CAR T-CELLS DYSFUNCTION IN THE CNS IS MEDIATED
BY AN AICD MECHANISM
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Yarden Aharony-Tevet1,2, Amilia Meir1,2, Orly Ravid3, Hadar Levi1,2, Daniel Burshtein1,2, Itzik Cooper3, Elad Jacoby1,2.
1 Laboratory of Cell Therapy, Sheba Medical Center
2 Faculty of Medicine, Tel-Aviv University
3 Sagol Neuroscience Center, Sheba Medical Center
Introduction: Chimeric Antigen Receptor (CAR) T-cells, approved for various hematological malignancies, have the capacity to eliminate leukemia and lymphoma within the central nervous system (CNS). Notably, CAR T-cells infiltrate the CNS independently of disease presence or ICANS development, 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-vivo and in-vitro.
Methods: Leukemic mice were injected with CD19-CAR T-cells, followed by phenotypic, functional analysis and RNAseq of CAR T-cells sorted from the CNS, bone marrow and spleen. For an in vitro blood-brain barrier (BBB) model, human endothelial cells were seeded on transwell inserts with bovine pericytes plated in the lower wells and co-cultured for 6 days. Upper compartment represents the blood-side and bottom represents the brain-side (Luminal and Abluminal, respectively). CAR T-cells were added to the luminal side and allowed to migrate for 4-hours. Cells were collected from both compartments for functional and phenotypic analysis.
Results: In both immunocompetent (B6) and humanized (NGS) mouse models CAR T-cells infiltrated the CNS with similar kinetics and expressed elevated PD-1 and CCR7 compared to CAR-T from the spleen or bone marrow. Isolated CNS-infiltrating CARs showed reduced target killing and IFNÉ£ secretion compared to bone marrow or spleen infiltrating CAR T-cells. Gene expression revealed lower expression of T-cells cytotoxic-related markers. Mechanistic studies utilizing a unique BBB model confirmed impaired cytotoxicity of BBB-crossing CAR T-cells, accompanied with increased IFNÉ£ secretion and annexin binding, suggesting BBB-mediated activation-induced cell death (AICD). Finally, CAR T-cells cultured in human cerebrospinal fluid showed impaired cytotoxic function and proliferative capacity compared to those in serum.
Conclusion: We describe a dysfunctional phenotype of CNS-infiltrating CAR T-cells, mediated by both nutrient-deprived CNS environment and interactions with BBB cells leading to AICD.