Fig. 5

Host endogenous CD8⁺ T cells are required to mediate antitumor immunity in AAA-CD4⁺ T-cell therapy. 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 were intratumorally injected. a-b: Before injection of the AAA-CD4⁺ T cells, an anti-mouse CD8-eliminating antibody or control IgG was intraperitoneally injected at a dose of 500 µg on day 8, and 250 µg on days 10 and 12. Tumor growth (a) and survival (b) are shown. The data are pooled from two independent experiments. c: Twenty-four hours after the intratumoral injection of AAA-CD4⁺ T cells, the host mice were euthanized, and draining lymph nodes (DLNs) were resected (three mice per group). After making a single-cell suspension of DLNs, CD8⁺ T cells were isolated and cultured in a medium containing human IL-2 (200 IU/mL) with lysates prepared from several B6-origin tumor cell lines. After 4 h of incubation with a protein transport inhibitor, the cells were recovered, stained with intracellular IFN-γ, and analyzed using flow cytometry. CD8⁺ T cells isolated from the spleens of naïve B6 mice were used as controls (three mice per group). d-e: Twenty-four hours after the intratumoral injection of AAA-CD4⁺ T cells, the host mice were euthanized and tumors were resected. Subsequently, the tumor cells and tumor-infiltrating mononuclear cells were isolated. Representative histograms (d) and mean fluorescent intensities (e) show the indicated immune checkpoint molecules on the surface of either in vivo isolated tumors or tumor-infiltrating host CD8⁺ TEM cells. Data are expressed as the mean ± SD. **P < 0.01, ***P < 0.001. Representative data from two independent experiments are shown