Necroptotic cell death and immunomodulator release induced by T-cell engaging anti-lymphoma therapies

Abstract Autologous cellular immunotherapies, which rely on cytotoxic T lymphocytes (CTLs), are increasingly applied in different B-cell malignancies. The general assumption is that CTLs exert their cytolytic function through granzymes that induce apoptosis in the target cell. However, the killing mechanism of immunotherapeutic CTLs is not clearly elucidated. Using T-cell redirecting bispecific antibodies (BsAbs) and chimeric antigen receptor (CAR) T-cells we assessed which cell death pathways were activated in B cell line models as well as in primary material from CLL patients. We demonstrate that for cytotoxic T-cell killing of malignant B-cells by any of the treatment strategies, caspase activity was not essential. We could also exclude a role for TNF or TRAIL-mediated pathways. Using electron microscopy, CAR T-cell and BsAb-mediated cell death showed a mixed apoptotic/necroptotic phenotype. This was corroborated by knockout and chemical inhibition of the essential necroptosis proteins RIPK1, RIPK3, and MLKL. Necroptotic death of target cells correlated with the release of HMGB1 in the supernatant as well as various immunomodulatory molecules from the T-cells. Besides known immune activators IFN-y, IL-17 and IL-6, also anti-inflammatory IL-10 and IL1-RA were released. Moreover, the quantity of these immunomodulators was differentially affected after application of apoptosis versus necroptosis inhibitors. Together, these data demonstrate that (CAR) T-cell-mediated killing of lymphoma cells has a necroptotic arm which is correlated with modulation of the wider immune response. They imply that manipulation of the apoptotic versus necroptotic balance in immunotherapy could affect engagement of the autologous immune response.