Chronic xenogeneic GVHD after CAR T cell therapy limits long-term safety assessment of CAR effects using NSG mouse model

The clinical successes of chimeric antigen receptor (CAR) T cells represent a major shift in immunotherapy. However, there is also increasing emphasis on potential long-term effects of CAR T cell therapy, especially using preclinical xenogeneic models. It has been previously demonstrated that only naïve, and not memory, peripheral blood human T cells can mediate a rapid and acute xenogeneic graft-versus-host disease (xenoGVHD). Here, we demonstrate that simply by altering the donor T cells in the process of generating CAR T cells, in which they are all memory phenotype, that the xenoGVHD outcome was markedly altered. Following tumor clearance with CAR T cell administration, we observed a significantly delayed (up to 200 days post-transfer with some donors) occurrence of lethal xenoGVHD, marked by profound scleroderma and multi-organ pathology consistent with chronic, not acute, GVHD. Notably, this novel chronic xenoGVHD occurred in the absence of B cells, which are classically associated with mediating chronic GVHD pathology. TCR-repertoire constriction during disease and the lack of disease using MHCI/II double-knockout NSG recipient mice confirmed the observed pathology was xenoGVHD and mediated by human-TCR:murine-MHC interactions. Interestingly, despite the consistent expansion of CAR-positive T cells during early tumor-clearance, a later emergence of CAR-negative populations during xenoGVHD also resulted. Our findings highlight xenoGVHD as a problem that makes long-term assessment of CAR T efficacy or toxicity highly problematic in xenograft models due to the artefact of xenoreactivity which is not representative of autologous clinical usage, and the profound effect that T cell alterations have in GVHD pathophysiology.