Tuning the show: Genetic engineering to unravel antigen presentation by the human leukocyte antigen class I

The HLA-I antigen presentation pathway is indispensable for T cell mediated tumor destruction. Disruption of this pathway is a key process for tumor immune escape. In this thesis, we sought to identify novel mechanisms of known and previously unknown factors in the antigen presentation pathway, with the ultimate goal to define targeted strategies to improve CD8+ T cell based anti-tumor therapy. We first describe the generation and validation of PAKC, a panel of ten human cell lines from a single genetic background. Each cell line is made knockout for a gene involved in the antigen presentation pathway using CRISPR/Cas9. Due to their identical background, these cell lines are ideal for comparative studies on the influence of each of the knocked-out factors. Using PAKC, we identified an antigen presented independently of TAP on both healthy and TAP-deficient cells. Our results emphasize the need for extensive testing to define clinically relevant antigens to safely target TAP-deficient tumors. Finally, a genome-wide haploid screening on HLA-I identified that the protease SPPL3 positively regulates accessibility of HLA-I for various of its ligands by inhibition of neolacto-series GSL (nsGSL) synthesis. Reactivity of CD8+ T cells towards cells overexpressing nsGSLs was decreased compared to cells lacking nsGSLs. Analysis of the TCGA glioma cohort revealed that there is a negative association between nsGSL synthesis genes and patient survival. Our data indicate that tumors can evade immunity through expression of nsGSLs