Abstract 4817: CAR negative T cells predominate during xenogeneic graft versus host disease after CAR T cell treatment in NSG mice
Authors: Wahed Firoz, Michael K. Sheng, Logan Vick, Craig P. Collins, Ryan Nielsen, Sean J. Judge, William J. Murphy
Journal: Cancer Research (2025)

Abstract Chimeric antigen receptor (CAR) T cells are currently FDA-approved and provide a curative solution for patients with select B-cell malignancies in the relapsed or refractory setting. However, there have been multiple reports of long-term toxicities associated with CAR T cell treatment, such as second primary malignancies, highlighting the importance of assessing the long-term effects of CAR T cells on the host in preclinical models. Since the study of CAR T cell efficacy is performed in in vivo xenograft mouse models, long-term study is limited by xenogeneic graft-versus-host disease (xenoGVHD) in which human T cells recognize foreign murine major histocompatibility complex, leading to immune-mediated pathology and eventual death of the animal. On the other hand, allogeneic CAR T cells are being increasingly investigated in the aims of lowering cost and increasing access of treatment, though poses the risk of GVHD. In this project, we aimed to investigate the cellular dynamics of CAR T cells in mediating xenoGVHD. To accomplish this aim, we utilized a tri-specific human CAR T cell product which targets CD19, CD20, and CD22 against the Raji human B-cell lymphoma cell line in the immunodeficient NSG mouse model. During the initial tumor clearance, the CAR-positive T cells preferentially expand and constitute upwards of 90% of CD3+ T-cells in the bone marrow of treated mice. However, after approximately 40 to 180 days post tumor clearance, we observed weight loss and outward signs of xenoGVHD. We found that the variability in xenoGVHD onset and occurrence is dependent on the human T cell donor used to make the CAR T cells. Additionally, we found that the CAR-negative T cells predominated in the spleen and bone marrow of Raji-bearing NSG mice treated with human CAR T cells. Using flow cytometry, we observed upwards of 85% CAR-negative T cells of CD3+ T-cells in the spleen and bone marrow of mice experiencing xenoGVHD across multiple experiments. This was an increase from the frequency of CAR-negative T cells at both the pre-infusion baseline timepoint and during tumor clearance, suggesting a preferential expansion of CAR-negative T cells in response to xenoantigens. In addition, we observed upregulation of markers associated with T-cell activation, such as PD-1 and HLA-DR, among the CAR-negative T cells during xenoGVHD, indicating TCR-mediated activation by xenoantigens. Overall, our findings suggest that the CAR-negative T cells predominate during xenoGVHD and play a role in mediating pathology in our tested model. The occurrence of xenoGVHD in these preclinical xenograft models of CAR T cell efficacy are a limitation and prevent the study of potential long-term effects of treatment. Our findings also give insights into the generation of allogeneic CAR T cell products, especially regarding the role of CAR-negative T cells in mediating allogeneic GVHD with off-the-shelf allogeneic CAR T cell products. Citation Format: Wahed Firoz, Michael K. Sheng, Logan Vick, Craig P. Collins, Ryan Nielsen, Sean J. Judge, William J. Murphy. CAR negative T cells predominate during xenogeneic graft versus host disease after CAR T cell treatment in NSG mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4817.