Abstract PR007: Mechanisms and impact of bystander killing by CAR T cells
Joanna Chorazeczewski, Matthew Lin, Alan Cooper, Lishi Xie, Xiufen Chen, Sidney Wang, Joshua Brody, Justin KlineAbstract
Background:
CD19-directed chimeric antigen receptor (CAR) T cell therapy has significantly improved outcomes for patients with relapsed/refractory diffuse large B cell lymphoma (r/r DLBCL), yet a majority of patients ultimately relapse. Understanding mechanisms of CAR T cell-mediated cytotoxicity is critical to improving therapeutic durability. CAR T cell killing is presumed to occur via CD19 recognition and activation of the perforin/granzyme axis. However, reports of CD19-independent bystander killing suggest additional mechanisms contribute to antitumor activity. Here, we investigated the role of Fas-mediated signaling in on-target and bystander killing by CD19-directed CAR T cells and evaluated whether this pathway can be exploited to enhance efficacy against antigen-heterogeneous tumors.
Methods:
We assessed CD19 and FAS gene expression in pre-treatment tumor biopsies from r/r DLBCL patients treated with axicabtagene ciloleucel (axi-cel) in two independent cohorts (ZUMA-1 and Stanford). Correlative findings were validated using flow cytometric phenotyping of DLBCL cell lines and in vitro cytotoxicity assays. CRISPR/Cas9 editing was used to generate isogenic CD19+FAS+, CD19+FAS–, CD19–FAS+, and CD19–FAS– human DLBCL cells. These populations were co-cultured with human CAR T cells to dissect the relative contributions of FAS to on-target (CD19+) and bystander killing (CD19–). In vivo function was tested using NSG mice bearing mixed-population DLBCL xenografts. Fas pathway modulation was evaluated by treating tumor cells with IAP inhibitors or using CAR T cells engineered to express membrane-bound Fas ligand (mFasL).
Results:
FAS expression was associated with improved survival in CD19-low DLBCL patients across multiple cohorts. In vitro, surface FAS expression strongly correlated with CAR T cell-mediated cytotoxicity, while CD19 levels showed only a weak correlation. Among the four engineered DLBCL subsets, CD19+FAS+ cells were killed most efficiently, followed by CD19+FAS– and CD19–FAS+ cells. CD19–FAS– cells were minimally susceptible. In vivo, CAR T cell-treated NSG mice bearing tumors composed of 80% CD19+FAS+ and 20% CD19–FAS+ cells had similar survival to mice with pure CD19+FAS+ tumors. NSG mice bearing 80% CD19+FAS+ and 20% CD19–FAS– cells, on the other hand, did not have any survival benefit from CAR T cell treatment. Fas pathway augmentation with IAP inhibition or mFasL-CAR T cells improved killing in vitro.
Conclusions:
FAS is required for optimal CAR T cell on-target and bystander killing. CD19 engagement is necessary to initiate bystander cytotoxicity through Fas signaling, and that CD19 and FAS need not necessarily be engaged on the same target cell. Our findings generate new insight into bystander killing and support leveraging Fas–FasL interactions to enhance CAR T cell efficacy in antigen-heterogeneous DLBCL.
Citation Format:
Joanna Chorazeczewski, Matthew Lin, Alan Cooper, Lishi Xie, Xiufen Chen, Sidney Wang, Joshua Brody, Justin Kline. Mechanisms and impact of bystander killing by CAR T cells [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr PR007.