Intra-tumoral T cell dysfunction, frequently referred to as T cell exhaustion, is considered a hallmark of cancer. Reinvigoration of T cell function by immune checkpoint inhibitors (ICI) can result in impressive clinical responses, but is effective only in a minority of cancer patients. The mechanisms involved in T cell dysfunction, and particularly its involvement in ICI therapy is still not fully understood. Moreover, the development of novel cancer immunotherapies urgently requires comprehensive understanding of molecular initiators and promotors of T cell dysfunction. Here, we have developed an ex vivo human model system to mimic tumor-specific T cell dysfunction in human cancers. To this end, circulating CD8 T cells from non-cancer individuals are transduced with a T cell receptor construct specifically recognizing a human tumor-associated antigen. Subsequently, these T cells are exposed to repetitive stimulation utilizing antigenic peptides with different affinities. We can demonstrate that this procedure stably renders CD8 T cells dysfunctional. The latter acquire a state which highly resembles CD8 T cells found in human tumors on a phenotypic and transcriptional level. Importantly, this model is capable to produce a high number of antigen-specific dysfunctional T cells sufficient to overcome restraints imposed by the limited availability of tumor-infiltrating T cells with unknown antigen specificity obtained from cancer patients. By taking advantage of this model, we aim to dissect individual genes or group of genes critical for the generation of T cell dysfunction and identify key genes and downstream pathways that could be targeted for T cell reinvigoration. To this end, we propose to perform an unbiased and comprehensive loss-of-function CRISPR screen with the following major research aims: (i) to discover genes involved in inducing and/or maintaining T cell dysfunction, and (ii) to mechanistically dissect how these genes are involved in T cell dysfunction. Experimentally, we will develop CRISPR-Cas9 mediated knockout systems scaled up to a genome-wide scale in antigen-specific CD8 T cells by co-transduction with a tumor antigen-specific T cell receptor and pooled single guide RNAs, combined with Cas9 protein electroporation. Initial experiments have already demonstrated the feasibility of the approach. After repetitive antigenic stimulation in compliance with our model system, the dysfunctional T cells will be restimulated and divided into functional/non-functional populations to systematically identify potential target genes that are critical in mediating T cell dysfunctionality. Validation of target gene knockouts will be accomplished in primary CD8 T cells and organotypic culture systems derived from cancer patients, utilizing proteomic and siRNA approaches. In addition, we will investigate the impact of target gene knockouts/inhibition using mouse tumor models of adoptive transfer and tumor-infiltrating lymphocytes (TIL) obtained from melanoma patients in the framework of a newly established clinical program for TIL transfer in our center. We expect that the findings advance our understanding of the molecular mechanisms that underpin T cell dysfunction in the context of cancer and in the future serve to optimize T cell-targeted immunotherapy for the benefit of patients.