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

CD4⁺ T cells responding to chronic viral infection often acquire a dysfunctional phenotype that is characterized by a progressive loss in Th1 differentiation and function, as well as an upregulation of multiple co-inhibitory receptors. Conversely, CD4⁺ T cells, and particularly Tfh cells, gradually increase their production of IL-21 during chronic viral infection, which is critical to sustain humoral immunity and also effector CD8⁺ T cell responses. Recent evidence further indicates that a memory-like CD4⁺ T cell population also develops in the face of persistent infection, although how the transcriptional landscape of this subset, along with the Th1 and Tfh cell subsets from chronic infection, differ from their acute infection counterparts remains unclear. Additionally, whether cell-intrinsic factors such as TCR usage influence CD4⁺ T cell fate commitment during chronic 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 chronic infection, including the presence of a previously unrecognized Slamf7⁺ subset with cytolytic features, and 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 viral infection. Importantly, a comparative analysis further reveals both subset-specific and core gene expression programs that are differentially regulated between CD4⁺ T cells responding to acute and chronic viral 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.