Fig 2: UTX demethylase activity is not required for effector CD8+ T cell function during chronic infectionWT, UTX-KI (catalytic dead knockin), and UTX-KO P14+CD8+ T cells were introduced into the same recipients, followed by CD4 depletion and infection with LCMV-A22. At day 8 post-infection, splenic donor P14T cells were analyzed for surface marker expression, transcription factor levels, and cytokine expression.(A) Illustration of the approach.(B) The three donor P14 T cell populations and host CD8+ T cells were identified by surface expression Ly5a, Ly5b, and Thy1.1.(C) Frequency of each donor P14 group among all donor CD8+Ly5a+ P14 T cells in spleen. Lines connect pairs of WT, UTX-KI, and UTX-KO P14T cells present in each recipient.(D) Histogram plots and gMFI for the transcription factors T-bet, Eomes, Tox, Tcf1, and Bcl2.(E and F) The fraction of donor P14 T cells producing granzyme (E) and the gMFI of granzyme expression among granzyme-positive cells (F).(G and H) The fraction of donor cells making IFN? (G) and the gMFI among IFN?-positive cells (H).(I) The distribution of IFN?+ (left) and IFN?- (right) donor cell populations after re-stimulation with GP33–41 peptide in an ICCS assay. The left graph shows the distribution of donor cells among the IFN?+ cells; the right graphs shows the distribution of donor cells that failed to make IFN?.(J) The histograms show several activation and inhibitory receptors expressed by IFN?+ donor cells.(K) The surface expression of Ly108 and CD69 on each donor cell population was used to identify Texprog1, Texprog2, Texint, and Texterm subsets.(L) Distribution of the four developmental stages of exhaustion for donor cell populations. Each circle represents 1% of the total population.Data show one experiment with five recipient mice. Samples from the five recipient mice were concatenated to generate histograms. Significance was determined by a paired Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).See also Figure S7.

Fig 3: Characterization of Tet2/3-deficient Treg cells. a Representative flow cytometry analyses of the percentage of Treg cells (% of Foxp3+ cells in gated CD4+ T cells) in spleen and pLN from WT and Tet2/3fl/flFoxp3Cre DKO mice (13–16 weeks-old). b Quantification of the percentages (left panel) and cell numbers (right panel) of Treg cells in spleen, pLN and mLN from WT and Tet2/3fl/flFoxp3Cre DKO mice (13-16 weeks old, n = 9). c–f Flow cytometry analysis of Tet2/3-deficient Treg cells from spleen and pLN for the expression of CD25 and Nrp1 (c); Helios, ICOS, CD103, GITR, CTLA4, PD1 and CD127 (d); CD62L versus CD44 (e); and CD69 (f). Shown are WT CD4+ T cells (shaded gray); WT Tregs (black line); DKO Tregs (red line). Statistical analysis was performed using two-tailed unpaired student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Error bars show mean ± s.d. from at least three independent experiments

Fig 4: Clotrimazole inhibited tumor growth by promoting antitumor immunity. (A) BMDCs were treated with clotrimazole (10 µM) or DMSO for 20 hours, then incubated with live B16-OVA cells for 24 hours, followed by co-culture with B3Z cells for an additional 24 hours. The LacZ activity and IL-2 production were measured. (B) B6 mice bearing MC38 tumors were treated with clotrimazole or vehicle on day 3–7 (40 mg/kg, i.p.), and tumor growth was monitored. n=5 per group. (C–F) B6 mice bearing MC38 tumors were treated with clotrimazole or vehicle on days 3–7 (40 mg/kg, i.p.), Tumors were isolated on day 13, and tumor-infiltrating immune cells were analyzed by flow cytometry. Shown are tumor weight (C), numbers of tumor-infiltrating CD4+ T cells (D), CD8+ T cells (E), CD11c+ dendritic cells (F), CD69 and PD1 expression on CD8+ T cells (G–H). (n=5 for C–F, n=4 for G–H). (I.) Balb/c nude mice bearing MC38 tumors were treated with clotrimazole or vehicle on days 3–7 (40 mg/kg, i.p.), and tumor growth was monitored. n=5 per group. (J) B6 mice were injected with anti-CD8 depletion antibody on days 3, 6, and 9 after MC38 tumor inoculation, followed by clotrimazole treatment on day 3–7 (40 mg/kg, i.p.), and tumor growth was monitored. n=5 per group. (K) B6 background WT or Batf3-/- mice bearing MC38 tumors were treated with clotrimazole or vehicle on day 3–7 (40 mg/kg, i.p.), and tumor growth was monitored. n=5 per group. Tumor volume is shown as mean±SD, *p<0.05, **p<0.01, ns=not significant, by two-way analysis of variance with Bonferroni’s post-test (B, I–J) or unpaired Student’s t test (A, C–H). BMDC, bonemarrow-derived dendritic cell; CLT, clotrimazole; DMSO, dimethyl sulfoxide.

Fig 5: High dimensionality analysis reveals KFE8-Ag85B-dependent shift in Trm surface marker profile of Ag85B-specific CD4+ T cells in BCG-primed and boosted mice.a We gated for tetramer+ CD4+ T cells in each group. Data shown as mean ± SE. Significance was determined by one-way ANOVA followed by a Benjamini and Hochberg test. b The tetramer+ cells from each group were analyzed as a whole and visualized in a tSNE plot. The cells isolated from each treatment group are highlighted c and heatmaps were generated to describe intensity of specific markers CD44, CD62L, CD69, CD45, and CXCR3. Area highlighted in light blue represents the cell populations most augmented by KFE8-Ag85B and KFE8-Ag85B-pulsed DCs. d Heat maps are representative of light blue population and include markers CD127, CD69, CXCR6, CD49a, and CD103. Dark blue regions of heat maps correspond with lowest the lowest intensity of fluorescent antibody staining, whereas red corresponds with the highest intensity and ranges from -1622 up to 262856.