Exposed phosphatidylserine is a non-classical inhibitory molecule during CD8 T cell exhaustion [scRAN-seq]

In cancer and chronic infection CD8 T cell exhaustion is hallmarked by expression of inhibitory surface receptors such as PD-1, TIM-3, CTLA-4, LAG-3, and TIGIT. Thus, inhibitory receptor focus has been limited to surface expressed proteins and an expansion into different classes of inhibitory molecules is needed. In this study we identify the lipid metabolite phosphatidylserine (PS) as a regulator of T cell exhaustion. PS localizes to the inner plasma membrane of live cells but is externalized to the outer membrane of apoptotic cells where it can function as a potent inhibitory ligand. However, the role of PS on live immune cells is less clear4. We show that live, exhausted, antigen specific CD8 T cells externalize PS during chronic lymphocytic choriomeningitis virus (LCMV) infection. PS exposure is initiated upon T cell activation and chronic antigen stimulation led to continued externalization. PD-1+ Tcf1+ stem-like, Tim-3+ transitory, and Tim3+ terminally differentiated/exhausted cells all expose PS. Furthermore, transcriptomic and lipidomic analyses identified increased PS metabolism during exhaustion. To evaluate the inhibitory function of exposed PS in exhaustion we treated chronically infected mice with the PS-targeting antibody (mch1N11)5 and found that monotherapy alone expanded the PD-1+ LCMV-specific CD8 T cell immune response. Most significantly, PD-1+ Tcf1+ stem-like CD8 T cells downregulated quiescence-associated gene modules and increased proliferation and self-renewal after treatment. Mechanistically, we found that exposed PS from exhausted T cells could suppress co-stimulatory molecule expression on neighboring dendritic cells to limit PD-1+ CD8 T cell proliferation. Combination of PS-targeting with aPD-L1 synergized to further increase the CD8 T cell effector response and improve viral control. Lastly, we identify that CD8 tumor infiltrating lymphocytes from human clear cell renal cell carcinoma (ccRCC) and human non-small cell lung cancer (NSCLC) patient tumors similarly expose PS indicating its translational potential. In fact, clinical trials combining PS-targeting antibodies and PD-L1 antibodies have recently been used in combination therapies. Therefore, our work details live PS CD8 T cell biology and provides a potential mechanism of action by which exposed PS facilitates suppressive T cell – DC interactions to function as a ‘non-classical’ inhibitory molecule during exhaustion.