LB915 A targeted CRISPR screen to identify essential genes for the TRM T cell response to cancer

Abstract

Bunkered in barrier tissue, tissue-resident memory T (T RM ) cells are strategically positioned to react quickly to tissue perturbations, such as infection, injury, or cancer. Recent studies have highlighted a potential role for T RM cells as a targetable T cell population due to their abundance in most tissues. To better understand the role T RM cells play in the antitumor immune response, we developed a targeted CRISPR screen to identify the essential genes regulating the T RM -response to tumors. We first developed a murine tumor model system in which OT-I cells could be adoptively transferred and re-isolated from tumors. Yumm1.7 tumor cells were transduced with OVA fused to mCherry. SIINFEKL reactivity to Yumm.OVAmCh tumors was confirmed by pentamer staining, ELISPOT, and OT-I proliferation in vivo. To promote T RM -responses we used a model of skin scarification where tumors develop in the uppermost layers of the skin. In this model, approximately 1/3 tumors are spontaneously rejected. On day 7 after tumor inoculation, skin-residing T cells expressed CD103 and CD69, which was not observed in tumors engrafted subcutaneously. Further, CD8 + T cells isolated from the skin of rejected tumors contained SIINFEKL-reactive T RM cells. Next, we optimized T cell transduction to achieve approximately 20% efficiency, which was enriched to 50% after puromycin selection. Finally, to achieve statistically powerful guide-RNA (gRNA) coverage we chose to target select genes expressed in T RM cells and CD8 + tumor-infiltrating lymphocytes (TILs). To this end, we compared gene expression profiles of T RM cells and TILs across several studies. From this cross-study comparison, we chose both up-and down-regulated genes (log2 fold-change > 1.5) totaling 1,469 genes. In conclusion, the epicutaneous tumor model system provides a vehicle to study T RM -responses to melanomas. Future studies will apply a targeted-deletion OT-I T cell library to these tumors to reveal genes critical for the T RM -response to tumors.