Fig. 2. Activated, OX40^hiGITR^hi T_reg are functionally suppressive and associate with nonresponse to PD-1 blockade.

(A) Functional analysis of OX40^loGITR^lo and OX40^hiGITR^hi TIL-T_reg–mediated suppression of conventional CD4⁺T cell (T_conv) proliferation. TIL-T_reg from patients MD017–0157, MD017–0092, MD017–0115, MD017–0124, and MD017–0116 were combined to ensure sufficient T_reg numbers for this experiment (n = 1). (B) Volcano plot showing differential expression between the Activated (1)/OX40^hiGITR^hi cluster (right) versus all other T_reg (left) when analyzing all tumor samples (n = 25). Each dot represents one gene. False discovery rate (FDR) < 0.05 is considered significant (blue/red dots). LAG3, TNFRSF18, and TNFRSF4 represent the top three most differentially expressed genes in the Activated (1)/OX40^hiGITR^hi T_reg (red dots). (C) Overlay of the Activated T_reg score on the TIL-T_reg UMAP for each response/treatment group. Red indicates higher expression; blue indicates lower expression. (D) The frequency of T_reg with a high T_reg activation score is shown for tumor (left) and adjacent NL (right) from nonresponders (NR; red), responders (R; blue), and treatment-naive patients (Tx-naive; green). Comparisons were performed at the individual patient level using Wilcoxon rank test. (E) Clonotype sharing pattern across T_reg subsets. The frequencies of T_reg TCR clones that were detected in at least two T_reg clusters were calculated and are shown on a heatmap. (F) Diffusion plot with RNA velocity for the Activated (1)/ OX40^hiGITR^hi and Activated (3) clusters (among which most clonotype sharing was observed). (G) Boxplots showing the relative proportion of Activated (1)/OX40^hi GITR^hi and Activated (3) clusters by R (blue), NR (red), and treatment-naïve status (green). (H) A violin plot shows TNFSF4 (OX40L) expression by neoantigen-specific (red) and flu-specific (blue) CD8 TILs from the same neoadjuvant-treated patients. (I) Violin plot comparing TNFSF4 (OX40L) expression by neoantigen-specific CD8⁺ TIL between nonresponding (red)and responding (blue) tumors. (J) Gene set enrichment analysis to evaluate differing biological functions of ex vivo OX40L, 41BBL, and GITRL agonism in sorted human TIL-T_reg. TIL-T_reg from patients MD043–011 and MD01–019 were combined to ensure sufficient T_reg numbers for this experiment (n = 1). NF-κB, nuclear factor κB.