Circadian rhythm disruption impairs CAR-T cell efficacy by promoting an immunosuppressive tumor microenvironment through enhancing monocyte-MDSC infiltration

Abstract Background: Circadian rhythm disruption is associated with various chronic diseases and cancers, and may influence cancer immunotherapy outcomes by modulating immune cell function. Timing CAR-T cell infusion to an optimal circadian phase can enhance CAR-T cell antitumor efficacy. However, the impact of circadian rhythm disruption on CAR-T therapy remains unexplored. High circulating levels of myeloid-derived suppressor cell (MDSC) are proved to be associated with a lack of response to CAR-T cell therapy. CAR-T non-response (NR) patients exhibit higher frequencies of MDSC and reduced circadian genes expression (BMAL1 and CLOCK) compared with response (R) patients (GSE197268), suggesting circadian rhythm disruption may influence CAR-T therapy efficacy by modulating MDSC frequency and function. Aims and Methods: This study aims to investigate the effects and underlying mechanisms of circadian disruption on CAR-T cell efficacy. In vivo, a circadian rhythm-disrupted mouse model was established by subjecting mice to chronic jet lag (CJL), involving repeated 6-hour phase advances of the light-dark cycle every 2–3 days. A syngeneic non-Hodgkin's lymphoma mouse model was established using CD19-targeted CAR-T cells. CAR-T antitumor efficacy was evaluated across different circadian conditions, and flow cytometry was used to characterize immune cell composition and function within the tumor microenvironment (TME) and spleen. MDSC suppressive function was evaluated in vitro by co-culturing MDSCs with CAR-T cells. Results: In mice, circadian disruption contributed to increased tumor burden. CJL mice showed increased levels of MDSC and M-MDSC in the spleen compared to control mice. However, under tumor-bearing conditions, a significant decrease in splenic MDSC and M-MDSC populations was observed in CJL mice, accompanied by increased accumulation of these cells within the tumor microenvironment. This redistribution suggests that circadian disruption may promote the mobilization of MDSCs from peripheral reservoirs such as the spleen to the tumor site. We next monitored anti-tumor responses following CAR-T cell infusion up to 21 days. CAR-T cells exhibited a significant tumor-killing effect in normal circadian mice by day 7 post-infusion. In contrast, circadian-disrupted mice displayed a markedly attenuated CAR-T cytotoxic response. Notably, this impaired efficacy was associated with an increased frequency of M-MDSCs expressing PD-L1, along with an increased proportion of CAR-T cells expressing inhibitory receptors such as TIM-3 and PD-1. M-MDSCs isolated from tumors of circadian-disrupted mice exhibited enhanced suppressive activity against CAR-T cell proliferation and function in vitro, indicating that circadian disruption potentiates the immunosuppressive capacity of M-MDSCs within the tumor microenvironment. Conclusion: Circadian rhythm disruption impairs CAR-T efficacy by forming an immunosuppressive tumor microenvironment through promoting the migration of M-MDSCs to the tumor and enhancing their immunosuppressive capacity. Modulating circadian rhythms may represent a novel strategy to optimize CAR-T therapy outcomes.