*Purpose: For most cell types, the pathways that regulate the levels of critical disease-relevant genes are not known. Recent advances in gene editing have enabled us to perform large-scale perturbation experiments in primary human T cells, allowing for identification of key regulators of disease relevant genes which may have applicability in modulating the transplant immune response.

*Methods: Using a CRISPR loss-of-function screen, we identified transcription factors that regulate levels of CTLA-4, a checkpoint inhibitor found on regulatory T cells as well as activate conventional T cells that is the target of Belatacept. We created a library of 6000 guide-RNAs which target 1349 genes (generally 4 guides per gene), and 600 control guides. Results were validated using ribonucleoprotein knockouts of individual genes of interest.

*Results: Fifty-nine genes significantly regulate the levels of CTLA-4 (FDR < 0.05, Figure 1), 23 of which decrease levels of CTLA-4, while 36 increase CTLA-4. The majority of these hits were validated using an independent arrayed validation strategy (Figure 2).

*Conclusions: CRISPR screens can be used to map regulatory networks that control protein levels of critical factors that mediate the immune response in primary human T cells. We identified both canonical and novel regulators of CTLA-4. These data may help elucidate variances in regulation that could explain phenotypic differences seen in the clinical setting among well-matched recipients who experience heterogeneous outcomes. As gene editing moves into clinical practice, understanding key regulators of the immune system will be critical in selecting appropriate targets for therapeutic intervention.

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