Figure S2.

Th17 CAR T cells are indispensable for DMXAA therapeutic efficacy, which enhances the number and cytotoxicity of Th17 CAR T cells in the TME. (A and B) Tables for the percentage of T cell subsets (A) and macrophage and myeloid cell subsets (B) calculated from scRNA-seq data. (C) Table for the percentage of CD8⁺ T cells expressing indicated genes calculated from scRNA-seq data. (D–F) Assessment of CAR expression on tumor-infiltrating T cells isolated 7 d after therapy by flow cytometry. (D) Determination of change in tumor area 7 d after injection of Tc17 or Th/Tc17 CAR T cells. (E) Assessment of intratumoral CD8⁺ CAR T cells was performed 7 d after therapy. (F) Absolute number of CD8⁺ CAR T cells/mm² tumor 7 d after therapy. Data in G–M data were derived from the characterization of T cells expressing CD4 from scRNA-seq result. (G) CD4⁺ T cells were subdivided based on absence of Cd44 (naive T cells), expression of Cd4 and Cd44 (activated CD4 T cells), Cd4 and Foxp3 (T reg cells). CD4⁺ T cells were compared between the +DMXAA and −DMXAA treatment groups, and each subpopulation was examined further as described below. (H) Table for the percentage of CD4⁺ T cells expressing the indicated genes from the scRNA-seq data. (I) t-SNE plot of CD4⁺ T cells for Th17-related genes. (J) t-SNE plot of CD4⁺ T cells for Th17/Th1-related genes. (K) Violin plot shows the distribution and change of indicated gene expression in B and C. (L) Validation of single-cell data using flow cytometry to detect CD4⁺ CAR T cells. (M) Analysis of the correlation between tumor growth change and absolute cell number of CAR-expressing CD4 T cells/cm² tumor. Data (excluding scRNA-seq) represent one of two independent experiments (n ≥ 5 mice per group). Single-cell sequencing data represent three mice/treatment group, with statistical significance determined by differential GSA with the Partek flow workstation. *, q < 0.05; **, q < 0.01, in the change of gene expression level. Fold change is +DMXAA versus −DMXAA. For percentage/number change, please refer to Fig. S2 H. Flow cytometry data were pooled from two independent experiments and shown as mean ± SD. Statistics analysis for cytometric analysis is determined by Student’s t test; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.