Figure 4.

Tumor-specific CD4⁺ T cells get exhausted during tumor progression. Tumor-bearing mice were treated with 2 × 10⁶ activated SMARTA cells eight days after tumor inoculation and sacrificed on Day 7 or Day 15 post-transfer. Cells are gated on live CD4⁺CD44⁺CD45.1⁺ cells. (A, B) Representative flow cytometry plots of intracellular cytokine staining of CD4⁺ SMARTA cells on Day 7 (n=4) and Day 15 (n=3) post-SMARTA transfer (A). The frequencies of IFN-γ⁺ TNF-α⁺ and IFN-γ⁺ IL-2⁺ cells of CD4⁺SMARTA cells (B). (C, D) In a separate experiment, representative flow cytometry plots of PD-1 expression in CD4⁺ SMARTA cells on Day 7 and Day 15 (n=3/group) post-transfer (C). The frequency of PD-1⁺ SMARTA cells and MFI of PD-1 (D). (E, F) Representative flow cytometry plots of CTLA4 expression in CD4⁺ SMARTA cells on Day 7 and Day 15 (n=3/group) post SMARTA transfer (E). The frequency of CTLA4⁺ SMARTA cells and MFI of CTLA4 (F). Statistical differences are calculated by unpaired t-test. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.