Eomesodermin+ CD4 T cells are critical for curative immunotherapy outcomes

IL-10-producing CD4+ type-1 regulatory T cells (Tr1) promote immune tolerance during chronic infection, autoimmunity, and transplantation. However, specific Eomes-dependent stages of Tr1 differentiation and function remain unclear. Using preclinical models of bone marrow transplantation (BMT) and chimeric antigen receptor (CAR) T cell immunotherapy, we demonstrate a Tr1 differentiation trajectory in vivo from Eomes+IL-10– to Eomes+IL-10+ subsets with the acquisition of cytokine, cytolytic and exhaustion features. The Eomes+CD4+ fraction represents the dominant cytotoxic subset after BMT, mediating graft-versus-leukemia effects while limiting inflammation. In CD19-targeted CAR T cell immunotherapy, Eomes drives the same CD4+ Tr1 phenotype that controls cytolysis, whilst mitigating immune toxicity and promoting persistence. In patients receiving commercial CD19-targeted CAR T cells with long term disease control, Eomes+ Tr1 cells represent a stable population comprising 40-80% of the CD4+ CAR T cell population. Hence, Eomes controls both regulatory and cytotoxic programs in CD4+ T cells, essential for curative immunotherapy outcomes. Overall design: B6D2F1 recipients were transplanted with TCD BM (5x106) and naive CD4+ T cells (0.5x106) from EomesmCherry/IL-10GFP donors. Spleens (n = 4) were taken on day +14, pooled, enriched to CD4+ T cells with MACS selection and FACS sorted to P1 (Eomes- IL10-), P2 (Eomes+ IL10-) and P3 (Eomes+ IL10+) based on the expression of mCherry and GFP. The sort-purified cells were subsequently processed for single cell RNAseq.