and plated in DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 μg/mL thioguanine for clonogenic growth. After 4 days, cells were fixed with methanol and stained with crystal violet staining solution for counting colonies.Single-cell RNA-Seq data analysis. Data for single-cell RNA-Seq analysis has been previously published (59). We compared CD40L expression among CD4 + T cells in dLN, spleen, and tumors from day 10 and day 20 to produce a heatmap of relative gene expression.Statistics. All the experimental data analyses were performed with GraphPad Prism statistical software and shown as mean ± SEM. P value was determined by 2-way ANOVA for tumor growth, log-rank test for survival, and 2-tailed t tests or 1-way ANOVA for other analyses. When multiple comparisons were done, significance was adjusted by Holm-Šidák method. P < 0.05 was considered statistically significant.Study approval. Animal experiments were conducted according to guidelines set by the IACUC of the UT Southwestern Medical Center. We performed no human research.Author contributions Conceptualization was contributed by CM, YXF, and JQ. Methodology was contributed by CM, LL, JB, and HL. Investigation was contributed by CM, JB, and HL. Writing of the original daft was contributed by CM. Review and editing of the manuscript were contributed by CM, LL, JB, YXF, and JQ. Funding acquisition was contributed by YXF, JQ. Supervision was contributed by YXF and JQ. CM and JB are co-first authors. CM initiated this study and is therefore listed first.
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