Cytotoxic CD4+ exTregs expressing KLRG1 retain immunosuppressive function in cancer

The role of cytotoxic CD4+ T cells in cancer remains controversial, with both pro- and anti-tumor functionality described. Based on a prior observation that putatively cytotoxic KLRG1+ FoxP3- conventional T cells (Tconv) associated with tumor progression, we deeply investigated the phenotype and function of KLRG1+ Tconv in multiple murine tumor models and in human clear cell renal cell carcinoma (ccRCC) specimens. We found KLRG1+ Tconv have a conserved, highly activated, terminally differentiated phenotype across species. In murine tumors, KLRG1+ Tconv resemble tumor-infiltrating regulatory T cells (Tregs) at both transcriptional and proteomic levels, upregulating a spectrum of exhaustion-related checkpoint receptors, producing IL-10 and granzyme B (GzmB), and silencing expression of IFNγ and TNFα. Functionally, KLRG1+ Tconv exhibited immunosuppressive function in ex vivo assays. Using an in vivo Treg fate mapping approach in tumor-bearing mice, we found a subset of KLRG1+ Tconv are derived from Tregs that have lost FoxP3 protein expression. Ex vivo functional assays demonstrated the exTreg component of the KLRG1+ Tconv compartment is responsible for IL-10 production and reduced pro-inflammatory cytokine expression. In treatment studies, we found that KLRG1-targeted antibody-mediated depletion was more efficient than anti-KLRG1 blockade at slowing tumor growth and improving survival, with responses associating with depletion of intratumoral KLRG1+ Tconv and Tregs. In sum, our study discovered a novel conserved pathway of CD4+ T cell plasticity, wherein intratumoral, cytotoxic KLRG1+ CD4+ Tconv cells can be derived from Tregs and maintain immunosuppressive function, despite loss of FoxP3, and that KLRG1-targeted depletion may outperform KLRG1 blockade as an immunotherapeutic strategy in cancer.