Fig. 3: Combined PD-L1 and TGFβ blockade allows T_SCL expansion in the tumor.

a UMAP as in Fig. 2a, here colored by cell density. Red indicates high cell density, blue low density. b Quantification of the frequency of cells in cluster T0 and T1 (y axis; n = 5 per group of treatment from one experiment) in each animal (dots) by treatment group (x axis). c Gating strategy for flow cytometry analysis of CD8 T cells at day 7 after initiation of treatment. Representative plots of control and combo samples. d Flow cytometry quantification of CD8 T_SCL (PD1^lowLAG3^-) and T_PEX cells (PD1^hiLAG3⁺TIM3^-) (cells per mg of tissue, fold change over average of the control group: y axis; groups: x axis; data from three independent experiments, ctrl n = 19, aPD-L1 n = 20, aTGFb n = 14, combo n = 17). e Heatmap of relative average expression of selected genes in CD8 T cells for each treatment group. f Signature score in cells (dots) from cluster T5 based on genes enriched in cluster T1. P value is from paired Wilcoxon rank-sum tests (two-sided) comparing between treatment conditions. Control not included due to low number of cells in cluster T5. (aPD-L1 n = 420 cells, aTGFb n = 326 cells, Combo n = 284 cells; all conditions include five animals). g Flow cytometry quantification of EdU⁺ T_SCL (cells per mg of tissue, fold change over average of the control group: y axis; groups: x axis; data from two independent experiments, ctrl n = 14, aPD-L1 n = 15, aTGFb n = 14, combo n = 12). h Group fit curves of EMT6 tumor growth treated with FTY720 in addition to control, anti-PD-L1 and combo. Two independent experiments (10 animals in each experiment, mean ± SEM (see Supplementary Fig. 4i for individual animal curves)). Source data are provided as a Source Data file. Ctrl = control (anti-GP120), aPD-L1 = anti-PD-L1, aTGFb = anti-TGFβ, combo = combination anti-PD-L1 + anti-TGFβ; Adj P values *<0.1, **<0.05, ***<0.01, ****<0.001 Dunn’s test (two-sided) with Benjamini–Hochberg multiple testing correction. Adjusted P values for (b): 0.005517513 (ctrl vs combo T0), 0.030889647 (aTgfb vs combo T0), 0.08364055 (ctrl vs combo T1). Adjusted P values for (d): 0.000512 (ctrl vs combo), 0.0679 (aPD-L1 vs combo), 0.00197 (aTGFb vs combo), 0.0841 (ctrl vs aPD-L1). Adjusted P values for (g): 0.00347 (ctrl vs combo), 0.00308 (aTGFb vs combo). b, d, f, g Whiskers represent the minimum and maximum (unless points extend 1.5 * IQR from the hinge, then shown as individual points), the box represents the interquartile range, and the center line represents the median.