Detection of PD-1 inhibitory signaling and its molecular relays in T cells: Implications for cancer immunotherapy - PD-1 blocking agents have achieved significant success as anti-cancer therapeutics. The mechanism(s) of how PD-1 compromises anti-tumor function remain poorly understood. Although in trans engagement of PD-1 expressed in T cells by its ligands expressed on APC or cancer cells inhibits T cell activation, evolving discoveries provide evidence that PD-L1:B7-1(CD80) in cis and PD-1:PD-L1 in cis non-canonical interactions occur when these molecules are co-expressed on APC, and disrupt the canonical interaction between PD-1 and PD-L1 in trans and T cell inhibitory signaling. Thus, expression of PD-L1 in the tumor microenvironment (TME) is not synonymous with PD-1-mediated T cell inhibition. We generated an antibody that recognizes PD-1pY248 (pPD-1), which is required for PD-1 inhibitory signaling, and detected pPD-1 in mouse and human. We found pPD-1+ T cells in cultures, in mouse tumor models and patient biopsies, but also in CD8+ T central memory (TCM) cells in the peripheral blood of healthy individuals. In tumor bearing mice, pPD-1 was expressed in tumor infiltrating CD8+ T lymphocytes but mostly in Treg. We generated mice with conditional targeting of Pdcd1 gene (PD-1f/f) and selectively eliminated PD-1 in Treg (Pdcd1f/fFoxP3cre). In tumor-bearing Pdcd1f/fFoxP3cre mice, Treg displayed enrichment in pathways regulating lipid metabolism, fatty acid oxidation, and production of monocyte/macrophage chemotactic protein-1 (MCP-1) and GABAergic neurotransmitter with known immunosuppressive function. These features correlated with a significant increase of B cells and M2-like macrophages, diminished activation of tumor infiltrating T cells, and increased tumor growth. Our results reveal a previously unappreciated network by which Treg-selective blockade of PD-1 signaling reshapes the immunological landscape and suggest that abrogation of PD-1 signaling in distinct cell types differentially impacts the TME. Our findings indicate that pPD-1 is a powerful marker to identify T cells subjected to PD-1 inhibitory signaling and support the novel hypothesis that cell-specific detection of PD-1 signaling by pPD-1 might predict the outcome of checkpoint immunotherapy. To investigate these, we will pursue the following specific aims: 1. To identify the immunological and biochemical properties of pPD-1+ T cells in the context of cancer. We will characterize pPD-1+ CD8+ TIL and Treg by single cell immunoprofiling, and investigate how T cell subset- specific PD-1 signaling reshapes the TME by using our Pdcd1f/fCD8cre and Pdcd1f/fFoxP3cre mice. 2. To identify the molecular and functional properties of pPD-1+ cells in healthy individuals. We will examine how PD-1 signaling shapes the properties of T cells in healthy individuals and in cancer patients and uncover why only cancer-mediated PD-1 signaling induces TEX. 3. To determine expression, function and prognostic significance of pPD-1+ TIL in cancer patients. We will examine cell-specific PD-1 expression and signaling in patient biopsies, co-expression of PD-1/pPD-1, PD- L1 and CD80, and determine their prognostic significance.
