Hyperthermia Combined with Anti-CTLA-4 Antibody Induces Tumor Microenvironment Remodeling Involving CD4+ T Cells in Local and Distant Antitumor Effects in a Murine Triple-Negative Breast Cancer

Background/Objectives: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Our previous study demonstrated that combination therapy with local hyperthermia (HT) and anti-CTLA-4 antibody (C4), an immune checkpoint inhibitor, induced regression of both local and distant tumors. However, tumor microenvironment (TME) changes in local and distant tumors following local HT and C4 remain unclear. Here, we aimed to evaluate TME changes in local and distant tumors induced by local HT + C4 therapy. Methods: Murine TNBC cells were inoculated in both legs of male BALB/cAJcl mice, and only one leg was treated with HT (42.5 °C for 20 min). C4 was administered intraperitoneally every 3 days for a total of 3 doses. For CD4+ T cell depletion experiments, anti-CD4 antibody (αCD4) was administered intraperitoneally every 3 days for a total of 12 doses. Tumor-infiltrating immune cells in locally heated tumors and unheated distant tumors were analyzed 9 days after the initial treatment. Furthermore, tumor growth in heated tumors and unheated distant tumors under αCD4 administration was evaluated. Results: HT + C4 therapy increased the proportion of helper T cells and elevated the ratio of cytotoxic T cells plus helper T cells to myeloid-derived suppressor cells in both heated tumors and unheated distant tumors. The HT + C4 + αCD4 group exhibited significantly larger tumor growth, compared with the HT + C4 group in both heated (p < 0.01) and unheated distant tumors (p < 0.01). Conclusions: These results suggest that combination therapy of HT and C4 favorably modulates the TME. CD4+ T cell infiltration may contribute to both local and distant antitumor effects.