Project Summary Multidrug resistance (MDR)-1 is a plasma membrane-associated, ATP-dependent efflux pump widely recognized—and named—for removing chemotherapeutic compounds from drug-resistant tumor cells. Whereas endogenous substrates and functions of MDR1 have remained enigmatic for nearly 50 years, both the presence of MDR1 orthologs in prokaryotes and an emerging body of literature suggest that MDR1 has broader and more fundamental functions in cell biology beyond simply interfacing with synthetic medicines. Using CRISPR/Cas9-mediated genome editing in mouse zygotes, we previously created a fluorescent MDR1 reporter mouse (Abcb1aAME/+) and showed that MDR1 is expressed in a variety of lymphocyte lineages at steady-state. We have gone on to show two examples of how endogenous MDR1 functions serve to support the establishment and maintenance of lymphocyte homeostasis in vivo. First, we have shown that MDR1 acts intrinsically in CD4+ T helper (TH) cells infiltrating the small intestinal mucosa to enforce homeostasis, limit pathogenic cytokine expression and suppress Crohn’s disease-like ileitis in the presence of naturally- circulating bile acids. More recently, we have found that MDR1 is constitutively expressed in cytolytic lymphocytes, including CD8+ cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, due to direct regulation by runt-related (Runx) transcription factors. In CTLs, MDR1 promotes the survival of cytolytic effector cells, such that loss or pharmacologic inhibition of MDR1 leads to increased CTL death, reduced expression of key cytolytic effector molecules (e.g., perforin, granzyme B), impaired CTL-dependent target cell killing in vitro, and diminished CTL-dependent anti-tumor immunity in vivo. As a whole, these innovative preliminary studies highlight diverse and important functions of MDR1 in normal immune physiology. Our long- term objectives are to elucidate additional cellular contexts where endogenous MDR1 functions regulate physiologic immune responses, whilst defining core classes of endogenous MDR1 transport substrates in vivo. Because existing genetic tools (e.g., RNAi, whole-body MDR1 knockout mice) are not sufficient to address these question, we will leverage our collective expertise in CRISPR/Cas9-based genome editing to generate the first conditional MDR1 (Abcb1a) knockout mouse. Successful generation of these mice will pave the way for myriad future studies utilizing conditional gene ablation to explore the diverse molecular functions of MDR1 in immune, parenchymal and malignant cells.