Pretreatment with BTK inhibitors could improve the sensitivity of DLBCL cells to CAR-T cells in co-culture system by down-regulating the polarization of M2 macrophages

Abstract The Tumor microenvironment (TME) of relapsed/refractory (R/R) diffuse large B-Cell lymphoma (DLBCL) patients is associated with resistance of DLBCL cells to CD19 CAR-T cells. How to improve TME in DLBCL and improve the efficacy of CAR-T cell therapy remains to be further explored. We observed the sensitivity of HBL-1/U2932 cells pretreated with BTK inhibitors to CAR-T cells with flow cytometry (FCM), then observed the sensitivity of HBL-1 cells extracted from the co-culture system to CAR-T cells. Effect of pretreatment of BTK inhibitors on the substitute activated M2 macrophages was observed with FCM, Real-time PCR and Western blot method. Then the expression consistency of Notch-1 and RBP-J in activated M2 macrophages was observed by siRNA transfection of Notch-1. After substitute activated M2 macrophages and HBL-1 cells were pretreated with ibrutinib/orelabrutinib respectively, the cytotoxicity of CAR-T cells to HBL-1 cells was higher than that of in substitute activated M2 macrophages pretreated with ibrutinib/orelabrutinib group, and higher than that of in HBL-1 cells pretreated with ibrutinib/orelabrutinib group. Pretreatment with BTK inhibitors could down-regulate the expression of CD206 and IL-10 in activated M2 macrophages. Pretreatment with BTK inhibitors down-regulated the expression of Arg-1 and up-regulated the expression of iNOS in activated M2 macrophages. The up-regulation polarization of M2 macrophages by Notch1 agonists could be reversed by BTK inhibitors. Expression of RBP-J protein decreased in substitute activated M2 macrophages by siRNA silencing Notch 1. Pretreatment with BTK inhibitors could down-regulate the polarization of M2 macrophages and reverse the resistance of DLBCL cells which were co-cultured with substitute activated M2 macrophages to CAR-T cells. This effect might be achieved by down-regulating the Notch-RBP-J pathway.