Fig. 5: RT + CTLA4i combination modulates the phenotype of intratumoral T cells.

BALB/c mice were injected with 4T1 tumors (n = 10 biologically independent mice/group except control n = 9), treated with RT (8 GyX3), CTLA4i or the combination as described in Supplementary Fig. 5. 2 days after the last dose of CTLA4i (day 22) tumors were collected and the immune infiltrate analyzed by flow cytometry. a Intra-tumoral density of CD8⁺ T cells and (b) CD44⁺ CD62L^- antigen-experienced CD8⁺ T cells. c Expression of activation markers by antigen-experienced CD8⁺ T cells in RT + CTLA4i treated tumors. Data are mean ± SD. d High dimensional analysis of the CD8⁺ T cell infiltrate of RT + CTLA4i treated tumors. CD8⁺ CD44⁺ CD62L^- PD1⁺ T cells were down sampled to 1000 cells per sample and concatenated. opt-sne was run using standard imputs (perplexity = 30, iterations = 1000) based on 4 markers (PD1, Lag3, TIGIT, Tim3). FlowSOM-based metaclusters (Mc) are overlaid on the opt-sne 2D plot as a color dimension. e Frequency of the 4 metaclusters among antigen-experienced-PD1⁺ CD8⁺ T cells. Boxplots show the median and interquartile intervals, whiskers indicate minimum and maximum values of 8 tumors from the RT + CTLA4i group. f Intra-tumoral density of CD4⁺ T cells; (g) conventional over regulatory (Foxp3⁺ CD25⁺) CD4⁺ T cells ratio. h Frequency of activated (CD69⁺) conventional CD4⁺ T cells out of intra-tumoral CD4⁺ T cells. i–l Percentage of OX40⁺ and GITR⁺ conventional (h, i) and regulatory (j, k) CD4⁺ T cells. Lines indicate median and each dot represent one animal. Kruskal–Wallis test and post-hoc Dunn’s test were performed on each individual panel *, **, ***, and ****, indicate p-values < 0.05, 0.01, 0.001, and 0.0001, respectively. Source data and exact p values are provided in the Source Data file.