Activation of CAR and non-CAR T cells within the tumor microenvironment following CAR T cell therapy.

Mechanisms of chimeric antigen receptor (CAR) T cell-mediated antitumor immunity and toxicity remain poorly characterized because few studies examine the intact tumor microenvironment (TME) following CAR T cell infusion. Axicabtagene ciloleucel is an autologous anti-CD19 CAR T cell therapy approved for patients with large B cell lymphoma. We devised multiplex immunostaining and ISH assays to interrogate CAR T cells and other immune cell infiltrates in biopsies of diffuse large B cell lymphoma following axicabtagene ciloleucel infusion. We found that a majority of intratumoral CAR T cells expressed markers of T cell activation but, unexpectedly, constituted ≤5% of all T cells within the TME 5 days or more after therapy. Large numbers of T cells without CAR were also activated within the TME after axicabtagene ciloleucel infusion; these cells were positive for Ki-67, IFN-γ, granzyme B (GzmB), and/or PD-1 and were found at the highest levels in biopsies with CAR T cells. Additionally, non-CAR immune cells were the exclusive source of IL-6, a cytokine associated with cytokine release syndrome, and were found at their highest numbers in biopsies with CAR T cells. These data suggest that intratumoral CAR T cells are associated with non-CAR immune cell activation within the TME with both beneficial and pathological effects.