Single-cell transcriptomics of pediatric B-ALL bone marrow to determine mechanisms of durable CD19 CAR T cell persistence

Abstract Chimeric antigen receptor T cell (CAR T) therapy has revolutionized the treatment of relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, approximately 50% of patients relapse following CD19-directed CAR T, in part due to early loss of CAR T persistence. Pre-infusion and peripheral blood CAR T cells from patients have been studied to infer potential mechanisms of in vivo CAR T persistence. Yet, the degree to which the bone marrow microenvironment – which is the primary site of leukemia residence and CAR T activity – impacts CAR T persistence and function is poorly understood. We hypothesize that the bone marrow microenvironment shapes CAR T fitness and longevity through tissue-specific factors and cell-cell interactions. Using our unique access to the Children's Hospital of Philadelphia biorepository, we analyzed bone marrow aspirates from two cohorts of pediatric B-ALL patients treated with CTL019 (i.e. CD19.BBz): 1) patients with durable CAR T persistence defined as disease remission and peripheral B cell aplasia (BCA) > 5 years, and 2) patients with poor CAR T persistence defined as peripheral B cell recovery (BCR) within 6 months. We performed single-cell RNA sequencing of bone marrow mononuclear cells and FACS-enriched bone marrow resident CAR T cells collected one month after infusion (n=12 BCA; n=12 BCR) to detect cell subsets, phenotypes, and biological pathways associated with CAR T persistence. Transcriptomic analyses revealed an enrichment of memory-associated signatures (e.g. LEF1, KLF2, FOXO1 regulon) in CAR T from BCA patients, while CAR T from BCR patients were enriched in CAR+ FOXP3+ regulatory T cells and exhaustion signatures (e.g. TOX, LAG3, HAVCR2, CTLA4), consistent with a model in which CAR T persistence is maintained by memory T cell programs and limited by immunosuppressive and exhaustion programs. Of note, bone marrow monocytes and macrophages displayed divergent transcriptional profiles between cohorts, whereby myeloid cells from BCA were enriched for costimulatory molecules (e.g. CD86) and memory-supporting cytokines (i.e. IL-15), suggesting that myeloid cells support CAR T persistence.Our study provides the first comprehensive transcriptomics analyses of CTL019 cells and other cell types in the bone marrow microenvironment. Our results show that patients with BCA are enriched in memory-like CAR T subsets, while patients with early BCR display CAR T with more immunosuppressive and exhausted phenotypes. Additionally, patients with BCA were enriched in bone marrow myeloid cells with immunoactivation signatures, consistent with the notion that myeloid cells directly govern CAR T persistence and function.