FITS: Framework for In vivo T cell Screens

In vivo T cell screens are a powerful tool for elucidating complex mechanisms of immunity, yet there is a lack of consensus on the screen design parameters required for robust in vivo screens: gene library size, cell transfer quantity, and number of mice. The goals of this study are to provide experimental and analytical guidelines to determine optimal parameters for diverse in vivo contexts. As a proof-of-concept, we optimized the parameters for a CD8+ T cell screen in the B16-OVA tumor model. We also included unique molecular identifiers (UMIs) in our screens to (1) improve statistical power and (2) track T cell clonal dynamics for distinct gene knockouts (KOs) across multiple tissues. These datasets provide an experimental and analytical framework for performing in vivo screens in immune cells and illustrate a case study for in vivo T cell screens with UMIs.