CD4+ T cell help is required for the formation of a cytolytic CD8+ T cell subset that protects against chronic viral infection and cancer

Abstract

Abstract CD8 T cells responding to chronic viral infection can be classified into two major subsets, with a TCF-1+ subset serving as a self-renewing progenitor population that can give rise to a more terminally exhausted PD-1hi subset. However, whether additional heterogeneity exists amongst virus-specific CD8 T cells remains unclear. Here, using single cell RNA-sequencing and flow cytometric approaches, we demonstrate that in addition to the progenitor and exhausted subsets, CD8 T cells responding to persistent LCMV infection also encompass a previously unrecognized T cell subset that exhibits potent cytolytic activity in vitro, and is required for viral control in vivo. This cytotoxic subset is characterized by high expression of killer cell lectin-like receptors, the chemokine receptor CX3CR1, and the transcription factors T-bet and Zeb2. Notably, we further demonstrate a critical role for CD4 help via IL-21 production in facilitating the differentiation of this cytotoxic subset, as depletion of CD4 T cells or deletion of IL-21R signaling in P14 cells (which express a transgenic TCR specific for GP33–41) wholly abrogated the development of CX3CR1+CD8 T cells. Moreover, PD-L1 blockade, an immunotherapy known to partially reverse T cell exhaustion, failed to rescue this differentiation defect of “un-helped” CD8 T cells. Lastly, using a B16 melanoma model, we found that provision of IL-21+CD4 T cells was sufficient to induce over a 2-fold increase in the development of CX3CR1+CD8 TILs, which was associated with enhanced control over tumor growth. Collectively, our findings demonstrate a critical role for CD4 help in the generation of protective CX3CR1+CD8 T cells, and that this differentiation checkpoint may be exploited for immunotherapy.