Abstract A075: Cytotoxic Tr1 CD4+ T cells suppress cancer immunotherapy in an antigen-specific manner

Abstract The induction of tumor-specific CD8+ cytolytic T cells (CTL) capable of destroying tumors is the ultimate outcome of cancer immunotherapy. While helper CD4+ T cells are often required to foster the induction of antitumor effector CTLs, CD4+ Foxp3+ regulatory T cells impede CTL induction and, consequently, inhibit anti-tumor immunity. Previous studies suggested that other CD4+ T cell populations might also suppress tumor immunity, but the identity, origins and function of these cells remained poorly defined. Herein, we show that FoxP3-negative type 1 regulatory T cells (Tr1) play a major role in suppressing naturally occurring and therapeutically induced tumor immunity. Using neoantigen (neoAg)-peptide-based cancer-specific vaccines, we confirmed that therapeutically effective vaccines required both MHC-I- and MHC-II-restricted neoAgs matched to those in the tumor but exhibited a complex dependency on MHC-II peptide dosage. Specifically, vaccines containing MHC-I neoAg in combination with low-dose MHC-II neoAg (LDVax) promoted tumor rejection in mice, whereas vaccines with the same amount of MHC-I neoAgs but high-doses of MHC-II neoAg (HDVax) were ineffective. Intriguingly, HDVax inhibited the antitumor efficacy of anti-PD-1 and anti-4-1BB therapies in an antigen-specific manner. These findings were consistent across three independent MCA-induced tumor cell lines (T3, F244 and 1956) and their respective independent MHC-I and MHC-II-restricted neoAgs. Interestingly, whereas LDVax-induced CD4+ T cells expressed high levels of IFN-γ, TNF-α, IL-2, and GM-CSF, HDVax-induced CD4+ T cells did not express these cytokines but rather expressed high levels of IL-10 and TGF-β. Adoptive transfer of HDVax-induced CD25-negative, peptide-I-Ab tetramer-positive CD4+ T cells into vaccine-naïve, tumor-bearing mice suppressed the efficacy of anti-PD1 treatment and LDVax. Using high-dimensional single-cell and bulk RNA sequencing, flow analyses, and protein multiplex assays, we identified the tumor-specific HDVax-induced inhibitory cells as Tr1 cells. HDVax-induced Tr1 cells express high levels of IL-10, LILRB4, Granzyme B, CCL5, and perforin. HDVax-induced Tr1 cells failed to provide help for CD8+ T cell expansion and function and inhibited tumor immunity by killing type 1 conventional dendritic cells (cDC1) in a Granzyme B-dependent manner, as evidenced by in vitro killing assays and by low recovery of cDC1 from tumors in HDVax-treated mice compared to LDVax-treated or control mice. Notably, the same Tr1 cells become detectable in progressively growing tumors as they become resistant to anti-PD-1 treatment. Finally, the suppressive capacity of Tr1 can be circumvented through LILRB4 blockade, treatment with CD8-targeted IL-2 mutein, and avoidance of antigen presentation by cDC2. Collectively, our findings indicate that HDVax-induced Tr1 cells, which are crucial for maintaining peripheral tolerance, also impede immune control of cancer. Citation Format: Hussein Sultan, Robert Schreiber. Cytotoxic Tr1 CD4+ T cells suppress cancer immunotherapy in an antigen-specific manner [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A075.