CAR T-Cells Are Dysfunctional In The CNS

Chimeric Antigen Receptor (CAR) T-cells can eliminate leukemia and lymphoma within the central nervous system (CNS). Infiltration to the CNS is independent of disease presence or neurotoxicity, yet the mechanisms driving their migration and functional state within the CNS remain poorly understood. Here, we characterize the unique phenotype and functionality of CNS-infiltrating CD19-CAR T-cells in-vitro and in-vivo using immunocompetent and humanized murine models. In leukemia-bearing B6 mice, CNS-infiltrating CAR T-cells displayed distinct exhausted phenotypes compared to those from the spleen or bone marrow. Findings confirmed in human CAR T-cells in NSG mice. CNS-infiltrating CARs showed reduced cytolytic activity when co-cultured with leukemia. RNAseq revealed lower expression of activation markers in CAR T-cells from the CNS compared to bone marrow or spleen residing CAR T-cells, indicating a less activated or exhausted state. To investigate the mechanism involved, we used an in vitro BBB model utilizing human CD34-endothelial cells and brain pericytes to demonstrate that CAR T-cells that cross the BBB exhibited diminished cytolytic capacity while showing a more apoptotic stage, suggesting an activation-induced cell death mechanism. Finally, CAR T-cells cultured in cerebrospinal fluid showed impaired cytotoxic and proliferative function compared to those in serum.

In summary, our comprehensive studies demonstrate dysfunctionality of CNS-infiltrating CAR T-cells potentially driven by interactions with BBB cells or the nutrient-deprived CNS environment.