Figure 5.

Tumor-specific CD4⁺ T cells have a synergistic therapeutic effect with PD-L1 blockade. (A) Representative image of lung samples harvested from tumor-bearing C57BL/6J mice (n=6/group) which were treated with 2 × 10⁶ activated SMARTA cells or PD-L1 blockade therapy alone or the two combined and sacrificed on Day 7 post transfer (endpoint). In PBS-treated group, a mouse died before the endpoint and metastatic foci on the mouse lung was calculated as the same value as the most severe one (similarly hereinafter). The numbers of the metastatic foci are summarized beside. (B) Representative image of lung samples harvested from tumor-bearing Cd8 ^–/– mice which were treated with PBS (n=6) or PD-L1 monoclonal antibody (n=5) or SMARTA cells alone (n=5) or in combination (n=5) on Day 8 post tumor inoculation and were sacrificed on Day 15. In PD-L1 blockade-treated group, a mouse died before the endpoint. The statistical analysis of the metastatic foci is shown beside. (C–E) Flow cytometry analysis of transferred CD45.1⁺ CD44⁺ SMARTA cells in Cd8 ^–/– mice with or without PD-L1 blockade therapy. The frequencies and absolute numbers of SMARTA cells (D) and the statistical analysis of cytokine production of SMARTA cells (E) in the two groups. (F, G) Tumor-bearing C57BL/6J mice (n=5/group) were transferred with SMARTA cells or PBS on Day 8 post tumor challenge, followed by three doses of FTY720 (25 µg intraperitoneal injection) or PBS treatment from Day 9 to Day 15. Representative flow cytometry plots of CD4⁺ and CD8⁺ T cells in the metastatic lung tissue after FTY-720 treatment analyzing the blocking efficacy of FTY-720 (F). The image of lung tissue harvested from mice of indicated groups with the statistical analysis of the metastatic foci shown beside (G). Statistical differences are calculated by one-way ANOVA (A, B, and G, number of metastasis), unpaired t test (D, E). ns, not significant, *p <0.05, ****p < 0.0001.