Disruption of IFNγ, PRF1, GZMB, or LYST results in reduced suppressive function in human CD8+ T cells

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Abstract Human CD8+ T-cells play an important immune regulatory role during autoimmune diseases, characterized by suppressing the activation and proliferation of pathogenic effector cells, mediated in part through cytotoxicity (PRF/GZM), IFNγ secretion or by Fas/FasL interactions. Recent studies from our group demonstrated that IL-12 exposure during in vitro differentiation of naïve CD8+ T-cells induced an immune-suppressive deficit, with Tc1 cells showing significant loss of suppressive ability against CD4+ T-cells. RNAseq analysis revealed that various cytokines, transcription factors, and other regulatory molecules were differentially regulated in these cells. In this study, we further investigated the importance of IFNγ-, PRF1-, GZMB- and LYST-associated pathways in the suppressive ability of CD8+ T-cells. Using the CRISPR-Cas9 RNP optimized transfection system in primary bulk human CD8+ T-cells. Knockout was confirmed through quantitative real-time PCR assays in all cases, combined with flow cytometry when possible, as well confirmation of indels at genomic target sites. We observed that knockout of each of the four molecules, but not the knockout of IL-4 or IL-5, resulted in significantly diminished suppressive ability, as measured through in vitro immune suppression assays. Interestingly, in some of the knockouts, we observed differential expression of non-target molecules indicating potential functional links between these pathways. Collectively, these results reveal a pivotal role for these pathways in CD8+ T cell-mediated immune suppression and provide important insights into the biology of human CD8+ T-cell-mediated suppression that could be targeted for immunotherapeutic intervention. This work was supported, in part, by grant awards to NJK from the NIH (R01 AI121567) and VA.