Fig. 4. CCR enhances tumor control and T cell survival in vivo.

A Representative of tumor burden measurements (left) for NSG mice engrafted with 1 × 10⁵ cells A375F and subsequently infused with a total of 5 × 10⁶ CD4 and CD8 T_{TCR-MA1-CD8αβ} CCR cells (T_{TCR-Irr.-CD8/CD28}, T_{TCR-MA1-CD8αβ}, T_{TCR-MA1-CD8/CD28)} at a ratio of 1:1 12 days post engraftment. Final tumor volume of tumor-bearing mice (right panel) between days 6–8 after infusion (n=9 mice/group, 2 tumors/mouse averaged, from 3 separate experiments). p value determined by Kruskal-Wallis with Dunn’s multiple comparison test. All data are presented as mean ± SEM. B Representative IHC images of A375F tumors resected from NSG mice (same experiment design as A) showing DAPI (blue), CD4 (red), and CD8 (green) T cells of 4 groups: No T cells, irrelevant TCR (T_{TCR-Irr.-CD8/CD28}), CD8αβ (T_{TCR-MA1-CD8αβ}), and CD8/CD28 CCR (T_{TCR-MA1-CD8/CD28}). 2X and 10X magnification. Scale bars represent 100 µm and 500 µm. The white box represents the area enlarged in the 500 µm images underneath the top images. C–F Tumor-infiltrating T cells to tumor cells ratio (C) from resected A375F tumors from NSG mice infused with T_{TCR-Irr.-CD8/CD28}, T_{TCR-MA1-CD8αβ}, or T_{TCR-MA1-CD8/CD28}. The indicated p-value was determined by a Mann–Whitney unpaired two-tailed t-test. CD4 TCR-T cells and tumor cells ratio (D), p value determined by Welch’s unpaired two-tailed t-test. CD8T cells and tumor cells ratio (E), p value determined by Mann-Whitney unpaired two-tailed t-test. CD4 and CD8 T cell ratio (F), p value determined by Welch’s unpaired two-tailed t-test. Analysis performed using HALO Link software on IHC slides. (n = 3 mice, 6 tumors/group, 2 tumors/mouse). All data are presented as mean ± SEM. G Bar graph showing PD-1⁺/Tim3⁺, CD39⁺/Tim3⁺, and PD-1⁺/TIGIT⁺ frequency of CD4 and CD8 TILs between T_{TCR-MA1-CD8αβ} and T_{TCR-MA1-CD8/CD28}. Single cell suspensions were obtained at sacrifice, 8 days after T cell infusion from (same experiment design as A). p value determined by unpaired two-tailed t-test. (n = 3 mice, 6 tumors/group, 2 tumors/mouse). The graph represents mean ± SD. H Quantification of IFNγ⁺/TNFα⁺ populations after 1 μM cognate peptide stimulation for 18 h ex vivo across the same experimental groups from (A) in CD4 (left panel) and CD8 (right panel) T_{TCR-MA1-CD8αβ} for the indicated conditions. Single cell suspensions were obtained at sacrifice, 6 days after T cell infusion. Graph represents mean ± SD. Unpaired two-tailed t-test. (n = 3 mice, 5-6 tumors/group, 2 tumors/mouse). I NSG mice were engrafted with 1 × 10⁵ cells A375F and subsequently infused with a total of 5 × 10⁶ CD4 and CD8 T_{TCR-MA1-CD8αβ} CCR cells (T_{TCR-Irr.-CD8/CD28}, T_{TCR-MA1-CD8αβ}, T_{TCR-MA1-CD8/CD28}) at a ratio of 1:1 6 days post engraftment. Survival analysis was carried out when tumor burden reached 500 mm³ and assessed tumor progression occurring 44 days vs. 23 days vs. 16 days post T cell infusion, respectively; the Kaplan-Meier test for analysis of survival was applied. p values determined by a log-rank test. (n = 6 mice/group). Panels A & I were created in BioRender. Tang, A. (2026) https://BioRender.com/6wg9k7f.