Abstract A031: Unhelpful CD4 help: Removal of conventional CD4+ T cells promotes priming of tumor antigen-specific CD8 T cells in the context of CD4-depletion therapy
Authors: Dragnev Christo P. C. , Ramirez Delaney E. , Searles Tyler G. , Spicer Nathaniel , Chen TIffany , Lines J. Louise , Hawkes Aaron R. , Davis Wilson L. , Mohamed Asmaa , Shirai Keisuke , Phillips Joesph D. , Rosato Pamela C. , Huang Yina H. , Turk Mary Jo 
Published: October 2024

Abstract
Preclinical and clinical studies have used antibodies to deplete CD4-expressing cells to promote anti-tumor immunity. Specifically, a monoclonal anti-CD4 antibody (anti-CD4) has been employed to remove FOXP3+ CD4+ regulatory T cells (Tregs), which are known to impede anti-tumor immunity. Our previous studies demonstrated that administering anti-CD4 during B16 melanoma tumor growth in mice leads to the generation of tumor Ag-specific CD8 T cells that disseminate and form memory following surgical tumor excision. However, the importance of co-depleting other CD4-expressing cell compartments for anti-CD4 treatment efficacy are unknown. We utilized flow cytometry and single-cell RNA sequencing (scRNAseq) to evaluate the primary response of tumor Ag-specific CD8 T cells (pmel) in B16 melanoma tumor bearing mice treated with anti-CD4. Responses were compared to those induced by anti-PD1 and anti-CTLA4 dual immune checkpoint blockade (ICB). To determine the importance of homeostatic space in priming, homeostatic expansion and priming were decoupled, and neutralizing antibodies were administered. Treg-depleted (Foxp3-DTR) mice were used for comparison, and Batf3-/- mice and IL-2R blocking antibodies were used to determine requirements for CD8 T cell priming. Finally, scRNAseq was conducted on CD4 T cells from human melanoma tumors previously treated with ICB to characterize transcriptional properties of intratumoral CD4 T cells. Compared to ICB, anti-CD4 treatment elicited a more robust, disseminated, and persistent primary tumor Ag-specific CD8 T cell response. ScRNAseq revealed that anti-CD4 treatment induced more stem-like (TCF1+) memory precursor cells, whereas ICB induced more differentiated effector (Gzmb+) cells. Removal of homeostatic space or blockade of IL-7R and IL-15 during priming did not diminish pmel responses in anti-CD4 treated mice, indicating that homeostatic space was not required. Importantly, pmel cells were shown to produce their own IL-2, and pmel priming was significantly lower after IL-2 receptor blockade, suggesting dependence on autocrine IL-2 signals. Using DT-treated FOXP3-DTR mice, we found that targeted Treg depletion was insufficient for the pmel priming that disseminated and formed memory, indicating that total CD4 T cell depletion was required for this systemic response. Depletion of total CD4 T cells uniquely increased cDC1 proportions in tumor-draining LNs as well as their expression of CD86. However, treatment of tumor-bearing RAG-/- mice with anti-CD4 confirmed that depletion of CD4-expressing APC compartments does not contribute to anti-CD4 treatment efficacy. ScRNAseq of CD4 T cells from human melanoma tumors showed dominance of Tr1-like cells and other subsets without transcriptional evidence of helper differentiation, despite prior ICB. In mice, combination of anti-CD4 with dual ICB restored the systemic response of tumor-specific CD8 T cells. This study underscores the importance of eliminating conventional CD4 T cells in tumor-bearing hosts to elicit systemic primary and memory CD8 T cell responses.
Citation Format: Christo P. C. Dragnev, Delaney E. Ramirez, Tyler G. Searles, Nathaniel Spicer, TIffany Chen, J. Louise Lines, Aaron R. Hawkes, Wilson L. Davis III, Asmaa Mohamed, Keisuke Shirai, Joesph D. Phillips, Pamela C. Rosato, Yina H. Huang, Mary Jo Turk. Unhelpful CD4 help: Removal of conventional CD4+ T cells promotes priming of tumor antigen-specific CD8 T cells in the context of CD4-depletion therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A031.