Project Grant F30CA310070

UNRAVELING THE IMMUNOBIOLOGY OF TUMOR-SPECIFIC T FOLLICULAR HELPER CD4 T CELLS - PROJECT SUMMARY WHILE IMMUNE CHECKPOINT BLOCKADE THERAPIES HAVE REVOLUTIONIZED THE TREATMENT OF CANCER FOR PATIENTS, MANY REMAIN UNRESPONSIVE. THERE HAS BEEN INCREASED FOCUS ON FACTORS OUTSIDE OF TUMOR-INFILTRATING CD8 T CELLS THAT CONTRIBUTE TO TREATMENT EFFICACY, PARTICULARLY THE ROLE OF HELPER CD4 T CELLS. IN INVESTIGATING THIS QUESTION, CURRENT STUDIES HAVE IDENTIFIED THE CD4 SUBSET, T FOLLICULAR HELPER CELLS AND THEIR CANONICAL CYTOKINE INTERLEUKIN (IL)-21, TO BE AN IMPORTANT DRIVER OF RESPONSE, PARTICULARLY TO THE CTLA-4 AXIS OF IMMUNOTHERAPY. THESE STUDIES, HOWEVER, FAIL TO DEFINE THE PRECISE LOCATION OF IL-21 DELIVERY FOR OPTIMAL CD8 T CELL HELP OR ADDITIONAL REQUIRED FACTORS, INCLUDING THE GENERATION TUMOR-SPECIFIC B CELLS. BASED ON PRELIMINARY DATA THAT TUMOR DRAINING LYMPH NODES (TDLNS) CONTAIN A RESERVOIR OF IL-21 PRODUCING CD4 T CELLS OUTSIDE OF B CELL FOLLICLES AND THAT CTLA-4-MEDIATED IL-21 PRODUCTION DEPENDS ON A B CELL ANTIGEN, I HYPOTHESIZE THAT IN ORDER TO OPTIMALLY PRIME CD8 T CELLS TO GENERATE ANTITUMOR EFFECTORS AND RESPOND TO CTLA-4, IL-21 PRODUCTION MUST OCCUR OUTSIDE OF THE B CELL FOLLICLE IN TDLNS AND THAT THIS DEPENDS ON THE PRESENCE OF TUMOR-SPECIFIC B CELLS. TO DIRECTLY TEST THIS, I WILL PURSUE THE FOLLOWING AIMS. FOR MY FIRST AIM, I WILL DEFINE THE FACTORS THAT GIVE RISE TO THE SUBSET OF IL-21+ CD4 T CELLS IN THE EXTRAFOLLICULAR ZONE OF TDLNS. TO ACCOMPLISH THIS, I WILL SPATIOTEMPORALLY TRACK THE LOCATION, PHENOTYPE, AND PATH OF DIFFERENTIATION OF IL-21-PRODUCING CELLS IN TDLNS BY CYCLIC IMMUNOFLUORESCENCE, FLOW CYTOMETRY, AND SINGLE CELL RNA SEQUENCING. I WILL TARGET GENES PREDICTED TO BE INVOLVED IN THIS DIFFERENTIATION PATH USING CAS9 RIBONUCLEOPROTEIN EDITING OF TUMOR-SPECIFIC CD4 T CELLS TO MODIFY THEIR DEVELOPMENT AND FUNCTION. FOR MY SECOND AIM, I WILL DETERMINE THE ROLE OF IL-21 PRODUCTION ON CD8 T CELL EFFECTOR FUNCTION AND ANTI-CTLA-4 THERAPEUTIC EFFICACY. TO DO THIS, I WILL ELIMINATE THE ABILITY TO PRODUCE IL-21 IN TUMOR-SPECIFIC CD4 T CELLS OR REMOVE B CELLS AND DETERMINE THE EFFECTS ON CD8 T CELL EFFECTOR FUNCTION AND CTLA-4 BLOCKADE EFFICACY. I WILL THEN USE ADOPTIVE TRANSFER STUDIES WITH A MODEL OF LUNG ADENOCARCINOMA THAT IS CAPABLE OF GENERATING IL-21+ TFH, HELLO, AND ONE THAT IS NOT, NINJA, TO ASSESS THE REQUIREMENT OF CD4 INTERACTIONS WITH TUMOR-SPECIFIC B CELLS FOR CTLA-4 IMMUNOTHERAPY RESPONSES. LASTLY, I WILL DIRECTLY DELIVER TUMOR-SPECIFIC CD4 T CELLS WITH ECTOPIC IL-21 EXPRESSION TO NINJA TUMOR-BEARING MICE TO OVERCOME THE REQUIREMENT OF B CELLS. I EXPECT THAT MY FINDINGS WILL UNCOVER THE NECESSARY FACTORS FOR PRODUCTIVE IL-21 DELIVERY WITHIN TDLNS AND IDENTIFY NEW STRATEGIES TO GENERATE IL-21 PRODUCING CELLS AND REINVIGORATE CD8 T CELLS IN ICB UNRESPONSIVE TUMORS.