Transcriptional programming of CD4 T differentiation in viral infection balances effector- and memory-associated gene expression.

After resolution of infection, T cells differentiate into long-lived memory cells that recirculate through secondary lymphoid organs or establish residence in tissues. In contrast to CD8 tissue-resident memory T cells (T), the developmental origins and transcriptional regulation of CD4 T remain largely undefined. Here, we investigated the phenotypic, functional, and transcriptional profiles of CD4 T in the small intestine (SI) responding to acute viral infection, revealing a shared gene expression program and chromatin accessibility profile with circulating T1 and the progressive acquisition of a mature T program. Single-cell RNA sequencing identified heterogeneity among established CD4 T, which were predominantly located in the lamina propria, and revealed a population of cells that coexpressed both effector- and memory-associated genes, including the transcriptional regulators Blimp1, Id2, and Bcl6. T1-associated Blimp1 and Id2 and T-associated Bcl6 were required for early T formation and development of a mature T population in the SI. These results demonstrate a developmental relationship between T1 effector cells and the establishment of early T, as well as highlighted differences in CD4 versus CD8 T populations, providing insights into the mechanisms underlying the origins, differentiation, and persistence of CD4 T in response to viral infection.