Delineating the transcriptional landscape and clonal diversity of virus-specific CD4 T cells during chronic viral infection.

Although recent evidence indicates that CD4 T cells responding to chronic viral infection are functionally heterogenous, our understanding of the developmental relationships between these subsets, and a determination of how their transcriptional landscape compares to their acute infection counterparts remains unclear. Additionally, whether cell-intrinsic factors such as TCR usage influence CD4 T cell fate commitment during persistent infection has not previously been studied. Herein, we perform single-cell RNA sequencing (scRNA-seq) combined with single-cell T cell receptor sequencing (scTCR-seq) on virus-specific CD4 T cells isolated from mice infected with chronic lymphocytic choriomeningitis virus (LCMV) infection. We identify several transcriptionally distinct states among the Th1, Tfh, and memory-like T cell subsets that form at the peak of infection, including the presence of a previously unrecognized Slamf7 subset with cytolytic features. We further show that the relative distribution of these populations differs substantially between acute and persistent LCMV infection. Moreover, while the progeny of most T cell clones displays membership within each of these transcriptionally unique populations, overall supporting a one cell-multiple fate model, a small fraction of clones display a biased cell fate decision, suggesting that TCR usage may impact CD4 T cell development during chronic infection. Importantly, comparative analyses further reveal both subset-specific and core gene expression programs that are differentially regulated between CD4 T cells responding to acute and chronic LCMV infection. Together, these data may serve as a useful framework and allow for a detailed interrogation into the clonal distribution and transcriptional circuits underlying CD4 T cell differentiation during chronic viral infection.