Extended Data Fig. 9: CD28 agonism and CTLA-4 blockade, but not PD-1 blockade, drive loss of Treg cell functional stability.
From: CTLA-4 blockade drives loss of Treg stability in glycolysis-low tumours
a, b, Representative flow cytometry plots of in vitro assays reported in Fig. 4i, j. a, Representative proliferation (CellTraceViolet dilution) by flow cytometry of activated CD8+ T cells cultured alone or in the presence of Treg cells at the indicated glucose concentrations and treated with anti-CD28 (2 μg ml−1) or IgG control. b, Representative CD86 staining by flow cytometry on B cells from co-cultures with CD8+ T cells and Treg cells treated as in a. c, Proliferation of CD8+ T cells cultured alone or with Treg cells in 0.5 mM (grey) or 10 mM glucose (black) and activated with increasing concentrations of anti-CD28 (0–0.2 μg ml−1) (n = 3, representative of two independent experiments). d, Quantification and representative plots showing suppression of CD4+ T cell proliferation (left) and CD86 expression on B cells (right) by flow cytometry in culture with Treg cells treated with anti-CTLA-4, anti-PD-1 or an IgG control in complete RPMI1640 containing 11 mM glucose. Percentage suppression was calculated relative to proliferation of CD4+ T cells cultured alone in the same treatment conditions (n = 3; n = 1 experiment with anti-PD-1). e, Suppression of proliferation of CD8+ T cells cultured at the indicated ratios with FOXP3–GFP+PD-1+ Treg cells (top) or FOXP3–GFP+PD-1− Treg cells (bottom) FACS-sorted from spleens of naive FOXP3–GFP mice and incubated with anti-PD-1 or IgG control for 48 h (representative results from one experiment conducted with CD8+ and CD4+ as target T cells with similar results). f, Quantification and representative plots of GlucoseCy3 staining by flow cytometry in Treg cells activated as in Fig. 4c and treated with anti-PD-1 or IgG control (n = 3, representative of two independent experiments). g, Flow cytometry quantification and phenotypic analysis of Treg cells from 4T1-KD tumours treated with anti-CTLA-4, anti-PD-1 or IgG control as indicated (n = 10 mice per group, representative of two independent experiments). Data are mean ± s.d. (c, d, f) or mean ± s.e.m. (g). **P < 0.01; ***P < 0.001. P values determined by two-sided unpaired t-test.
