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Abstract

Immune checkpoint blockade has revolutionized immunotherapy against cancer with tremendous clinical benefits for patients. Despite these achievements, tumors utilize a plethora of suppressive mechanisms to evade immune destruction which are yet to be understood and matched by today’s immunotherapy. Our group developed a high-throughput RNAi screening to unravel the arsenal of immune checkpoints of cancer. We screened a siRNA library (around 2880 kinases and surface-associated genes) with patient-derived tumor cells and HLA-matched tumor-infiltrating lymphocytes (TILs). The library was reverse-transfected into M579-A2 melanoma cells and these were co-cultured with MART1- and gp100-specific TILs to determine TIL-mediated lysis. We identified 75 genes in tumor cells that impaired TIL-mediated cytotoxicity. Interestingly, we found that several genes and their associated pathways were found in pancreatic adenocarcinoma and multiple myeloma as well. This suggests that different cancer entities might share inhibitory modes of action. In order to distinguish between genes altering tumor susceptibility towards TIL-mediated killing and those impairing TIL activity, we established a secondary screening assaying multiple T cell activation marker, including effector cytokines. The olfactory receptor OR10H1 was one of the strongest candidates from our primary screening as its knockdown increased TIL-mediated killing in melanoma, PDAC and colorectal carcinoma. Furthermore, TILs were activated stronger after interaction with OR10H1-deficient cells as sensed by the increased secretion of type 1-associated cytokines and a reduced T cell apoptosis. We confirmed the role of OR10H1 as an immune checkpoint in vivo using a xenograft mouse model in combination with adoptive T cell transfer. We performed mode of action analyses in order to understand how OR10H1 affects T cell activity. These analyses revealed that tumor-associated OR10H1 controls cAMP-dependent signaling inside T cells. Inside TILs, cAMP activates protein kinase alpha (PKA) and PKA in turn activates C-terminal Src kinase (Csk. Csk phosphorylates an inhibitory tyrosine residue of Lck impairing its activity and shutting down TCR-associated signaling. Furthermore, PKA activates CREB and thus induces an anergy-associated gene expression profile in TILs. Our data suggest that OR10H1 alters the balance between the inhibitory (GαI) and the stimulatory/olfactory G-Protein alpha (GαS/Olf) inside tumor cells depending on the encounter of TILs. This results into increased production of cAMP in tumor cells and its subsequent transport into T cells. In summary, we established a discovery platform aiding the search for immune checkpoints in cancer. We identified OR10H1 and its associated olfactory receptor signaling as a novel pathway inhibiting TIL responses by inducing cAMP-dependent Lck inhibition.