CD3 bispecific antibodies (CD3BiAbs) physically link CD3 on T cells to target tumor antigens, which directs T cells to kill tumors. CD3BiAbs have shown high therapeutic potency against hematologic malignancies and are also being developed to target solid tumors. In clinical settings, CD3BiAbs causes cytokine release syndrome (CRS), a difficult-to-manage side effect associated with elevated levels of circulating cytokines, which has proven be a dose-limiting toxicity (DLT) and can be life-threatening. Premedication with corticosteroid, anti-IL-6 therapy, and intra-patient dose escalation have been reported to mitigate CRS, but more is needed to successfully control CRS. In an in vivo cynomolgus monkey study, a daily ascending dosing of a surrogate CD3BiAb mitigated CRS and the tolerance to CRS remained in effect for the following weekly doses. Antibodies having ADCC also cause CRS, and CRS typically occurs with the first dose and rarely with following doses. However, the mechanisms underlying the mitigation of CRS are not fully understood. We established an in vitro model with human lymphocytes showing that the cytokine release potential was decreased by the priming treatment. Cytokine mRNA levels after the repeated treatment were lower than those after the first treatment, but CD3 signal transduction was similar. ATAC-seq analysis showed that the priming treatment altered T-cell chromatin states, suggesting that epigenetic changes played an important role in the mitigation of cytokine release. In this presentation, we will introduce these studies on the mitigation of CRS by CD3BiAbs.