| CAR-T cell therapy is a revolutionary anticancer therapy that brings many hopes to help cure cancer,especially hematological malignancy.During CAR-T therapy,the release of cytokines is considered to be the hallmark of CAR-T cell efficacy,but the excessive release of these cytokines can/may lead to severe cytokine release syndrome(s CRS).s CRS can rapidly lead to tissue or organ damage,or even death,indicating the unpredictable risks of CAR-T cell treatment.At present,the anti-IL-6receptor antibody Tocilizumab and glucocorticoids are two standard drugs for the management of s CRS,but there are still many shortcomings during the clinical application,besides not all patients are effectively responded to these two medicines.Therefore,it is urgent to find new drugs and therapeutic methods for the treatment/management of severe CRS in order to reduce the CAR-T treatment risk of patients with severe CRS.Recently,ruxolitinib has been shown to significantly reduce the severity of CRS induced by CAR-T cells in clinical.Nevertheless,the exact mechanism of the effects of ruxolitinib on CAR-T cells is still unclear and lead to the lack of appropriate scientific data for clinical medication guidance.In this study,we systematically explored and investigated the role of ruxolitinib in the treatment of s CRS and its effect on CAR-T cell function.Firstly,we successfully designed,optimized and constructed a CAR plasmid vector with CAR gene expression cassette targeting the CD19 antigen,produced lentiviral vectors using a four-plasmid lentiviral packaging system after sequencing validation,and successfully transduced and expressed CAR on primary T cells.The effects of different concentrations of ruxolitinib on CAR-T cells and other related immune cells in terms of the proliferation,function and subtypes profiles were systematically investigated in vitro,as well as the investigation of the recovery of cellular function after withdrawal of the drug.In the in vivo study,we simulated the clinical CAR-T treatment,and compared the effect of different doses of CAR-T treatment in terms of the proliferation and the secretion of the cytokines of CAR-T cells.Finally,we provided the important study data and successfully help to design and conduct the clinical trials.From the systematic study,first,we revealed that ruxolitinib significantly inhibited the expansion of CAR-T-CD19 cells without affecting their activity.In addition,ruxolitinib could make the phenotypic characteristics of CAR-T cells more biased toward a differentiated state enriched in memory cells favorable for clinical efficacy,once the addition of the ruxolitinib,the antigen-specific killing ability and cytokine release ability of CAR-T cells are temporarily suppressed.At the same time,unexpectedly,it also showed significant inhibitory effects on other immune cells.However,the cancer-killing ability of CAR-T cells could be rapidly restored,but still maintain their suppression of cytokine release.,which could be a well explanation of the role of reducing the s CRS reaction by ruxolitinib.Furthermore,in mice study,we found that ruxolitinib could significantly slow down the proliferation of CAR-T cells at certain doses,but showed no significant effect on their anti-tumor effects/properties.In clinical validation trials,ruxolitinib could effectively control CAR-T-induced s CRS and significantly reduce the secretion level of immune cytokines,and timely withdrawal of ruxolitinib after remission of CRS had no significant effect on the therapeutic efficacy of CAR-T.Our study first demonstrated that ruxolitinib could not only inhibit the activities of CAR-T cells,but also of other immune cells,as well as CAR-T cells;ruxolitinib inhibited the function of CAR-T cells in a dose-dependent manner,but the cytotoxic properties of CAR-T cells could be restored once ruxolitinib was withdrawn.This work provides useful and important data for clinicians when applying ruxolitinib in the treatment of s CRS to address the issue of consideration of loss of CAR-T cell function. |
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