Fig 2: Expression of the PD-1 and CTLA-4 checkpoints in each subpopulation of different immune tissues at each stage of tumor progression. The different colors represent different levels of expression. The differential expression of PD-1 and CTLA-4 in immune cell subpopulations in different immune tissues isolated from the Colon26 animal model. Different expression levels of PD-1 and CTLA-4 in each subpopulation in TILs (A), lymph nodes (B), spleen (C), and peripheral blood samples (D) in the Colon26 animal model. The statistical analysis in this figure was obtained by analyzing the raw data. The dendrogram was plotted from the raw data of each subpopulation and biomarker using the R language.

Fig 3: Elevated Inhibitory Receptor Expression Corresponds with Decreased Glucose Uptake in CD8+ T Cells(A and B) Proportion of total or Mtb-specific (TB10.4-TET+) CD8+ T cells expressing (A) PD-1 or (B) CTLA-4 over the course of Mtb or BCG infection.(C) Proportion of cells co-expressing PD-1 and CTLA-4 at 12 weeks post-infection.(D) Proportion of KLRG-1-expressing cells during infection.(E and F) Glucose uptake measured by incorporation of the glucose analog 2-NBDG in total or TB10.4-TET+ CD8+ T cells (E) along with representative histograms (F).(G and H) 2-NBDG uptake in CD8+ T cells expressing (or not expressing) PD-1 (G) with representative histogram (H) at 12 weeks post-infection.All data represent CD8+ T cells purified from the lungs of uninfected (UI), Mtb-infected, or BCG-infected mice; TB10.4-TET+ cells are from Mtb-infected mice. 2-NBDG, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose. Statistics, unless otherwise indicated, are relative to UI. Data are representative of three independent experiments (n = 5 mice per group). “p = 0.05, #p = 0.01 8p = 0.005, and *p = 0.001 by one-way ANOVA or unpaired Student’s t test. Error bars are mean ± SD.

Fig 4: Expression of PD-1 and CTLA-4 checkpoints in each subpopulation of different immune tissues at different stages of the tumor progression. The different colors represent the different levels of expression. The differential expression of PD-1 and CTLA-4 in immune cell subpopulations in different immune tissues isolated from the CT26 animal model in Balb/c female mice. (A) Different expression levels of PD-1 and CTLA-4 in each subpopulation in TILs. (B) Different expression levels of PD-1 and CTLA-4 in each subpopulation in the lymph nodes. (C) Different expression levels of PD-1 and CTLA-4 in each subpopulation in the spleen (no sufficient data could be gathered for the MFI of CTLA-4 in the different subpopulations when the tumor size was 400 mm3 and 800 mm3). (D) Different expression levels of PD-1 and CTLA-4 in each subpopulation in the peripheral blood samples in the CT26 animal model. The statistical analysis in this figure was obtained by analyzing the raw data. The dendrogram was plotted from the raw data of each subpopulation and biomarker using the R language.

Fig 5: Expression of suppressive markers is upregulated on CD4+ Tregs and is downregulated on CD4+CD8+ Tregs in the thymus of VISTA-/- mice. Median fluorescence intensity (MFI) of (A) Foxp3, (B) CTLA4, (C) CD25, and (D) CD69 on CD4+ Tregs in the thymus. Median fluorescence intensity (MFI) of (E) Foxp3, (F) CTLA4, (G) CD25, and (H) CD69 on CD4+CD8+ Tregs in the thymus. Summary graphs show mean ± SEM [WT-sham: n = 3, WT-CLP: n = 3, VISTA-/-sham: n = 4, VISTA-/- -CLP: n = 4]; significance *p < 0.05; **p < 0.01; ***p < 0.001.