Fig 2: 6-Phosphogluconate dehydrogenase (6PGD) blockade in regulatory T cells (Tregs) prevents their suppressive function and induces potent anti-tumor responses.(A–B) Wild-type (WT) Foxp3Cre naïve CD4+ T cells (CD62Lhigh CD44low) were driven toward Tregs in vitro (as inducible Treg [iTreg]) along with treatment with 6-aminonicotinamide (6-AN) or vehicle DMSO and Treg drive efficacy was evaluated as CD25+YFP+ cells. YFP+ percentage in CD4+ T cells (A) and YFP gMFI (B) are demonstrated. (C) Real-time PCR analysis on derived Tregs demonstrate lower expression of Foxp3 transcription factor and higher expression of Gata3 transcription factor and IL-5 under treatment with 6-AN versus vehicle DMSO. (D–E) 6-AN treatment of driven Tregs demonstrate lower suppressive capacity evaluated in in vitro suppression assay. YFP+ cells were sorted for the suppression assay. (F) Tamoxifen treatment schedule and tumor induction by implanting B16F10 cells in Pgdfl/fl Foxp3EGFP-Cre-ERT2 and WT mice. (G) Flow cytometry analysis show the same frequency of Tregs (EGFP+) in CD4+ cells in spleen of both mouse groups 10 days post tamoxifen treatment. (H) Suppression assay on sorted EGFP+ Tregs as described in supplemental information. (I–J) 6PGD blockade in Tregs resulted in lower tumor volume (I) and tumor weight (J). (K–L) Tumor of the mouse models from (F) were evaluated for infiltration of both CD4+ and CD8+ T cells on day 16 post implantation, which were higher in 6PGD blocked mice, as shown by representative flowgram (K) and bar graphs (L). (M–O) Tumor infiltrating CD4+ (M) and CD8+ (N) T cells showed higher capacity of IFN-? production on day 16. Results are from two independent experiments with N = 8 per group. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: Treg expansion results from a T cell-intrinsic action of Ikbkbmut.Reciprocal bone marrow chimera experiment (Ikbkbmut/mut recipients, n = 4; Ikbkb+/+ recipients, n = 9), showing experimental design (a), proportions of CD25+Foxp3+ Tregs in indicated chimeric strains (b), serum IL-17A, TNFα and IFNγ concentrations (c) (p values from Student’s t-tests), and representative histology of tail, tarsals, and ankles of chimeras (scale bars, 1000 μm for tails and tarsals, 500 μm for ankles) (d). The transparent bar in b shows mean Treg proportions observed in intact Ikbkb+/+ mice (grey) and Ikbkbmut/mut mice (red) for comparison (as shown in Fig. 1e). Adoptive transfer experiment showing experimental design (e), Kaplan-Meier plots of mice remaining free of ear dermatitis after adoptive transfer of sorted Ikbkbmut/mut x Foxp3-GFP Tregs (f). Representative images (g) and sections of ears and digits stained by H&E after second phase transfer of Ikbkbmut/mut Tregs (h). Scale bars, ears, 200 μm; digits, 500 μm. n = 3 per group. Comparison of Kaplan Meier curves was performed using a log-rank test.

Fig 4: Knockdown of EphB4 in cancer cells combined with radiation treatment promotes suppressive intratumoral immune populations.A Experimental design for C57BL/6 J mice implanted with MOC2 cancer cells. The buccal tumor received single dose of radiation treatment (10 Gy) on day 7 after implantation. B–G Flow cytometric quantification of CD4 + T cells (B), regulatory T cells (Tregs) defined by CD4 + CD25+ (C) and CD4 + CD25+ Foxp3+ expression (D), myeloid-derived suppressor cells (MDSCs) defined by CD45 + Ly6G + Ly6C+ (E), CD45 + CD11b+ Ly6Ghigh Ly6Clow (F), and CD45 + CD11b+ Ly6Glow Ly6Chigh (G) in the tumor microenvironment (TME) (Ctrl sh, n = 5; EphB4 sh n = 7). H Experimental design for coculture of LY2 Ctrl or EphB4 shRNA cancer cells with CD4 + T cells. Cancer cells were incubated with interferon-gamma (IFNg) for 48 h, then OVA peptide overnight. CD4 + T cells from DO11.10 BALB/c mice were then cocultured with the cancer cells for 72 hours and subsequently harvested for flow cytometry. I Flow cytometry quantification of Tregs defined by CD4+ Foxp3+ and CD4 + CD25+ Foxp3+ expression (n = 6 per group). J Flow cytometry quantification of Treg immunosuppressive function defined by IL-10 expression (n = 6 per group). K Flow cytometry quantification of CD4 + T cells treated with conditioned media from MOC2 control or EphB4 shRNA cells showing PD-1, CD69, and CD25 expression (n = 5 per group). The experiments were performed once with their own biological replicates. Comparison between control and experimental groups was done using a Mann Whitney test for the in vivo experiment and a two-sided student’s t-test for the in vitro experiments. Significance was determined if the p-value was < 0.05*, < 0.01**, < 0.001***, and < 0.0001****. p-values are indicated for the figures (B) ns p = 0.3636, (C) **p = 0.0025, (D) **p = 0.0051, (E) *p = 0.0177, (F) *p = 0.0101, (G) *p = 0.0101, (I) CD3 + CD44+Foxp3+ ****p < 0.0001; CD3 + CD4 + CD25+Foxp3+ *p = 0.0394, (J) **p = 0.0027, (K) ****p < 0.0001. The error bars represent the standard error of the mean ( ± SEM).

Fig 5: Acute induction of Ezh2Y641F in Treg cells increases their activation and effector differentiation state(A) Generation of Treg.iEzh2Y641F model.(B) Normalized H3K27me3/histone H3 levels of CD4+Foxp3+ Treg cells in LN.(C) Frequencies (left) and numbers (right) of Treg cells in LN.(D–F) Frequencies of TIGIT+ or PD1+ (D); CD103+, CD69+, or CD25+ (E); and CXCR3+, CCR8+, and CCR6+ (F) Treg cells in LN.Data are mean ± SEM (n = 6–18 mice per group pooled from two independent experiments). Unpaired two-tailed Student’s t test was used; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.See also Figure S4.