Fig. 7. The gene expression profile of T_{TCR-MA1CD8/CD28mut} identified a balance between functional, exhaustion and persistence.

A GSEA pathway analysis of cytokine pathways (Biocarta), cytokine activity (GOMF), cytokine and inflammatory response (WP), and the Th1/Th2 pathway (Biocarta) of CD4 T_{TCR-MA1-CD8/28}, T_{TCR-MA1-CD8/28mut} normalized by T_{TCR-MA1-CD8αβ}. T_{TCR-MA1-CD8αβ}, T_{TCR-MA1-CD8/CD28}, and T_{TCR-MA1-CD8/CD28mut} were stimulated with 1 µM cognate peptide for 4 h and further analyzed by bulk RNA sequencing. p values were determined by two-sided Mann–Whitney U-test. B Schematic of experimental layout showing CD4 T_{TCR-MA1-CD8/28} or T_{TCR-MA1-CD8/28mut} incubated with irradiated ME275 for 21days, with tumor rechallenge every 7 days. Cells were stimulated with 1 µM cognate peptide for 18 h or without stimulation and further analyzed by flow cytometry and bulk RNA-seq downstream analysis. Image created in BioRender. Tang, A. (2026) https://BioRender.com/f64l203. C PCA plot of bulk RNA-seq data, showing the first two principal components (PC1 and PC2). Data points represent individual samples, color-coded by condition to visualize any separation based on gene expression profiles. Ellipses, drawn at a 90% confidence level, indicate the range of variation within each condition, highlighting how closely related or distinct the three groups are from each other. D Heatmap showing the expression levels of differentially expressed genes across constructs (T_{TCR-MA1-CD8/CD28}, T_{TCR-MA1-CD8/CD28mut}) and stimulations (unstimulated vs. stimulated) in the rlog-transformed data. Rows represent genes, and columns represent individual samples (3 biological replicates per condition). Expression values are color-coded from low (blue) to high (red) based on z-scores. The dendrograms on the axes indicate hierarchical clustering of genes and samples. E Boxplots showing the z-scores for the expression of manually curated gene signatures of proliferation in unstimulated and stimulated conditions. The z-scores were calculated from the averaged normalized counts of significant genes (p_adj <0.05, log₂ fold-change > 0.5). Each box represents the distribution of z-scores for a specific gene signature in the unstimulated and stimulated groups for T_{TCR-MA1-CD8/CD28} and T_{TCR-MA1-CD8/CD28mut}. Boxplots show the IQR; lines denote medians. The bounds of the box represent the 25th and 75th percentiles; whiskers span 1.5x IQR. F Boxplots showing the z-scores for the expression of manually curated gene signatures of exhaustion across unstimulated and stimulated conditions. The z-scores were calculated from the averaged normalized counts of significant genes (padj <0.05, log2 fold-change > 0.5). Each box represents the distribution of z-scores for a specific gene signature in the unstimulated and stimulated groups for T_{TCR-MA1-CD8/CD28} and T_{TCR-MA1-CD8/CD28mut}. Boxplots show the IQR; lines denote medians. The bounds of the box represent the 25th and 75th percentiles; whiskers span 1.5x IQR. G Dot plot summarizing GSEA results for the ‘Apoptosis’ pathway across multiple comparisons. The x-axis represents the pathway of interest, while the y-axis denotes the pairwise comparisons between experimental conditions. Dot size corresponds to the -log10-transformed p-value, indicating the significance of enrichment, and dot color represents the normalized enrichment score (NES), with red indicating positive enrichment and blue indicating negative enrichment. Enrichment p-values were calculated by two-sided permutation testing. H Flow cytometry quantification of Tim3⁺ PD-1⁺ double positive populations from T_{TCR-MA1-CD8/28} and T_{TCR-MA1-CD8/28mut} groups was measured at the end of the 21 days stimulation with irradiated ME275 incubation. The graph represents mean ± SD. p-value determined by a two-tailed unpaired t-test. (n = 3 wells/group). I Flow cytometry quantification of IFNγ⁺ and TNFα⁺ expression in T_{TCR-MA1-CD8/28} and T_{TCR-MA1-CD8/28mut} group was measured at the end of the 7 days stimulation with irradiated ME275 incubation. The graph represents mean ± SD. p value determined by one-way ANOVA with Tukey’s multiple comparison test. (n = 3 wells/group).