms anti-IL-2/IL-2 complexes that augment IL-2 binding to IL-2Ra but block IL-2-binding to IL-2R|3, activated donor T cells increase expression of PD-1 and Tcf1 (Ly 108) and decrease production of GM-CSF. Some donor CD8 ⁺ T cells differentiate into PD-1 ^hi Ly108 ⁺ T effector memory (Tern) progenitors. In the lymphoid tissues, the Tern progenitors and their derivatives Teff are well preserved due to lack of PD-1 interaction with PD-L1 expressed by host-tissues, leading to strong GVL activity. In contrast, in the GVHD target tissues, plenty of host-tissue PD-L1 interaction with PD-1 on donor T cells results in depletion of GM-CSF ⁺ Th1/Tc1 as well as expansion of Foxp3 ⁺ Treg and Foxp3- Tr1 cells, leading to effective prevention of acute GVHD.

[0010] Figures 2A-2G show that tolerogenic anti-IL-2 mAb (JES6-1A12) but not non-tolerogenic anti-IL2 mAb (S4B6) prevents acute GVHD and preserves GVL activity more effectively than tacrolimus. Figures 2A-2D: Lethally irradiated WT BALB/c recipients were given splenocytes (5x10 ⁶ ) with or without TCD-BM (2.5x10 ⁶ ) from WT C57BL/6 donors. Recipients were given a total of 4 i.p. injections of rat-IgG, anti-IL-2 mAb (JES6-1A12) or anti-IL-2 mAb (S4B6) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT. Figures 2A and 2C: Plots of % Original Body weight, diarrhea and % survival are shown. n=7-8 combined from 2 replicated experiments. Figures 2B and 2D: Mean ± SEM of histopathology scores of liver, small intestine and colon are shown; n=4 per group. Combined from 2 replicated experiments. Figure 2E: WT BALB/c recipients given splenocytes and TCD-BM cells from WT C57BL/6 donors and injected IgG or anti-IL-2 mAb (JES6-1A12) as described in Figures 2A-2D. Recipients were challenged with i.p. injection of BCLIZLuc cells (5x10 ⁶ /mouse) on day 0. Mice were monitored for tumor growth using in vivo bioluminescent imaging (BLI), clinical signs of GVHD and survival. One representative BLI image from each time point is shown for each group and summary of photons/sec, diarrhea and tumor-free survival of recipients. n=8 combined from 2 replicated experiments. Figure 2F: Lethally irradiated WT BALB/c recipients were given splenocytes (2.5x10 ⁶ ) and TCD-BM (2.5x10 ⁶ ) from WT C57BL/6 donors. Recipients were challenged with i.p. injection of BCLI/Luc cells (5 x 10 ⁶ /mouse) on day 0 and were given a total of 4 i.p. injections anti-IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT, or daily i.p. injections of tacrolimus (0.75 mg/Kg) at days 0-21 after HCT. One representative BLI image from each time point is shown for anti-IL-2 mAb (JES6-1A12) and tacrolimus group and summary of photons/sec, body weight change, as well as the tumor free survival of recipients. n=8-10 combined from 2 replicated experiments. Figure 2G: Lethally irradiated BALB/c recipients were given splenocytes (1.25x10 ⁶ ) and TCD-BM (2.5x10 ⁶ ) from C57BL/6 donors. Recipients were challenged with i.p. injection of BCLI/Luc cells (10x10 ⁶ /mouse) on day 0 and were given a total of 4 i.v. injections IL-2 mAb (500 pg/mouse) at days 0, 2, 4 and 6 after HCT, or i.p. injections of tacrolimus (0.75 mg/Kg) once daily until moribund with tumor growth. One representative BLI image from each time point is shown for IL-2 mAb (JES6-1A12) and tacrolimus group and summary of photons/sec and tumor-free survival of recipients. n=8-10 combined from 2 replicated experiments. “+” indicates death. Data represent mean ± SE. Pvalues were calculated by ordinary one-way ANOVA (Figures 2B & 2D), two-tailed Student t tests (Figures 2F & 2G) or log-rank test for survival comparison (Figures 2A, 2E, 2F and 2G) (*p<0.05, ** p<0.01 , *** p<0.001 , **** p < 0.0001).

[0011] Figures 3A and 3B show prevention of aGVHD by tolerogenic anti-IL-2 mAb (JES6-1A12) but not by non-tolerogenic anti-IL-2 mAb (S4B6). Lethally irradiated WT BALB/c recipients were given splenocytes (5x10 ⁶ ) and TCD-BM (2.5x10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 4 i.v. injections of control IgG, anti- IL-2 mAb (JES6-1A12) or anti-IL-2 mAb (S4B6) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT. At 7 days after H CT, histopathology of liver, small intestine and colon was evaluated. A representative photomicrograph is shown, original magnification x100 (liver), x 200 (small intestine and colon).

[0012] Figures 4A and 4B show that tolerogenic anti-IL-2 mAb (JES6-1A12) preserves GVL activity against BC-CML tumor cell. Lethally irradiated C57BL/6 recipients were given TCD-BM (2.5x10 ⁶ ) with or without splenocytes (2.5x10 ⁶ ) from A/J donors. Recipients were challenged with i.v. injection of GFP+ BC-CML cells (1x10 ⁶ /mouse) at day 0 and were given at total of 4 i.v. injections anti-IL-2 mAb (JES6- 1A12) (500 pg/mouse) or control IgG at days 0, 2, 4 and 6 after HCT. Figure 4A: Plots of % Original body weight, % mice without diarrhea and % survival. n=8 from two replicate experiments. Figure 4B: Moribund mice with or without GVHD during observation and mice at day 40 after HCT were checked for BC-CML tumor cells in the spleen, liver and bone marrow. Representative flow cytometry patterns and percentages of BC-CML cells in the spleen, liver and bone marrow from each group are shown. n=6 combined from duplicated experiments. Data represent mean ± SE. Nonlinear regression (curve fit) was used for body weight comparisons. Log-rank test was used for survival comparisons. 2-way ANOVA was used for BC-CML cell comparison (*p<0.05, ** p<0.01 , ****p<0.0001 ).

[0013] Figures 5A-5D show that tolerogenic anti-IL-2 mAb (JES6-1A12) augments NK cells expansion. Figures 5A-5B: Lethally irradiated WT BALB/c recipients were given splenocytes (5x10 ⁶ ) and TCD-BM (2.5x10 ⁶ ) from WT C57BL/6 donors. Recipients were given a total of 4 i.v. injections of control IgG or anti-IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT. At day 6 after HCT, NK cells were enumerated in the spleen and liver. Representative flow cytometry pattern (Figure 5A), percentage and yield of donor NK cells are shown (Figure 5B), n=4-6 from 2 replicates. Figures 5C-5D: WT BALB/c recipients were engrafted with splenocytes and TCD-BM cells from WT C57BL/6 donors as described in Figure 2D. Recipients were given a total of 4 i.v. injections of anti-IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6 with anti-NK1.1 (200ug/mouse) or control IgG once a week after HCT. Figure 5C: Day 24 after HCT, NK cells were enumerated in the spleen and liver from recipients given anti-NK1.1 treatment. Representative flow cytometry patterns are shown. Figure 5D: Representative BLI image from each time point of one experiment is shown for each group. Summaries of photons/second and % survival of recipients are shown. n=7 combined from duplicated experiments. Data represent mean ± SE. P values were calculated by unpaired two-tailed Student t test (* p<0.05, ** p<0.01).

[0014] Figures 6A-6B show that tolerogenic anti-IL-2 mAb (JES6-1A12) prevents aGVHD and preserves GVL activity more effectively than tacrolimus. Lethally irradiated BALB/c recipients were given splenocytes (1.25x10 ⁶ ) and TCD-BM (2.5x10 ⁶ ) from C57BL/6 donors. Recipients were challenged with i.p. injection of BCL1/Luc cells (5x10 ⁶ /mouse) at day 0 and were given a total of 4 i.v. injections anti- IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT or i.p. injections of tacrolimus (0.75 mg/Kg) daily until moribund with tumor growth. Figure 6A: Representative BLI image from each time point of one experiment is shown for IL-2 mAb and tacrolimus group. Figure 6B: Summary of photons/second and survival curve of recipients are shown. n=10 combined from duplicated experiments. Data represent mean ± SE. P values were calculated by log-rank test (*p<0.05).

[0015] Figures7A-7J show that prevention of aGVHD by tolerogenic anti-IL-2 mAb (JES6-1A12) requires PD-L1 expression by GVHD target tissues. Lethally irradiated WT or PD-L - BALB/c recipients were given splenocytes (2.5x10 ⁶ ) and TCD-BM (2.5x10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 4 i.p. injections of rat-IgG, anti-IL-2 mAb (JES6-1A12) or anti-IL-2 mAb (S4B6) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT. Recipients given TCD-BM cells (2.5x10 ⁶ ) alone were used as controls. Figure 7A: Plots of % original body weight, diarrhea and % survival are shown. n=8 per group. Combined from 2 replicated experiments. Figure 7B: Mean ± SEM of histopathology scores of liver, small intestine and colon are shown; n=4 per group. Combined from 2 replicated experiments. Figures 7C-7F: At day 6 after HCT, spleen, liver and colon of recipients were harvested for analysis of donor CD4 ⁺ and CD8 ⁺ T cell percentage and yield. Mean ± SEM of the percentage and yield of H-2K ^b+ TCR[3 ⁺ CD4 ⁺ or CD8 ⁺ T cells are shown; n=4-11 per group. Combined from two to three replicated experiments. Figures 7G-7I: Day 6 post HCT, percentage of Eomes ⁺ PD1 ⁺ cells among donor CD4 ⁺ and CD8 ⁺ T cells in liver and colon of WT or PD-L1- ⁷ - recipients treated with anti-IL-2 mAb (JES6-1A12), anti-IL-2 mAb (S4B6) or control IgG; n=4-5 per group. Figure 7J: Day 6 post HCT, percentage of Eomes ⁺ PD1 ⁺ cells among donor CD4 ⁺ and CD8 ⁺ T cells in liver and colon of WT and PD-LT ⁷ - recipients treated with anti-IL-2 mAb (JES6-1A12); n=4 per group. Data represent mean ± SE combined from two replicated experiments. P values were calculated by log-rank test (Figure 7A) or one-way ANOVA (Figure 7B) or 2-way ANOVA (Figures 7C-7J) (*p<0.05, **p<0.01 , ***p<0.001 , **** p < 0.0001 ).

[0016] Figure 8 shows that prevention of aGVHD by tolerogenic anti-IL-2 mAb (JES6-1A12) requires PD-L1 expression by GVHD target tissues. Lethally irradiated WT or PD-L - BALB/c recipients were given splenocytes (5x10 ⁶ ) and TCD-BM (2.5x10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 4 i.v. injections of control IgG or anti-IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT. At 7 days after HCT, histopathology of liver, small intestine and colon was evaluated. A representative photomicrograph is shown, original magnification x100 (liver), x 200 (small intestine and colon).

[0017] Figures 9A-9D show that prevention of GVHD by tolerogenic anti-IL-2 mAb (JES6-1A12) but not non-tolerogenic anti-IL-2 mAb (S4B6) is associated with tissue PD-L1 -dependent depletion of GM-CSF-producing Th1 and Tc1 cells. Lethally irradiated WT and PD-LT ⁷ - BALB/c recipients were given splenocytes (2.5 x 10 ⁶ ) and TCD-BM (2.5 x 10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 3 i.p. injections of rat-IgG, anti-IL-2 mAb (JES6-1A12) or anti-IL-2 mAb (S4B6) (500 pg/mouse) at days 0, 2, and 4 after HCT. On day 6, donor cells in the spleen, liver and colon were analyzed for cytokine profile. Figures 9A-9B: Percentage and yield of GM-CSF ⁺ cells among donor IFN-y ⁺ CD4 ⁺ and CD8 ⁺ T cells in spleen, liver and colon of WT recipients treated with anti-IL-2 mAb (JES6-1A12 or S4B6) or control IgG. Figure 9C: Percentage and yield of GM-CSF ⁺ cells among donor IFN-y ⁺ CD4 ⁺ and CD8 ⁺ T cells in spleen, liver and colon of PD-L1- ⁷ - recipients treated with anti-IL-2 mAb (JES6-1A12) or control IgG; n=4 per group. Figures 9D: Percentage and yield of GM- CSF ⁺ cells among donor IFN-y ⁺ CD4 ⁺ and CD8 ⁺ T cells in spleen, liver and colon of WT or PD-L - recipients treated with anti-IL-2 mAb (JES6-1A12); n=5 per group. Data represent mean ± SE combined from two replicate experiments. P values were calculated by unpaired two-tailed Student t tests (*p<0.05, ** p<0.01 , ***p<0.001 , **** p < 0.0001 ).

[0018] Figure 10 shows representative flow cytometry patterns of Figure 9A.

[0019] Figure 11 shows representative flow cytometry patterns of Figure 9B.

[0020] Figure 12 shows representative flow cytometry patterns of Figure 9C.

[0021] Figure 13 shows representative flow cytometry patterns of Figure 9D.

[0022] Figures 14A-14J show that tolerogenic anti-IL-2 mAb (JES6-1A12) results in the reduction of donor myeloid cells. Lethally irradiated WT BALB/c recipients were given splenocytes (2.5x10 ⁶ ) and TCD-BM (2.5x10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 4 i.v. injections of control IgG or anti-IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6. At day 12 after HCT, cells from the spleen (SPL), liver and colon were analyzed by flow cytometry. Figures 14A-14B: Example gating (14A) and quantification of myeloid cell populations (14B) in the liver. Figures 14C- 14D: Example gating (14C) and frequency of pro-IL-1 |3-producing neutrophils and monocytes within the H2Kb ⁺ CD45 ⁺ population from liver (14D). Figures 14E-14F: Example gating (14E) and frequency of pro-IL-1 |3-producing neutrophils and monocytes within the H2Kb ⁺ CD45 ⁺ population from colon (14F). Figures 14G-14H: Example gating (14G) and frequency of pro-IL-1 |3-producing neutrophils and monocytes within the H2Kb ⁺ CD45 ⁺ population from spleen (14H). Figures 14I-14J: Flow cytometric analysis of ROS (CellROX reagent) in the spleen showing representative histograms (141) and quantification of median fluorescence intensity (MFI) for neutrophils, monocytes, and MDCs (14J). Data represent two experiment with n = 4 to 6 per group. Data represent mean ± SE. P values were calculated by unpaired two-tailed Student t tests (*p < 0.05, **p < 0.01 ).

[0023] Figures 15A-15C show that tolerogenic (JES6-1A12) and non-tolerogenic (S4B6) anti-IL-2 mAb exhibit different impact on Foxp3 ⁺ Treg cells. Lethally irradiated WT (Figures 15A-15B) or PD-LT ⁷ - BALB/c (Figure 15C) recipients were given splenocytes (2.5 x 10 ⁶ ) and TCD-BM (2.5 x 10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 3 i.v. injections of rat-IgG or 500 pg/mouse anti-IL-2 mAb (JES6- 1A12) (15A) or anti-IL-2 mAb (S4B6) (15B) at days 0, 2, and 4 after HCT. At day 6 after HCT, FoxP3 ⁺ Treg cells were measured in the spleen, liver and colon. n=4-5 per group. Combined from two experiments. Data represent mean ± SE. P values were calculated by 2-way ANOVA (* p < 0.05, **p < 0.01 ).

[0024] Figures 16A-16G show that prevention of acute GVHD by tolerogenic anti- IL-2 mAb (JES6-1A12) but not non-tolerogenic anti-IL-2 mAb (S4B6) requires PD-L1- dependent expansion of Trl cells. Figures 16A-16E: Lethally irradiated WT or PD- LT ^Z - BALB/c recipients were given splenocytes (2.5 x 10 ⁶ ) and TCD-BM (2.5 x 10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 3 i.p. injections of rat-IgG or anti-IL-2 mAb (JES6-1A12 or S4B6) (500 pg/mouse) at days 0, 2, and 4 after HCT. Day 6 after HCT, donor-type T cells from live and colon were analyzed for Tr1 cells. Figures 16A-16B: Representative staining pattern with percentage and yield of donor IL-10 ⁺ Foxp3- CD4 ⁺ T cells in liver and colon of WT recipients treated with anti-IL-2 mAb (JES6-1A12 or S4B6) or control IgG; n=5 per group. Figure 16C: Representative staining pattern with percentage and yield of donor IL-10 ⁺ Foxp3- CD4 ⁺ T cells in liver and colon of PD-L - recipients treated with anti-IL-2 mAb (JES6-1A12) or control IgG; n=4 per group. Figures 16D-16E: Blimp-1 and Eomes expression on of donor CD4 ⁺ T cells in liver and colon of WT recipients treated with anti-IL-2 mAb (JES6-1A12 or S4B6) or control IgG; n=4 per group. Figure 16F: Blimp-1 and Eomes expression on of donor CD4 ⁺ T cells in liver and colon of PD-LT ⁷ - recipients treated with anti-IL-2 mAb (JES6-1A12) or control IgG; n=4 per group. Figure 16G: Lethally irradiated WT BALB/c recipients were given T cells (1.0 x10 ⁶ ) from WT or IL-1 Q- ¹ - C57BL/6 donors and TCD-BM (2.5 x 10 ⁶ ) from WT C57BL/6 donors. Recipients were given a total of 4 i.p. injections of anti-IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT, plots of % original body weight, diarrhea and survival are shown. n=8 per group. Data represent mean ± SE combined from two replicate experiments. P values were calculated by 2way ANOVA (Figures 16A-16F) or log-rank test (Figure 16G) (*p<0.05, ** p<0.01, ***p<0.001 , ****p < 0.0001 ).

[0025] Figure 17 shows that H2Kb ⁺ TCR|3 ⁺ IL-10 ⁺ FoxP3- CD4 ⁺ Tr1 cells are IFN- y ⁺ but GM-CSF-. One representation is shown from four replicate experiments.

[0026] Figure 18 shows that IL-10 deficiency in donor T cells does not affect the percentages or yields of GM-CSF ⁺ Th/Tc1 cells. Lethally irradiated WT BALB/c recipients were given T cells (1.0 x10 ⁶ ) from WT or IL-10- ⁷ - C57BL/6 donors with TCD- BM (2.5 x 10 ⁶ ) from WT C57BL/6 donors. On day 6 after HOT, GM-CSF+ Th/Tc1 cells were enumerated in the spleen, liver and colon by flow cytometry. % and yield of GM- CSF+ Th/Tc1 are shown. n=4 per group. Data represent mean ± SE combined from two replicate experiments.

[0027] Figure 19 shows that tolerogenic anti-IL-2 mAb (JES6-1A12) decreases the percentage of donor G-MDSC in the liver. Lethally irradiated WT BALB/c recipients were given splenocytes (2.5 x 10 ⁶ ) and TCD-BM (2.5 x 10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 4 i.v. injections of control IgG or anti-IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6. At day 12 after HCT, cells from the spleen (SPL), liver and colon were analyzed by flow cytometry. Percentages of G-MDSC and M-MDSC are shown. n=4, data represent mean ± SE combined from two replicate experiments. P values were calculated by 2-way ANOVA (**p<0.01).

[0028] Figures 20A-20E show that prevention of GVHD by tolerogenic anti-IL-2 mAb (JES6-1A12) but not non-tolerogenic anti-IL-2 mAb (S4B6) requires expression of PD-L1 by GVHD target tissues to inhibit activation of IL-2-Stat-5 and AKT-mTOR pathways in donor T cells. Lethally irradiated WT and PD-L1 ⁷ - BALB/c recipients were given splenocytes (2.5 x 10 ⁶ ) and TCD-BM (2.5 x 10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 3 i.v. injections of rat-IgG or anti-IL-2 mAb (JES6-1A12 or S4B6) (500 pg/mouse) at days 0, 2, and 4 after HCT. At day 6 after HCT, spleen and colon were harvested for analysis. Figure 20A: Representative GSEA plots are shown of IL-2-STAT5 pathway-related gene sets expression on CD4 ⁺ T and CD8 ⁺ T cells in the spleen or colon of the WT recipients treated with anti-IL-2 mAb (JES6- 1A12) versus IgG cohorts. P values were calculated by bioconductor package “clusterProfiler” v3.10.1 , * p <0.05, **p <0.01. Figures 20B-20C: pAKT and pMTOR expression on of donor CD4 ⁺ T cells in spleen and colon of WT recipients treated with anti-IL-2 mAb (JES6-1A12 or S4B6) or control IgG; n=5 per group. Figure 20D: pAKT and pMTOR expression on of donor CD4 ⁺ T cells in spleen and colon of PD-L1- ⁷ - recipients treated with anti-IL-2 mAb (JES6-1A12) or control IgG; n=4 per group. Figure 20E: pAKT and pMTOR expression on donor CD4 ⁺ T cells in spleen and colon of WT or PD-LT ⁷ - recipients treated with anti-IL-2 mAb (JES6-1A12); n=4-5 per group. Data represent mean ± SE combined from two replicate experiments. P values were calculated by unpaired two-tailed Student t tests (*p<0.05, **p<0.01 , ****p < 0.0001).

[0029] Figure 21 shows the comparison of the percentage of host parenchymal cells versus donor T cells. Lethally irradiated WT BALB/c recipients were given splenocytes (2.5 x 10 ⁶ ) and TCD-BM (2.5 x 10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 4 i.v. injections of anti-IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT. Day 7 after HCT, spleen and colon were harvested for analysis. The percentage of H2Kd ⁺ CD45- cells versus H2Kb ⁺ TCR|3 ⁺ cells is shown. n=5 per group. Data represent mean ± SE combined from two replicate experiments. P values were calculated by unpaired two-tailed Student t tests (** p<0.01).

[0030] Figures 22A-22D show that tolerogenic anti-IL-2 (JES6-1A12) but not non-tolerogenic anti-IL-2 (S4B6) treatment inhibits activation of AKT-m TOR pathway in CD8 ⁺ T cells in a host-tissue PD-L1 -dependent manner. Lethally irradiated WT or PD-L - BALB/c recipients were given splenocytes (2.5 x 10 ⁶ ) and TCD-BM (2.5 x 10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 3 i.v. injections of rat-IgG or anti-IL-2 mAb (500 pg/mouse) at days 0, 2, and 4 after HCT. At day 6 after HCT, spleen and colon were harvested for analysis. Figure 22A: pAKT and pMTOR expression on of donor CD8 ⁺ T cells in spleen and colon of WT recipients treated with anti-IL-2 (JES6-1A12) or control IgG; n=5 per group. Figure 22B: pAKT and pMTOR expression on of donor CD8 ⁺ T cells in spleen and colon of WT recipients treated with anti-IL-2 (S4B6) or control IgG; n=5 per group. Figure 22C: pAKT and pMTOR expression on of donor CD8 ⁺ T cells in spleen and colon of PD-LT ⁷ - recipients treated with anti-IL-2 or control IgG; n=4 per group. Figure 22D: pAKT and pMTOR expression on of donor CD8 ⁺ T cells in spleen and colon of WT or PD-L1- ⁷ - recipients treated with anti-IL-2; n=4-5 per group. Data represent mean ± SE combined from two replicate experiments. P values were calculated by 2-way ANOVA (*p<0.05, ** p<0.01 , ***p<0.001 ).

[0031] Figures 23A-23D show that tolerogenic anti-IL-2 mAb (JES6-1A12) and tacrolimus induce distinct T cell subpopulations and T cell transcriptional signatures. Figure 23A: t-SNE plot displaying clusters identified in spleen cells from recipients treated with anti-IL-2 mAb (JES6-1A12) or tacrolimus on day 7 after HCT. Figure 23B: Violin plots showing CD3e, CD4 and CD8a distribution on individual clusters. Figure 23C: Violin plot showing Tcf7 expression level in individual clusters under anti-IL-2 mAb (JES6-1A12) or tacrolimus treatment. Figure 23D: Compared T cell percentage in individual clusters under anti-IL-2 mAb (JES6-1A12) or tacrolimus treatment.

[0032] Figure 24 shows that scRNA-Seq reveals distinct subpopulations and transcriptional signatures of CD8 T cells after treatment with tolerogenic anti-IL-2 (JES6-1A12) mAb or tacrolimus. Heatmap showing differentially expressed coding genes for the 9 clusters in Figure 23A.

[0033] Figures 25A-25L show that tolerogenic anti-IL-2 mAb (JES6-1A12) preserves CD8 ⁺ T memory progenitors and functional effectors that mediate GVL activity in lymphoid tissues more effectively than tacrolimus. Lethally irradiated WT BALB/c recipients were given splenocytes (2.5 x 10 ⁶ ) and TCD-BM (2.5 x 10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 4 i.p. injections of anti-IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT, or once daily i.p. injections of tacrolimus (0.75 mg/Kg) at days 0-6 after HCT. On day 7 after HCT, mesenteric lymph node (MLN), spleen (SPL), liver and colon were harvested for analysis. Figure 25A: Representative pattern of gating strategy in recipients given anti-IL-2 treatment. Figure 25B: Representative flow cytometry pattern showing the expression of Granzyme B and CD107a on CD8 ⁺ T cells and % of CD107a ⁺ GranzymeB ⁺ CD8 ⁺ T cells in the spleen from IL-2 mAb or tacrolim us-treated recipients are showed, n=4-6, combined from 2 experiments. Figures 25C-25H: Percentage of Tmp (CD39’Ly108 ⁺ ), Teff (CD39 ⁺ Ly108 ⁺ ) and Tex (CD39 ⁺ Ly108-) among donor CD8 ⁺ T cells in MLN, spleen, Liver and colon of WT recipients treated with anti-IL-2 mAb (JES6-1A12), tacrolimus or control IgG; n=4-6 per group. Figure 25I: Percentage of GranzymeB ⁺ CD107a ⁺ CD8 ⁺ T, IFN-y ⁺ CD8 ⁺ T cells and MFI of perforin among Teff, Tex and Tmp in spleen and MLN of WT recipients treated with anti-IL-2 mAb (JES6-1A12). n=4-6 per group. Figures 25J-25K: Expression of T-bet, CD127, BrdU and Tim -3 on Teff, Tex and Tmp in spleen and MLN of WT recipients treated with anti-IL-2 mAb (JES6-1A12). n=4-6 per group. Figure 25L: Percentage of IL-2 ⁺ CD4 ⁺ T cells in spleen and MLN of WT recipients treated with anti-IL-2 mAb (JES6-1A12) or tacrolimus. n=4 per group. P values were calculated by unpaired two-tailed Student t tests, one-way ANOVA or two-way ANOVA (*p<0.05, ** p<0.01 , ***p<0.001, **** p < 0.0001 ).

[0034] Figure 26 shows gating strategy showing the Teff, Tex, Tex and Ter-Teff CD8 ⁺ T cell subsets in the spleen of the WT recipients treated with tacrolimus.

[0035] Figures 27A-27C show that tolerogenic anti-IL-2 (JES6-1A12) treatment is more effective than TAC treatment for preserving CD8 ⁺ T memory progenitors and functional effectors that mediate GVL effect in lymphoid tissues. Lethally irradiated WT BALB/c recipients were given splenocytes (2.5 x 10 ⁶ ) and TCD-BM (2.5 x 10 ⁶ ) from C57BL/6 donors. Recipients were given a total of 4 i.v. injections of anti-IL-2 mAb (JES6-1A12) (500 pg/mouse) at days 0, 2, 4 and 6 after HCT or i.v. injections of tacrolimus (0.75 mg/Kg) at days 0-6 after HCT. On day 7 after HCT, mesenteric lymph node (MLN), spleen (SPL), liver and colon were harvested for analysis. Figure 27A: Yield of total donor CD8 ⁺ T cells and % of KLRG1 ⁺ CD39 ⁺ CD8 ⁺ T cells in the spleen from anti-IL-2 or tacrolim us-treated recipients are showed, n=4-9, combined from 2 experiments. Figure 27B: Representative flow cytometry patterns showing the expression of Ly108 and CD39 expression in mesenteric lymph node, spleen, liver and colon from anti-IL-2 or tacrolim us-treated recipients. Figure 27C: Representative flow cytometry pattern showing CD107a, Granzyme B expression among Teff, Tex, Tmp and Ter-Teff cells in the spleen from anti-IL-2 or tacrolim us-treated recipients.

DETAILED DESCRIPTION

[0036] This disclosure is directed to a method of preventing or treating GVHD while preserving GVL activity in vivo in a subject receiving a hematopoietic cell transplantation (HCT). The method entails administering to the subject an effective amount of an anti-IL-2 antibody. In certain embodiments, the subject receives an allogeneic HCT. In certain embodiments, the GVHD is acute GVHD. In certain embodiments, the anti-IL-2 antibody augments IL-2 binding to IL-2Ra and blocks IL-2 binding to IL-2R|3 in vivo. In certain embodiments, the anti-IL-2 antibody is a monoclonal antibody. In certain embodiments, the anti-IL-2 antibody is a recombinant antibody. In certain embodiments, the anti-IL-2 antibody is a human antibody. In certain embodiments, the anti-IL-2 antibody is a humanized antibody. In certain embodiments, the anti-IL-2 antibody is anti-IL-2-JES6 antibody.

[0037] “Treating” or “treatment” of a disease or a condition may refer to preventing the disease or condition, slowing the onset or rate of development of the disease or condition, reducing the risk of developing the disease or condition, preventing or delaying the development of symptoms associated with the disease or condition, reducing or ending symptoms associated with the disease or condition, generating a complete or partial regression of the disease or condition, or some combinations thereof.

[0038] As used herein, the term “subject” refers to mammalian subject, preferably a human. The phrases “subject” and “patient” are used interchangeably herein.

[0039] An “effective amount,” “therapeutically effective amount” or “effective dose” is an amount of a composition (e.g., an antibody or a pharmaceutical composition) that produces a desired therapeutic effect in a subject, such as preventing or treating a target disease or condition, or alleviating symptoms associated with the disease or condition. The precise therapeutically effective amount is an amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the active agent (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject’s response to administration of an active agent and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy 21st Edition, Univ, of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0040] The administration schedule and doses of the anti-IL-2 antibody can be determined based on the need of the subject. For example, the anti-IL-2 antibody is administered to the subject immediately before or on the same day of receiving HCT. In certain embodiments, the anti-IL-2 antibody is administered to the subject after receiving HCT. In certain embodiments, the anti-IL-2 antibody is administered to the subject receiving HCT at the onset of GVHD. In certain embodiments, multiple doses of the anti-IL-2 antibody are administered after HCT. In certain embodiments, a single dose of the anti-IL-2 antibody is administered each day. In certain embodiments, the anti-IL-2 antibody is administered every other day fora week, for two weeks, for three weeks, or for a month after HCT. One of ordinary skill in the art would understand that when multiple doses of the anti-IL-2 antibody is administered, each dosage may be the same or different. For example, a higher dosage may be administered immediately after HCT and followed by a lower dosage at a later time, e.g., after a week of administration on every other day. Alternatively, a lower dosage may be administered first, followed by a higher dosage.

[0041] Any suitable administration route of the anti-IL-2 antibody may be chosen. For example, the anti-IL-2 antibody can be administered to the subject by intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, intranodal, or intrasplenic administration.

[0042] Administration of anti-IL-2 mAb may not simply neutralizing IL-2 in vivo. Certain anti-IL-2 mAbform complexes with IL-2 (IL-2C) that modulate IL-2 interactions with IL-2Ra and IL-2R0 [26], Anti-IL-2 from hybridoma clone JES6-1 (anti-IL-2-JES6) forms an IL-2C that enhances IL-2 interaction with IL-2Ra and augments IL-2Ra ^hi Foxp3 ⁺ Treg expansion, while blocking IL-2 interaction with IL-2R0 on conventional T cells [26, 27]. In contrast, anti-IL-2 from hybridoma clone S4B6 (anti-IL-2-S4B6) forms an IL-2C that blocks IL-2 interaction with IL-2Ra and augments IL-2 interaction with IL- 2R0, leading to expansion of conventional T cells [26, 27]. Thus, anti-IL-2-JES6 has tolerogenic effects, while anti-IL-2-S4B6 does not [26], Surprisingly, administration of anti-IL-2-JES6 can prevent GVHD while preserving GVL activity; whereas other anti- IL-2 antibodies may not have such effects. Accordingly, disclosed herein is a novel approach for preventing or treating GVHD in a subject receiving HCT using an antibody that preferentially blocks IL-2-binding to IL-2R|3 and augments IL-2-binding to IL-2Ra such that GVHD is prevented or treated while strong GVL effect is preserved. Administration of the anti-IL-2 antibody depletes pathogenic GM-CSF-producing Th1/Tc1 cells in the GVHD target tissues but expands IL-10-producing Tr1 cells, leading to effective prevention or treatment of GVHD. Administration of the anti-IL-2 antibody also preserves CD8 ⁺ memory T progenitors and effectors cells in the lymphoid tissues where they mediate GVL effect.

[0043] As demonstrated in the working examples, administration of tolerogenic anti-IL-2 mAb early after allo-HCT in mice markedly attenuates acute GVHD while preserving GVL activity that is dramatically stronger than observed with tacrolimus (TAC) treatment. In certain embodiments, the anti-IL-2 antibody is administered to the subject on the same day of receiving HCT, within 1 day of receiving HCT, within 2 days of receiving HCT, and within 3 days of receiving HCT. In certain embodiments, the anti-IL-2 antibody is administered to the subject receiving HCT before or immediately after the onset of GVHD. The anti-IL-2-treatment down-regulated activation of IL-2-Stat5 pathway and reduced production of GM-CSF. In GVHD target tissues, enhanced T cell PD-1 interaction with tissue-PD-L1 led to reduced activation of AKT-m TOR pathway and increased expression of Eomes and Blimp-1 , increased T cell anergy/exhaustion, expansion of Foxp3 ⁺ Treg and Foxp3’IL-10-producing Tr1 cells, and depletion of GM-CSF-producing Th1/Tc1 cells. In recipient lymphoid tissues, lack of donor T cell PD-1 interaction with tissue-PD-L1 preserved donor PD-1 ⁺ TCF- 1 ⁺ Ly108 ⁺ CD8 ⁺ T memory progenitors (Tmp) and functional effectors that have strong GVL activity. The anti-IL-2 and TAC treatment have qualitatively distinct effects on donor T cells in the lymphoid tissues, and CD8 ⁺ Tmp cells are enriched with the anti- IL-2 treatment compared to TAC treatment. Thus, administration of tolerogenic anti- IL-2 mAb early after Allo-HCT represents a novel approach for preserving GVL activity while preventing acute GVHD.

[0044] Immunosuppressive medications such as TAC are routinely used to prevent GVHD in patients after Allo-HCT, but they can also inhibit GVL activity [34, 35], Disclosed herein is a novel approach of preventing GVHD while preserving GVL activity by administration of tolerogenic anti-IL-2-JES6 early after HCT. Surprisingly, the anti-IL_2 JES6 antibody enables GVHD target tissue PD-L1 to effectively tolerize infiltrating T cells, leading to effective prevention of aGVHD, while preserving strong GVL activity that is much more effective than TAC under conditions where anti-IL-2 and TAC have similar effects on GVHD. Other anti-IL-2 antibodies such as anti-IL-2- S4B6 did not achieve the tolerogenic effects.

[0045] The mechanisms whereby tolerogenic anti-IL-2-JES6 treatment preserves GVL activity while preventing GVHD involve multiple steps is illustrated in Figure 1. First, anti-IL-2-JES6 treatment inhibits activation of IL-2-Stat5 signaling pathway in the donor T cells and reduces their production of GM-CSF. Second, in the lymphoid tissues, due to relative lack of PD-L1 -expression by recipient cells, the PD- 1 ⁺ TCF-1 ⁺ Ly108 ⁺ CD4 ⁺ and CD8 ⁺ Tmp cells are well preserved. Moreover, CD4 ⁺ T cells help the CD8 ⁺ Tmp differentiate into CD39 ⁺ Ly108 ⁺ functional Teff cells to mediate persistent GVL activity. Third, in GVHD target tissues, donor T cell PD-1 interacts with host-tissue PD-L1 , leading to down-regulated activation of the AKT-m TOR pathway and upregulated activation of anergy/exhaustion related nuclear factors, including Eomes and Blimp-1. Consequently, many donor T cells in GVHD target tissues become anergic, exhausted or apoptotic, with depletion of GM-CSF ⁺ T cells; at the same time, some T cells differentiate into Foxp3 ⁺ Treg cells or Foxp3-IL-10-producing Tr1 cells, and anti-IL-2-JES6 may augment IL-2 binding to IL-2Ra on the regulatory T cells to augment their expansion. The regulatory T cells further suppress the pathogenic T cell function in the GVHD target tissues.

[0046] Tolerogenic anti-IL-2-JES6 treatment can affect both naive and memory T cells in the graft. Conventional memory T cells express IL-2R0, and naive T cells upregulate IL-2R0 expression after activation [36], Memory T cells in the graft have reduced GVHD capacity with preserved GVL activity in mice [37-39] and in patients [40], Although not wishing to be bound by theory, administration of tolerogenic anti- IL-2 mAb may prevent GVHD while preserving GVL effects mediated by both naive and memory T cells in the graft.

[0047] The experimental data disclosed herein provide new insights into how to separate GVHD from GVL activity mediated by the same alloreactive donor T cell population. Anti-IL-2-JES6-treatment reduced IL-2-Stat5 activation independent of host tissue PD-L1 , but the treatment upregulated T cell expression of PD-1 and reduced activation of AKT-mTOR pathways in the T cells from GVHD target tissues in the host-tissue PD-L1 -dependent manner. Therefore, simultaneous reduction of activation by blocking IL-2 effect and augmentation of inhibition by PD-1 signaling enable inhibition of AKT-mTOR pathway in the T cells in GVHD target tissues, leading to prevention of GVHD; lack of PD-1 interaction with host tissue PD-L1 in the lymphoid tissues allows better alloreactive T cell survival, leading to stronger GVL effect.

[0048] As demonstrated in the working examples, PD-L1/PD-1 interaction augments differentiation and expansion of Foxp3-IL-10-producing Tr1 cells. IL-10 ⁺ Tr1 cells represent the major regulatory T cell population in allo-HCT recipients; moreover, Eomes is required for donor T cell differentiation into FoxP3-IL-10 ⁺ Tr1 cells, and Blimp-1 augments expansion of Tr1 cells [17], The observations that anti-IL-2-JES6 treatment upregulated donor CD4 ⁺ T expression of Eomes in both WT and PD-L - recipients, but upregulated expression of Blimp-1 only in WT but not in PD-LT ⁷ - recipients suggest that reduction of AKT-mTOR activation by blocking IL-2 effect alone is able to upregulate Eomes in the absence of PD-1 signaling; but simultaneous reduction of AKT-mTOR activation by blocking IL-2 effect and inhibition of AKT-mTOR activation by PD-1 signaling triggered by tissue PD-L1 is required to upregulate expression of Blimp-1. Therefore, anti-IL-2-JES6 enables tissue PD-L1 to mediate differentiation and expansion of Tr1 cells in GVHD target tissues.

[0049] Anti-IL-2-JES6 treatment exploits differences in expression of PD-L1 by recipient GVHD target and lymphoid tissues that affect the ability of PD1 ⁺ TCF- 1 ⁺ Ly108 ⁺ CD8 ⁺ Tmp cells to cause GVHD and mediate GVL activity. Blockade of PD- 1 interaction with PD-L1 can revive the function of TCF-1 ⁺ Tmp and Teff cells [33], The paucity of PD-L1 -expressing cells in recipient lymphoid tissues preserves donortype PD-1 ⁺ TCF-1 ⁺ Ly108 ⁺ CD8 ⁺ Tmp and their derivatives locally where they mediate GVL activity. In contrast, the abundance of PD-L1 -expressing cells in the GVHD target tissues such as colon and liver tolerizes donor-type PD-1 ⁺ TCF-1 ⁺ Ly108 ⁺ Tmp and their derivative Teff cells locally, thereby preventing GVHD. Thus, anti-IL-2-JES6 treatment allows donor-type PD-1 ⁺ TCF-1 ⁺ Ly108 ⁺ CD8 ⁺ Tmp cells to mediate GVL activity in lym pho-hematopoietic compartment without causing aGVHD in parenchymal tissues, even though CD8 ⁺ Tmp cells can mediate persistence of GVHD [24],

[0050] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. The examples are set forth to aid in understanding the invention but are not intended to, and should not be construed to, limit its scope in any way. The examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art and are described in numerous publications. All references mentioned herein are incorporated in their entirety.

Example 1. Materials and Methods

[0051] Induction and assessment of GVHD, measurement of cytokines in serum, flow cytometry analysis and sorting, histopathology and histoimmunofluorescent staining, single cell RNA sequencing library construction using the 10x genomics chromium platform, and statistical analysis are described in previous publications [13] and detailed below.

[0052] Mice: BALB/c (H-2 ^d ) and C57BL/6 (H-2 ^b ) mice were purchased from National Cancer Institute (Frederick, MD). IL-10 ⁷ - C57BL/6 mice (H-2 ^b ) were purchased from the Jackson Laboratory. PD-L - BALB/c breeders were provided by Dr. L. Chen (Yale University, New Haven). All mice were maintained in a pathogen- free room in City of Hope Animal Research Center. All experiments were approved by IACUC at City of Hope.

[0053] Induction and assessment of GVHD: BALB/c recipients were exposed to 850 cGy total body irradiation (TBI) with the use of a [137Cs] source 8 hours before HCT, and then given C57BL/6 donor spleen cells (2.5x10 ⁶ -5.0 x 10 ⁶ ),Thy1.2 ⁺ cells (1 x 10 ⁶ ) and T cell-depleted BM (TCD-BM) cells (2.5 x 10 ⁶ ) by tail vein injection. C57BL/6 recipients were exposed to 1300 cGy total body irradiation (TBI) with the use of a [137Cs] source 8 hours before HCT, and then given A/J donor spleen cells (2.5x10 ⁶ ) and T cell-depleted BM (TCD-BM) cells (2.5 x 10 ⁶ ) by tail vein injection. The bone marrow was depleted of T cells by using biotin-conjugated anti-CD4 and anti- CD8 mAbs, and streptavidin Microbeads (Miltenyi Biotec, Germany), followed by passage through an autoMACS Pro cell sorter (Miltenyi Biotec, Germany). For GVL experiments, Luc ⁺ B-cell leukemia/lymphoma 1 (BCL1) cells (5-10* 10 ⁶ ) were injected intraperitoneally at the same time when donor bone marrow and spleen cells were injected intravenously. GFP+ blast crisis chronic myeloid leukemia (BC-CML) cells (1x10 ⁶ ) were injected intravenously at the same time when donor bone marrow and spleen cells were injected. In vivo imaging of tumor growth was monitored by using Lago IVIS100 charge-coupled device imaging system. The assessment and scoring of clinical signs of acute GVHD has been described previously [42],

[0054] Cell lines: Luciference transfected BCL-1 cell line was provided by Dr. Christopher Contag at Stanford University (Stanford, CA). GFP ⁺ blast crisis chronic myeloid leukemia (BC-CML) cell line was provided by Dr. Warren Shlomchik at Pittsburgh University (Pittsburgh, PA).

[0055] Antibodies and FACS analysis: Anti-IL-2 mAb (JES6-1A12, that is, JES6-1), anti-IL-2 mAb (S4B6) and anti-NK1.1 (PK136) for in vivo treatment was purchased from Bio X Cell (West Lebanon, NH). ChromPure Rat IgG (012-000-003) was purchased from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA, USA). mAbs specific for MHCII (M5/114.15.2) and T-bet (4B10) were purchased from BD Bioscience. mAbs specific forTCRp (H57-597), H-2Kb (AF6-88.5), CD4 (RM4-5), CD8a (53-6.7), CD11 b (M1/70), CD11c (N418), Ly6G (RB6-8C5), Ly6C (HK1.4), pMTOR (MRRBY), CD39 (24DMS1) were purchased from ThermoFisher Bioscience. mAbs specific for CD24 (M1/69), Pro-IL-1|3 (NJTEN3), B7H1 (H1M5), pAKT473 (SDRNR), KLRG1 (2F1/KLRG1), CD107a (1 D4B), GranzymeB (QA16A02), ly108 (330-AJ), TIM3 (RMT3-23), IFN-y (XMG1.2), TNF-a (MP6-XT22), Eomes (Dan 11 mag), GM-CSF (MP1-22E9), IL-10 (JES5-16E3), Blimp-1 (5E7), perforin (eBioOMAK- D), CD127 (SB/199), IL-2 (JES5-5H4), Foxp3 (FJK-16s), NKp46 (29A1.4) and mouse Breg staining kit (anti-mouse CD19/CD5/CD1d) were purchased from Biolegend (San Diego, CA). CellROX™ Green Reagent (for detection of oxidative stress), anti-mouse CD3e (145-2C11), anti-mouse CD19 (eBio1 D3) and anti-mouse NK1.1 (PK136) was purchased from ThermoFisher Scientific. Flow cytometry analyses were performed with an Attune NxT Cytometer (ThermoFisher Scientific) and BD LSRFortessa (Franklin Lakes, NJ), and the resulting data were analyzed with FlowJo software V10 (Tree Star, Ashland, OR).

[0056] Isolation of cells from spleen, mesenteric lymph node, liver and large Intestine: Spleen, lymph node and liver tissue were mashed through a 70 pm cell strainer, and MNC were isolated from the cell suspensions with percoll. Intestine was cut first longitudinally and then laterally into pieces of approximately 0.5 cm length. Tissue pieces were incubated with 20 mL of predigestion solution (1 x HBSS without containing 5 mM EDTA, 5% fetal bovine serum (FBS), 1 mM DTT) for 20 minutes at 37 °C under continuous shaking, then passed through 100 pm strainer and held for at least 10 minutes on ice. Intestine epithelia lymphoid cells in the supernatant were collected. Then tissue pieces were digested with enzyme to isolate the lamina propria cells, following the protocol of Lamina Propria Dissociation Kit (Miltenyi Biotec).

[0057] Histopathology: Tissue specimens were fixed in formalin before embedding in paraffin blocks, sectioned and stained with H&E. Slides were examined at 100x (liver) or 200x (small intestine and colon) magnification and visualized with Zeiss Observer II. Tissue damage was blindly assessed according to a defined scoring system, as described previously [42], Liver GVHD was scored by the severity of lymphocytic infiltrate, number of involved tracts and severity of liver cell necrosis; the maximum score is 9. Gut GVHD was scored by mononuclear cell infiltration and morphological aberrations (e.g. hyperplasia and crypt loss), with a maximum score of 8.

[0058] Bioluminescent imaging: Mice were given with luciferase* BCL1 cells (BCL1/ Luc ⁺ ) by i.p. injection. For in vivo imaging of tumor growth, 200 pl firefly luciferin was injected i.p. (Caliper Life Sciences, Hopkinton, MA), and mice were anesthetized for analysis of tumor cell burden by using an IVIS100 (Xenogen) and AmiX (Spectral) imaging system. Data were analyzed by using Amiview software purchased from Spectral Instruments Imaging (New York, NY).

[0059] In vivo BrdU labeling: T cell proliferation was measured with a single i.p. injection of Brdll (2.5 mg/mouse, 100 mg/g) 3 hours before tissue harvesting. Analysis of donor CD8 ⁺ T cells for Brdll incorporation was performed according to the manufacturer’s instructions (BD Pharm ingen).

[0060] mRNA sequencing library preparation and sequencing: RNA concentration was measured by NanoDrop 1000 (Thermo Fisher Scientific, Waltham Massachusetts, US), and RNA integrity was determined using Bioanalyzer (Agilent). Library construction of 280 ng total RNA for each sample was made by using KAPA Stranded mRNA-Seq Kit (Illumina Platforms) (Kapa Biosystems, Wilmington, USA) with 10 cycles of PCR amplification. Libraries were purified using AxyPrep Mag PCR Clean-up kit (Thermo Fisher Scientific). Each library was quantified using a Qubit fluorometer (Life Technologies), and the size distribution was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Sequencing was performed on an Illumina® Hiseq 2500 (Illumina, San Diego, CA, USA) instrument using the TruSeq SR Cluster Kit V4-cBot-HS (Illumina®) to generate 51 bp single-end reads sequencing with v4 chemistry. Quality control of RNA-Seq reads was performed using FastQC. Bioconductor package “clusterProfiler” v3.10.1 was used for GSEA analysis to generate the NES and P value, while “enrichplot” v1.2.0 was used to generate the GSEA plot [43],

[0061] Single-cell sequencing library construction using the 10x Genomics Chromium Platform: Library preparation was done with the Chromium Single Cell 5' Reagent Kits from 10x Genomics according to manufacturer's protocol. Cellular suspensions were loaded on a Chromium Controller instrument (10x Genomics) to generate single-cell gel bead-in-emulsions (GEMs). GEM-reverse transcriptions (GEM-RTs) were performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific). After reverse transcription, GEMs were harvested, and the cDNAs were amplified and cleaned with the SPRIselect Reagent Kit (Beckman Coulter). Indexed sequencing libraries were constructed using the Chromium Single-Cell 5' Library Kit (10x Genomics) for enzymatic fragmentation, end-repair, A-tailing, adaptor ligation, ligation cleanup, sample index PCR, and PCR cleanup. The purity and library size were validated by capillary electrophoresis using 2,100 Bioanalyzer (Agilent Technologies). The quantity was measured fluorometrically using Qubit dsDNA HS Assay Kit from Invitrogen.

[0062] Libraries were sequenced with a NovaSeq 6000 instrument (Illumina) to a depth of 35k-40k reads per cell. Raw sequencing data were processed using the 10x Genomics’ Cell Ranger pipeline (version 3.1.0) to generate FASTQ files and aligned to mm 10 genome to generate gene expression counts. The subsequent data analysis was performed using “Seurat v3.0” package and R scripts. Cells with mitochondrial read >10% and <200 detectable genes were considered as low -quality and filtered out. Normalized and scaled data were clustered using the top significant principal components of 2000 highly variable genes and resolution of 0.4 using “Seurat”. The t-distributed stochastic neighbor embedding (t-SNE) algorithm was used to visualize the resulting clusters. Cluster specific markers were identified using “Seurat” to generate the heatmap of marker genes in these cell clusters. Genes were compared between different clusters using Bioconductor package “Lim ma” and Iog2 normalized data. Gene Set Enrichment analysis (GSEA) v3 was performed using genes ranked by the — Iog10 of “Lim ma” comparison P value to evaluate the significant activation or inhibition of the Hallmark gene sets in MSigDb (www.gsea- msigdb.org/gsea/msigdb/genesets.jsp?collection=H). All plots were generated using either “Seurat” or “ggplot2” package in R.

[0063] Statistical analysis: Data were displayed as mean ± SEM. Mortality rates in different groups were compared by log-rank test. Comparison of body weight in different groups was analyzed by nonlinear regression (curve fit). Comparison of means for more than two groups was analyzed by 1-way AN OVA or 2-way AN OVA multiple comparisons, while comparison of two means was analyzed by unpaired two- tailed Student t-test (Prism, version 8.0; GraphPad Software), P less than 0.05 was considered as statistically significant (*p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 ).

Example 2. Administration of tolerogenic anti-IL-2-JES6 mAb prevents aGVHD and preserves GVL activity more effectively than tacrolimus

[0064] It was proposed that IL-2 from donor CD4 ⁺ T cells may make alloreactive donor T cells resistant to induction of tolerance (i.e. anergy, exhaustion and apoptosis) by host-tissue PD-L1 [13], Thus, it was tested whether adm inistration of tolerogenic anti-IL-2-JES6 (JES6-1A12) mAb that block IL-2 interaction with IL-2R0 on conventional T cells [27] could prevent GVHD and preserve GVL activity. Accordingly, irradiated BALB/c recipients were engrafted with splenocytes (5x10 ⁶ ) and TCD-BM cells (2.5x10 ⁶ ) from MHC-mismatched C57BL/6 donors. Recipients given TCD-BM alone were used as GVHD-free controls. Recipients were treated with rat anti-IL-2- JES6 or control rat IgG at a dose of 500 pg/mouse i.p. on days 0, 2, 4 and 6 after HCT. As compared to IgG treatment, anti-IL-2-JES6 treatment limited loss of body weight and completely prevented diarrhea, and all recipients survived for more than 30 days. Moreover, anti-IL-2-JES6 treatment prevented GVHD target tissues (liver, small intestine and colon) damage (Figures 2A-2B and 3A). In contrast, administration of non-tolerogenic anti-IL-2-S4B6 that augment IL-2 interaction with IL-2R0 on conventional T cells did not prevent acute GVHD (Figures 2C-2D and 3B). These results indicate that administration of tolerogenic anti-IL-2-JES6 but not non- tolerogenic anti-IL-2-S4B6 greatly attenuated the seventy of aGVHD.

[0065] To assess the effect of tolerogenic anti-IL-2-JES6 treatment on GVL activity, BALB/c recipients were inoculated with luciferase-transfected BCL1 cells (BCL1/Luc, 5x10 ⁶ /mouse, i.p.) before HCT. BCLIZLuc cell growth was monitored by in vivo bioluminescent imaging (BLI). BCLI/Luc tumor cells grew rapidly in recipients engrafted with TCD-BM; anti-IL-2-JES6-treatment did not have significant impact on the tumor growth; and all recipients died within 20 days after HCT (Figure 2E). BCLI/Luc tumor cells grew transiently in IgG-treated recipients engrafted with splenocytes and TCD-BM cells, but all recipients died with GVHD within 10 days after HCT. BCLI/Luc tumor cells also grew transiently in anti-IL-2-JES6-treated recipients and were eliminated by day 11 , but all mice survived for more than 30 days without tumor relapse (Figure 2E). Treatment with anti-IL-2-JES6 also eliminated GVL- resistant BC-CMLtumor cells [28], while preventing aGVHD (Figure 4). Although anti- IL-2-JES6 treatment augmented NK cell expansion, depletion of NK cells had no impact on GVL activity (Figure 5). Taken together, these results show that treatment with tolerogenic anti-IL-2-JES6 effectively controlled GVHD while preserving strong GVL activity mediated by alloreactive T cells.

[0066] The calcineurin inhibitor tacrolimus (TAC) is widely used clinically to prevent aGVHD, in part by inhibiting endogenous IL-2 production in alloactivated donor T cells. Therefore, it was of interest to compare the effects of anti-IL-2-JES6 and tacrolimus regarding their respective abilities to prevent aGVHD while preserving GVL activity. BALB/c recipients engrafted with spleen (2.5x10 ⁶ ) and TCD-BM (2.5x10 ⁶ ) cells from C57BL/6 donors and challenged with 5x10 ⁶ BCL1/Luc cells on day 0 were treated with anti-IL-2 on days 0, 2, 4, and 6 after HCT or with i.p. injection of TAC (0.75 mg/kg) daily for up to 21 days. The two groups showed similar loss of body weight, and survival was not statistically different between the 2 groups during the first 30 days after HCT. Both groups cleared tumor cells by 12-17 days after HCT (Figure 2F).

[0067] In further experiments, BALB/c recipients engrafted with C57BL/6 BM cells and a lower number of spleen cells (1.25x10 ⁶ ) were challenged with i.p. inoculation of 5 or 10 x10 ⁶ Luc/BCLI cells, with the same regimen of anti-IL-2-JES6 or TAC. In recipients challenged with 5x10 ⁶ BCL1/Luc cells, tumor cells disappeared before day 12 in all anti-IL-2-JES6 treated, but only 60% of TAC -treated recipients cleared tumor by day 17, while the other 40% died with progressive tumor growth by 30 days after HCT (Figure 6). In recipients challenged with10x10 ⁶ BCL1/Luc cells, all anti-IL-2-JES6-treated recipients cleared tumor cells by day 12 after HCT, but all TAC- treated recipients died with progressive tumor growth by 9 days after HCT (Figure 2G). Thus, under the conditions used for these experiments, anti-IL-2-JES6 and TAC had comparable ability to prevent acute GVHD, but anti-IL-2-JES6 treatment preserved GVL activity, while TAC did not.

Example 3. Prevention of aGVHD by tolerogenic anti-IL-2-JES6 requires PD-L1 expression by GVHD target tissues

[0068] Prevention of GVHD by depleting donor CD4 ⁺ T cells that produced IL-2 was host-tissue PD-L1 -dependent [13], Therefore, it was tested whether prevention of GVHD by tolerogenic anti-IL-2-JES6 also depends on recipient PD-L1. Anti-IL-2- JES6 attenuated the severity of GVHD in WT recipients but not in PD-L1- ⁷ - recipients (Figures 7A, 7B and 8). Anti-IL-2-JES6 treatment markedly ameliorated tissue damage in the liver, small intestine and colon of WT recipients as compared with IgG- treated recipients. In contrast, anti-IL-2-JES6 treatment did not ameliorate tissue damage at all in PD-L1- ⁷ - recipients (Figures 7B and 8). As compared to IgG treatment, anti-IL-2-JES6-treatment reduced the yield of donor CD4 ⁺ T cells although not CD8 ⁺ T cells in the liver and colon at day 7 in WT recipients (Figure 7C). In contrast, anti- IL-2-S4B6-treatment expanded donor-type CD4 ⁺ and CD8 ⁺ T cells in the spleen but produced no significant changes in the liver or colon (Figure 7D). As compared to IgG treatment, the effect of anti-IL-2-JES6 treatment was not apparent PD-L1- ⁷ - recipients (Figure 7E). But as compared with WT recipients, the yield of CD4 ⁺ T cells in the liver and colon at day 7 was higher in PD-LT ⁷ - recipients (Figure 7F). These results also indicate that tolerogenic anti-IL-2-JES6 augments host-tissue PD-L1-m edated protection against aGVHD.

[0069] Upregulation of expression of PD-1 and Eomes is a feature of anergic/exhausted T cells [29], Anti-IL-2-JES6-treatment markedly increased the percentages of PD-1 ⁺ Eomes ⁺ CD4 ⁺ and CD8 ⁺ T cells in the GVHD target tissues liver and colon in a time-dependent manner in the WT recipients (Figure 7G), while anti-IL- 2-S4B6 did not have these effects (Figure 7H). As compared with IgG treatment, anti- IL-2-JES6 treatment also increased the percentage of PD-1 ⁺ Eomes ⁺ CD4 ⁺ and CD8 ⁺ T cells in the liver and colon tissues at day 6 after HCT in the PD-LT ⁷ - recipients (Figure 7I). Later time points were not available forevaluation, due to death of most PD-L ⁷ - recipients by 7 days after HCT. The percentages of PD-1 ⁺ Eomes ⁺ CD4 ⁺ and CD8 ⁺ T cells in the liver and colon tissues of anti-IL-2-JES6 treated PD-L1- ⁷ - recipients were markedly lower than in anti-IL-2-JES6-treated WT recipients at day 6 after HCT (Figure 7J). These results indicate that tolerogenic anti-IL-2-JES6 treatment and tissue PD- L1 interaction with PD-1 synergistically augment T cell anergy/exhaustion of tissue infiltrating T cells, leading to prevention of aGVHD.

Example 4. Prevention of GVHD by tolerogenic anti-IL-2-JES6 is associated with tissue PD-L1 -dependent depletion of GM-CSF-producing Th1 and Tc1 cells [0070] GM-CSF-producing Th1 and Tc1 cells play an essential role in aGVHD pathogenesis [30, 31], GM-CSF-producing (GM-CSF ⁺ ) Th1 and Tc1 cells were evaluated at day 6 after HCT. As compared with IgG treatment, anti-IL-2-JES6 treatment reduced the percentages and yield of GM-CSF ⁺ IFN-y ⁺ Th1 in the liver and colon of WT recipients and reduced the percentages but not the yields of Tc1 in those issues (Figures 9A and 10), while anti-IL-2-S4B6 did not have these effects (Figures 9B and 11). As compared to IgG treatment, anti-IL-2-JES6 treatment also reduced the percentage of GM-CSF ⁺ Th1 and Tc1 cells in the liver tissues although not in the spleen or colon tissues in the PD-L - recipients (Figures 9C and 12). The percentage and yield of GM-CSF ⁺ Th1 and Tc1 cells in the spleen, liver and colon of anti-IL-2 - JES6-treated PD-LT ⁷ - recipients were markedly higher than in anti-IL-2-JES6-treated WT recipients (Figures 9D and 13). The lower percentages of Th1 and Tc1 cells expressing GM-CSF induced by anti-IL-2-JES6 treatment in WT recipients were associated with lower infiltration of neutrophils and monocytes in the liver and colon and with their lower production of pro-IL-1 p and ROS (Figure 14). These results indicate that administration of tolerogenic anti-IL-2-JES6 and tissue PD-L1 interaction with PD-1 on T cells synergistically augment depletion of GM-CSF-producing Th1 and Tc1 cells in GVHD target tissues.

Example 5. Prevention of aGVHD by anti-IL-2-JES6 requires PD-L1 -dependent expansion of T r1 cells

[0071] Anti-IL-2-JES6 but not anti-IL-2-S4B6 increased the percentage of Foxp3 ⁺ Treg cells in the liver of WT recipients (Figures 15A-15B). Neither anti-IL-2 had effect on the percentage or yield of Foxp3 ⁺ Treg cells in the colon (Figures 15A-15B). Anti- IL-2-JES6 treatment did not have a significant effect on Treg expansion in PD-L - recipients (Figure 15C).

[0072] Foxp3-IL-10 ⁺ Tr1 cells represent the majority of regulatory T cells in allo- HCT recipients, and Tr1 cell expression of Eomes and Blimp-1 are required for Trl cell differentiation [17], Since tissue infiltrating CD4 ⁺ T cells expressed higher levels of Eomes (Figure 7G), the impact of anti-IL-2-JES6 treatment on Foxp3-IL-10 ⁺ Tr1 cell expansion at day 6 after HCT was evaluated. As compared with IgG treatment, anti- IL-2-JES6, but not anti-IL-2-S4B6, significantly increased the percentage of IL-10 ⁺ Tr1 cells among donor-type CD4 ⁺ T cells in the liver and colon of WT recipients (Figures 16A-16B). However, anti-IL-2-JES6 treatment did not increase the percentage of Tr1 cells in PD-LT ⁷ - recipients (Figure 16C). The IL-10 ⁺ Tr1 cells were also IFN-y ⁺ but GM- CSF- (Figure 17).

[0073] As compared to IgG treatment, anti-IL-2-JES6, but not anti-IL-2-S4B6, upregulated expression of Eomes and Blimp-1 by CD4 ⁺ T cells in the liver and colon of WT recipients (Figures 16D-16E). Anti-IL-2-JES6 did not upregulate expression of Eomes or Blimp-1 in PD-LT ⁷ - recipients (Figure 16F). Finally, anti-IL-2-JES6 treatment did not prevent aGVHD induced by IL-10 ⁷ - donor T cells (Figure 16G); although IL-10 deficiency in donor T cells did not expand GM-CSF ⁺ Th1 or Td cells (Figure 18). In addition, anti-IL-2-JES6 reduced the percentages of G-MDSC in the liver but not in the gut or on the percentages of M-MDSC in the gut or liver (Figure 19). Therefore, anti- IL-2-JES6 treatment augments T cell expression of Eomes and Blimp-1 and T cell differentiation into IL-10-producing Tr1 cells in a tissue PD-L1 -depedent manner; and that IL-10 from Tr1 cells also play an important role in anti-IL-2-JES6-mediated prevention of GVHD.

Example 6. Prevention of GVHD by tolerogenic anti-IL-2-JES6 requires expression of PD-L1 by GVHD target tissues to inhibit activation of IL-2-Stat-5 and AKT-mTOR pathways in donor T cells

[0074] Gene Set Enrichment Analysis (GSEA) at day 6 showed inhibition of the IL-2-Stat5 pathway in the CD4 ⁺ T and CD8 ⁺ T cells from the spleen and colon of recipients treated with anti-IL-2-JES6 compared to control IgG (Figure 20A). Anti-IL- 2-JES6 but not anti-IL-2-S4B6 decreased the expression of pAKT and pmTOR in CD4 ⁺ T cells from the colon tissue but not the spleen of WT recipients (Figures 20B-20C). Anti-IL-2-JES6 treatment did not change pAKT or pmTOR expression in PD-L ⁷ - recipients (Figure 20D). Consistent with PD-1 -mediated inhibition in WT recipients, AKT activation was higher in donor CD4 ⁺ T cells from the colon and spleen of anti-IL- 2-JES6-treated PD-L1- ⁷ - recipients than in anti-IL-2-JES6-treated WT recipients (Figure 20E).

[0075] As shown in Figure 20B, mTOR activation was not inhibited in WT CD4 ⁺ cells from the spleen of anti-IL-2-JES6-treated recipients, most likely reflecting the lack of PD-1 interaction with PD-L1 expressed by host parenchymal tissues, because the ratio of CD45- parenchymal cells to donor T cells was more than 50-fold lower in the spleen than in the colon (Figure 21 ). Similar results were observed with the CD8 ⁺ T cells (Figure 22). Taken together, anti-IL-2-JES6 treatment inhibits IL-2-Stat5 signaling in donor T cells from both spleen and GVHD target tissues and inhibits AKT- mTOR signaling in donor T cells only in GVHD target tissues via PD-1 interaction with the tissue PD-L1.

Example 7. Anti-IL-2-JES6 and tacrolimus induce distinct T cell subpopulations and T cell transcriptional signatures

[0076] Anti-IL-2-JES6 treatment prevented aGVHD while preserving strong GVL effect that was markedly better than observed with TAC treatment (Figure 2). Single cell RNA-seq was used to characterize the donor T cell subsets from the spleen of BCL1/luc tumor-bearing recipients treated with anti-IL-2-JES6 or TAC at day 7 after HCT. As visualized by t-distributed stochastic neighbor-embedding analysis (t-SNE), donor CD4 ⁺ and CD8 ⁺ T cells were grouped in 10 distinct clusters (Figure 23A). Clusters 0, 1 , 2, 5, 6 contain only CD8 ⁺ T cells, while Cluster 4 contains only CD4 ⁺ T cells. Cluster 3 contains both CD4 ⁺ and CD8 ⁺ T cells in anti-IL-2-JES6-treated recipient but only CD4 ⁺ T cells in TAC-treated recipients (Figure 23B). The general cluster distribution of splenic T cells was similar in recipients treated with anti-IL-2 - JES6 or TAC, and CD8 ⁺ T cells were more heterogeneous than CD4 ⁺ T cells.

[0077] Gene expression profiles differed between the CD8 ⁺ T cell-enriched clusters. Clusters 0 and 6 had high expression of G2/M phase markers, while clusters 1 and 2 had high expression of S phase markers (Figure 24). Cluster 3 had high expression of Il7r, Tcf7 (encoding TCF-1 ) (Figure 23C) and Bcl-2, with low expression of Gzma, Gzmk and markers of the cell cycle (Figure 24), suggesting that this cluster is enriched for newly described TCF-1 ⁺ memory progenitor (Tmp) cells with unequivocal self-renewal potential [32], Cluster 5 had high expression of Klrdl , Gzma, Gzmk, Id2, and Itgax, suggesting that Teff cells are enriched in this cluster (Figure 24). Although the gene expression profiles of different clusters in anti-IL-2-JES6-treated and TAC-treated groups were similar, relative cluster sizes differed in the two groups. Clusters 0, 1 and 2 containing CD8 ⁺ T cells in S phase were larger in the TAC-treated group than in the anti-IL-2-treated group, while Clusters 3 and 4 containing TCF-1 ⁺ CD4 ⁺ and CD8 ⁺ self-renewing memory progenitors were larger in the anti-IL-2-J ESS- treated group than in the TAC group (Figures 23C-23D). TCF-1 ⁺ CD8 ⁺ T progenitors can further differentiate into cytolytic CD8 ⁺ T cells under CD4 ⁺ T cell help [32], These results suggest that self-renewing CD8 ⁺ Tmp cells in lymphoid tissues are better preserved during anti-IL-2-JES6 treatment compared to TAC -treatment.

Example 8. Anti-IL-2-JES6 preserves CD8 ⁺ T memory progenitors and functional effectors that mediate GVL activity in lymphoid tissues more effectively than tacrolimus

[0078] Ly108 can be used as surrogate to identify TCF-1 ⁺ CD8 ⁺ T progenitors [33] and CD8 ⁺ T cells can be divided into KLRG1 ⁺ PD1 ⁺ and KLRGTPD1 ⁺ subsets [33], The KLRG1 ⁺ PD-1 ⁺ subset are mostly KLRG1 ⁺ CD39 ⁺ terminally differentiated CD8 ⁺ T effectors (Ter-Teff) [33], The KLRGTPD-1 ⁺ subset can be further divided into CD39- Ly108 ⁺ CD8 ⁺ Tmp, CD39 ⁺ Ly108 ⁺ CD8 ⁺ T effector cells (Teff), and CD39 ⁺ Ly108-CD8 ⁺ exhausted T effector cells (Tex) [33], All subsets were observed in the spleen of recipients treated with anti-IL-2 (Figure 25A) or TAC (Figure 26) at day 7 after HCT. As compared to TAC treatment, anti-IL-2-JES6-treatment reduced numbers of total donor CD8 ⁺ T cells and Ter-Teff cells in the spleen (Figure 27A), but anti-IL-2- treatment markedly increased the percentage of CD107a ⁺ Granzyme B ⁺ subset that have stronger cytolytic function among Ter-Teff cells (Figure 25B).

[0079] Consistent with single cell RNA-seq analysis, as compared to TAC- treated recipients, the percentage of Tmp cells within the CD8 ⁺ T cell population was higher in the LN, SPL, liver and colon of anti-IL-2-JES6 treated recipients at day 7 after HCT than in TAC-treated recipients (Figures 25C and 27B). The percentage of Teff cells within the CD8 ⁺ population was higher in the LN and SPL of anti-IL-2-J ESS- treated recipients than in TAC-treated recipients, with no differences in the liver or colon between the 2 groups (Figures 25D and 27B). The percentage of Tex cells within the CD8 ⁺ population in the LN and SPL of anti-IL-2-JES6-treated recipients was significantly lower than in TAC-treated recipients, with no difference in the liver or colon between the 2 groups (Figures 25E and 27B).

[0080] As compared to anti-IgG-treatment, anti-IL-2-JES6 treatment increased the percentages of Tmp and Teff cells within CD8 ⁺ cells in lymphoid tissues at day 7, while the percentage of Tex cells was lower in anti-IL-2-JES6-treated recipients than in IgG-treated controls (Figures 25F-25H). As compared with Teff cells, Tex cells and Tmp cells had lower expression of granzyme b, CD107a, perforin, IFN-y, T-bet, CD127 and lower Brdll-labeling (Figures 25I-25J). Tim3 expression was higher in Tex than in Teff and Tmp (Figure 25K). In addition, the percentage of IL-2-producing CD4 ⁺ T cells in SPL and LN was higher at day 7 after HCT in recipients treated with anti-IL-2- JES6 than in those treated with TAC (Figure 25L). Taken together, these results indicate that anti-IL-2-JES6 treatment augments donor CD8 ⁺ T differentiation into Tmp cells and their Teff progeny; and that anti-IL-2-JES6 treatment is more effective than TAC-treatment for preserving the function of donor CD8 ⁺ T cells in the lymphoid tissues of recipients.

REFERENCES

1. Li JM, Giver CR, Lu Y, Hossain MS, Akhtari M, Waller EK. Separating graft- versus-leukemia from graft-versus-host disease in allogeneic hematopoietic stem cell transplantation. Immunotherapy. 2009; 1 (4): 599-621.

2. Sung AD, Chao NJ. Acute graft-versus-host disease: are we close to bringing the bench to the bedside? Best Pract Res Clin Haematol. 2013;26(3):285-292.

3. Ebens CL, Maillard I. Notch signaling in hematopoietic cell transplantation and T cell alloimmunity. Blood Rev. 2013;27(6):269-277.

4. Kanakry CG, Fuchs EJ, Luznik L. Modem approaches to HLA-haploidentical blood or marrow transplantation. Nat Rev Clin Oncol. 2016; 13(1 ): 10-24.

5. N egrin RS. Graft-versus-host disease versus graft-versus-leukemia. Hematology Am Soc Hematol Educ Program. 2015;2015:225-230.

6. Chang YJ, Zhao XY, Huang XJ. Strategies for Enhancing and Preserving Antileukemia Effects Without Aggravating Graft-Versus-Host Disease. Front Immunol. 2018;9:3041.

7. Sofi MH, Wu Y, Schutt SD, et al. Thioredoxin-1 confines T cell alloresponse and pathogenicity in graft-versus-host disease. J Clin Invest. 2019;129(7):2760-2774.

8. Choi J, Cooper ML, Staser K, et al. Baricitinib-induced blockade of interferon gamma receptor and interleukin-6 receptor for the prevention and treatment of graft- versus-host disease. Leukemia. 2018;32(11 ):2483-2494.

9. Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ. Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity. 2007;27(1):111-122.

10. Cassady K, Martin PJ, Zeng D. Regulation of GVHD and GVL Activity via PD- L1 Interaction Wth PD-1 and CD80. Front Immunol. 2018;9:3061.

11. Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000; 192(7): 1027-1034.

12. Sharpe AH, Pauken KE. The diverse functions of the PD1 inhibitory pathway. Nat Rev Immunol. 2018; 18(3): 153-167.

13. Ni X, Song Q, Cassady K, et al. PD-L1 interacts with CD80 to regulate graft- versus-leukemia activity of donor CD8+ T cells. J Clin Invest. 2017; 127(5): 1960-1977. 14. Carter L, Fouser LA, Jussif J, et al. PD-1 :PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2. Eur J Immunol. 2002;32(3):634- 643.

15. Sykes M, Romick ML, Sachs DH. Interleukin 2 prevents graft-versus-host disease while preserving the graft-versus-leukem ia effect of allogeneic T cells. Proc Natl Acad Sci U S A. 1990;87(15):5633-5637.

16. Andolfi G, Fousteri G, Rossetti M, et al. Enforced IL-10 expression confers type 1 regulatory T cell (Tr1 ) phenotype and function to human CD4(+) T cells. Mol Then 2012;20(9): 1778-1790.

17. Zhang P, Lee JS, Gartlan KH, et al. Eomesodermin promotes the development of type 1 regulatory T (TR1) cells. Sci Immunol. 2017;2(10).

18. Blazar BR, MacDonald KPA, Hill GR. Immune regulatory cell infusion for graft- versus-host disease prevention and therapy. Blood. 2018;131 (24):2651-2660.

19. Schneidawind D, Pierini A, Negrin RS. Regulatory T cells and natural killer T cells for modulation of GVHD following allogeneic hematopoietic cell transplantation. Blood. 2013;122(18):3116-3121.

20. Koreth J, Matsuoka K, Kim HT, et al. Interleukin-2 and regulatory T cells in graft- versus-host disease. N Engl J Med. 2011 ;365(22):2055-2066.

21. Hongo D, Tang X, Dutt S, Nador RG, Strober S. Interactions between NKT cells and Tregs are required for tolerance to combined bone marrow and organ transplants. Blood. 2012; 119(6): 1581 -1589.

22. Xhaard A, Moins-Teisserenc H, Busson M, et al. Reconstitution of regulatory T- cell subsets after allogeneic hematopoietic SCT. Bone Marrow Transplant. 2014;49(8): 1089-1092.

23. Amarnath S, Mangus CW, Wang JC, et al. The PDL1-PD1 axis converts human TH1 cells into regulatory T cells. Sci Transl Med. 2011 ;3(111 ): 111 ra120.

24. Zhang Y, Joe G, Hexner E, Zhu J, Emerson SG. Host-reactive CD8+ memory stem cells in graft-versus-host disease. Nat Med. 2005; 11 (12): 1299-1305.

25. He S, Liu Y, Meng L, et al. Ezh2 phosphorylation state determines its capacity to maintain CD8(+) T memory precursors for antitumor immunity. Nat Commun. 2017;8(1 ):2125. 26. Spangler JB, Tomala J, Luca VC, et al. Antibodies to lnterleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms. Immunity. 2015;42(5):815-825.

27. Boyman 0, Kovar M, Rubinstein MP, Surh CD, Sprent J. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science. 2006;311 (5769): 1924- 1927.

28. Matte-Martone C, Venkatesan S, Tan HS, et al. Graft-versus-leukemia (GVL) against mouse blast-crisis chronic myelogenous leukemia (BC-CML) and chronicphase chronic myelogenous leukemia (CP-CML): shared mechanisms of T cell killing, but programmed death ligands render CP-CML and not BC-CML GVL resistant. J Immunol. 2011 ;187(4):1653-1663.

29. Wherry EJ, Kurachi M. Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol. 2015; 15(8):486-499.

30. Tugues S, Amorim A, Spath S, et al. Graft-versus-host disease, but not graft- versus-leukemia immunity, is mediated by GM-CSF-licensed myeloid cells. Sci TransI Med. 2018; 10(469).

31. Gartlan KH, Koyama M, Lineburg KE, et al. Donor T-cell-derived GM-CSF drives alloantigen presentation by dendritic cells in the gastrointestinal tract. Blood Adv. 2019;3(19):2859-2865.

32. Zander R, Schauder D, Xin G, et al. CD4(+) T Cell Help Is Required for the Formation of a Cytolytic CD8(+) T Cell Subset that Protects against Chronic Infection and Cancer. Immunity. 2019;51 (6): 1028-1042 e1024.

33. Chen Z, Ji Z, Ngiow SF, et al. TCF-1 -Centered Transcriptional Network Drives an Effector versus Exhausted CD8 T Cell-Fate Decision. Immunity. 2019;51(5):840- 855 e845.

34. Gallon L, Traitanon O, Yu Y, et al. Differential Effects of Calcineurin and Mammalian Target of Rapamycin Inhibitors on Alloreactive Th1 , Th17, and Regulatory T Cells. Transplantation. 2015;99(9): 1774-1784.

35. Rosenbeck LL, Kiel PJ, Kalsekar I, et al. Prophylaxis with sirolimus and tacrolimus +/- antithymocyte globulin reduces the risk of acute graft-versus-host disease without an overall survival benefit following allogeneic stem cell transplantation. Biol Blood Marrow Transplant. 2011 ;17(6):916-922. 36. Malek TR, Castro I. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity. 2010;33(2):153-165.

37. Zheng H, Matte-Martone C, Li H, et al. Effector memory CD4+ T cells mediate graft-versus-leukem ia without inducing graft-versus-host disease. Blood. 2008;111 (4):2476-2484.

38. Zheng H, Matte-Martone C, Jain D, McNiff J, Shlomchik WD. Central memory CD8+ T cells induce graft-versus-host disease and mediate graft-versus-leukem ia. J Immunol. 2009; 182(10): 5938-5948.

39. Zhang P, Wu J, Deoliveira D, Chao NJ, Chen BJ. Allospecific CD4(+) effector memory T cells do not induce graft-versus-host disease in mice. Biol Blood Marrow Transplant. 2012; 18(10): 1488-1499.

40. Bleakley M, Heimfeld S, Loeb KR, et al. Outcomes of acute leukemia patients transplanted with naive T cell-depleted stem cell grafts. J Clin Invest. 2015;125(7):2677-2689.

41. Trotta E, Bessette PH, Silveria SL, et al. A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism. Nat Med. 2018;24(7):1005-1014.

42. Chakraverty R, Cote D, Buchli J, et al. An inflammatory checkpoint regulates recruitment of graft-versus-host reactive T cells to peripheral tissues. J Exp Med. 2006;203(8):2021-2031.

43. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16(5):284-287.