Background Distinct subsets of CD4⁺ T cells can either promote tumor immunity by facilitating priming and expansion of cytolytic CD8⁺ T cells or, as Tregs, inhibit anti-tumor activity. Recently, other immunosuppressive CD4⁺T cell populations have been reported in mice and humans. However, the roles, origins and physiologic targets of the latter remain poorly defined. Our previous studies showed that, under defined conditions, synthetic long peptide (SLP) neoantigen (neoAg) cancer vaccines induced therapeutic rejection of established tumors, but efficacy was variable and depended on the quantity and quality of MHC-II neoAgs in the vaccine.¹ Herein we identify a population of tumor-specific inhibitory non-Treg cytolytic CD4⁺ T cells induced by high MHC-II neoAg doses in vaccines, define their biology and mechanism of action, and present strategies to circumvent their formation/inhibitory function during immunotherapy.

Methods Different methylcholanthrene-induced mouse sarcoma cell lines that never express MHC-II were implanted into syngeneic mice that were then treated with tumor specific SLP neoAg vaccines containing varying amounts of MHC-II neoAgs.

Results Vaccines containing tumor specific MHC-I neoAgs plus low doses of MHC-II neoAg (LDVax) promote tumor rejection. In contrast, similar vaccines containing high doses of MHC-II neoAg (HDVax) were ineffective. HDVax inhibited antitumor efficacy of anti-PD-1 and anti-4–1BB in an antigen-specific manner but not anti-CTLA4 indicating that HDVax induced an inhibitory immune cell population. Inhibition could also be achieved by adoptive transfer of antigen-specific HDVax-induced CD4⁺ T cells into vaccine-naïve, tumor-bearing mice. Single-cell and bulk RNA sequencing and multicolor flow cytometry revealed that HDVax-induced CD4⁺ T cells express high levels of LILRB4, Granzyme-B (GZMB), Perforin and CCL5. TCRSeq and scRNAseq showed that the HDVax-induced inhibitory CD4⁺T cells were distinct from Foxp3⁺ Tregs. HDVax-induced CD4⁺ T cells inhibited tumor immunity by killing type 1 conventional dendritic cells (cDC1), 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. Killing was dependent on GZMB protein expression in the CD4⁺ T cells. Treatment of these cells with blocking anti-LILRB4 reduced cellular expression of GZMB and circumvented HDVax-induced inhibition. A similar reprogramming occurred following genetic depletion of cDC2 or treatment with a novel cis-targeted IL-2 mutein (CD8-IL2) that selectively activates CD8⁺ T cells.²

Conclusions These results provide a roadmap to improve efficacy of cancer vaccines and potentially other immunotherapies by circumventing the generation/function of a heretofore unrecognized inhibitory CD4⁺ T cell population.

Acknowledgements We thank all members of the Schreiber laboratory for discussions and technical support. We thank Likui Yang of the Immunomonitoring Laboratory (IML) at Washington University in St.Louis, who provided tetramers for MHC-I and MHC-II neoAgs. This work was supported by grants to Robert Schreiber from the National Cancer Institute of the National Institutes of Health (R01CA190700), the Parker Institute for Cancer Immunotherapy, and a Stand Up to Cancer-Lustgarten Foundation Pancreatic Cancer Foundation Convergence Dream Team Translational Research Grant and postdoctoral training grant (T32CA009547) from the National Cancer Institute to Hussein Sultan.

References

1. Gubin MM. et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature 2014;515:577–581.

2. Moynihan K, et al. Selective activation of CD8+ T cells by a CD8-targeted IL-2 results in enhanced anti-tumor efficacy and safety. JITC 2021.

Ethics Approval All in vivo experiments were performed in our specific-pathogen-free facility using procedures approved by the AAALAC-accredited Animal Studies Committee of Washington University in St Louis and followed all relevant ethical regulations.