Reinvigorated exhausted T cells exhibit distinct features within 24 hours after PD-1 therapy

Abstract CD8 T cells are potent cytolytic immune cells. However, persistent antigens during chronic infections and cancers drive differentiation of exhausted T cells (T EX). T EXdo not generate optimal immune protection. Immunotherapy such as anti-PD-L1 provides a partial T EXreinvigoration. Some T EXregain effector functions, enter cell cycle and start proliferation by PD-1 therapy. However, the underlying molecular and epigenetic mechanisms of T EXreinvigoration are not well understood. Moreover, a key unknown question is why only some T EXgain reinvigoration. Here, we used proliferation as a surrogate for T EXreinvigoration. A transgenic mouse model carrying a cell cycle reporter (Fucci) was used. Fucci LCMV-specific CD8 T cells were transferred to mice that were then infected with chronic LCMV to induce exhaustion. Recipients were treated with anti-PD-L1 at d21 p.i. Fucci staining (cell cycle) and CTV dilution (cell division) allowed us to isolate reinvigorated T EXbefore first cell division and ask what molecular signals induce T EXreinvigoration. Thus, we found that T EXreinvigoration starts as early as 24 hours after a single anti-PD-L1. Proliferating cells upon treatment contained an increased progenitor-like population and a reduced terminally exhausted population compared to control, whereas non-proliferating cells were similar between groups. Single cell transcriptomics data further identified unique transcriptional signatures in reinvigorated T EXupon anti-PD-L1 compared to untreated T EXat 48 hours post treatment. Therefore, we identified the molecular signatures of T EXthat have the potential to become reinvigorated upon immunotherapy and suggest novel molecular targets to enhance the potency of PD-1 therapy.