Project Summary Killer cell immunoglobulin-like receptors (KIR) are mainly expressed by NK cells, although their expression has also been described in CD4+, CD8+ and γδ T cells. Within CD8 T cells, KIR expression is induced at later stages of lymphocyte maturation and is thought to regulate specific T cell effector functions. Within KIR receptors, KIR2DL2 modulates T cell effector functions, as KIR2DL2+ CD8+ T cells present reduced level of activation-induced cell death, and poor IFN-γ secretion after T cell receptor (TCR) stimulation. The notion of a suppressive function of KIR2DL2 expression in CD8+ T cells is supported by the observation that patients that express its cognate ligand, HLA-C1, showed decreased overall survival and could not control tumor growth. Our preliminary data show that KIR2DL2 expression increases in vivo in adoptively transferred T cells in patients and in preclinical models of adoptive immunotherapy. Using a pancreatic tumor model, we found that chimeric antigen receptor (CAR)-T cells expressing KIR2DL2 were significantly less cytotoxic than their KIR2DL2- counterparts in presence of KIR2DL2’s ligand. Furthermore, KIR2DL2 expression in CAR-T cells was associated with reduced antitumor efficacy, in an HLA-I-dependent manner, in a murine model of pancreatic cancer. Based on these preliminary findings we hypothesize that KIR2DL2 behaves as a T cell immune checkpoint, modulating T cell effector function and leading to an ineffective immunosurveillance. Therefore, targeting KIR2DL2 during T cell manufacturing may improve T cell performance after cell infusion. We will test our hypothesis by 1) Defining the modulatory mechanisms whereby KIR2DL2 shapes CAR- and TCR-transgenic T cell antitumoral effector function. We will determine the overall effect of KIR2DL2 engagement in TCR-transgenic and CAR-T cell effector function. Additionally, we will determine which regions within KIR2DL2 are responsible for its modulatory function. Finally, we will characterize both the KIR2DL2 signaling interactome and the downstream events triggered by its ligand interaction by immunoprecipitation and proteomic analyses. 2) Improving T cell performance for the enhancement of adoptive cell immunotherapies (ACTs) by abrogating KIR2DL2 function. To prevent its inhibitory effect, manipulation of KIR2DL2 expression and/or signaling will be conducted and adapted to the current protocols for CAR-T cell manufacturing. Based on the anticipated results, we will link for the first time the biological and molecular function of KIR2DL2 within therapeutic T cells. The proposed studies will increase our mechanistic understanding of KIR2DL2 biology and will generate novel cell products with high translational potential.

Project Narrative KIR2DL2 is an inhibitory receptor whose role in T cell biology has been poorly characterized. The proposed research aims to validate KIR2DL2 as a new T cell immune checkpoint and to elucidate the signaling mechanism underlying its modulatory functions. The knowledge acquired through this project will be used to design novel and more efficient immunotherapies, including TIL, TCR-transgenic, and CAR-T cells, through manipulation of KIR2DL2 expression or function.