Exposed phosphatidylserine is an inhibitory molecule in T cell exhaustion

In cancer and chronic infection, CD8 T cell exhaustion is hallmarked by expression of inhibitory receptors such as PD1, TIM3, LAG3 and others^1-3. Thus, inhibitory molecule focus has been limited to cell-surface proteins. Here we evaluate the surface lipid metabolite phosphatidylserine (PS) as a regulator of exhaustion. PS primarily localizes to the inner plasma membrane of live cells but is well known to be externalized to the outer membrane during cell death. The role of exposed PS on live immune cells is less clear. We show that viable, antigen-specific CD8 T cells externalize PS during lymphocytic choriomeningitis virus (LCMV) infection. T cell activation induced initial PS exposure, and chronic antigen stimulation sustained externalization. Transcriptomic and lipidomic analyses also identified PS accumulation in exhausted CD8 T cells. To evaluate a role for exposed PS in exhaustion, we treated LCMV chronically infected mice with a PS-targeting antibody (mch1N11)⁴ and found that it expanded LCMV-specific CD8 responses. PD1⁺TCF1⁺ stem-like CD8 T cells downregulated quiescence-associated gene modules and increased proliferation after antibody treatment, highlighting an inhibitory role for PS. Mechanistically, exposed PS on T cells functioned extrinsically to suppress dendritic cell immunostimulatory phenotypes, in turn limiting CD8 T cell responses. PS-targeting antibody with anti-PDL1 synergized to increase CD8 responses and improve viral control. Finally, we show that PD1⁺ CD8 T cells from human tumours can also expose PS. In summary, we detail CD8 T cell PS biology and provide insight into a mechanism by which exposed PS functions as a 'non-classical' extrinsic inhibitory molecule in exhaustion.