Fig. 3

Increased expansion and Th1-type inflammation of AAA-CD4⁺ T cells in the tumor after injection. AAA-CD4⁺ T cells were produced from BALB/c mouse CD4⁺ T cells in cultures activated with B6-derived GM-DCs. Host B6 mice were subcutaneously injected with 1 × 10⁶ B16F1 cells on day zero. Nine days after B16F1 inoculation, AAA-CD4⁺ T cells that had been labeled with CFSE or Far Red were intratumorally injected (AAA-CD4⁺ group). B6-derived DC-activated B6-derived CD4⁺ T cells (auto-CD4⁺ group) and PBS-injected mice were used as controls (three mice per group). a, g: Representative confocal images of the indicated markers in tumors isolated 24 h after immunotherapy are shown. Bar, 20 μm. b, c: Representative scatterplots of the donor CD4⁺ T cells before in vivo injection (b) and those isolated from the tumors (c) are shown. d, e: The proportions of CD62L^hi/CD44^hi central memory and CD62L^low/CD44^hi effector memory populations (d) and the number of CD44^hi/CD62L^low effector memory cells (e) in the donor CD4⁺ T cells isolated from the tumor. f: Graph shows the Intranuclear staining of Ki-67 in donor CD4⁺ T cells isolated from the tumor. h, j: Results show the relative expression levels of the indicated genes in the donor CD44^hi/CD4⁺ T cells isolated from the tumors, normalized to those in naïve CD4⁺ T cells of non-treated BALB/c mice. i: Results show the Intracellular staining of indicated cytokines in donor CD4⁺ T cells isolated from the tumor and cultured for 10 h in a medium containing human IL-2 (200 IU/mL) with a protein transport inhibitor. k: Seven days after immunotherapy, peripheral blood, spleen, and draining lymph nodes were isolated. Representative scatterplots of chimerism analysis of donor CD4⁺ T cells are shown. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Representative data from two independent experiments are shown. Abbreviations: TV: tumor volume