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

Background: 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.

Methods: 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.

Results: 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. The cytotoxicity of CAR-T cells to HBL-1 cells in co-culture system (HBL-1 cells and substitute activated M2 macrophages) was down-regulated by Notch1 agonist. The drug resistance of HBL-1 cells to CD19 CAR-T cells induced by Notch1 agonist could be reversed by replacing the M2 macrophages (M2 macrophages after 48h pretreatment with BTK inhibitors) in co-culture system (P_ibrutinib<0.001 and P_{orelabrutinib}<0.001). In co-culture system of HBL-1 cells and activated M2 macrophages, the expression of CD206 and IL-10 in activated M2 macrophages were up-regulated. 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 expression of Notch1 protein in activated M2 macrophages was down-regulated by pretreatment with ibrutinib/orelabrutinib (P_ibrutinib<0.001 and P_{orelabrutinib}<0.001). The up-regulation polarization of M2 macrophages by Notch1 agonist could be reversed by BTK inhibitors. The expression of RBP-J protein in activated M2 macrophages was down-regulated by pretreatment with ibrutinib/orelabrutinib (P_ibrutinib<0.001 and P_{orelabrutinib}<0.001). RBP-J has Notch 1 binding site and is regulated by Notch 1. The expression of Notch 1 protein decreased in substitute activated M2 macrophages by siRNA silencing Notch 1 (P<0.001). At the same time, the expression of RBP-J protein decreased in substitute activated M2 macrophages by siRNA silencing Notch 1 also (P<0.001). We also demonstrated our results in lymphoma mouse model. The expression of CD206 and IL-10 in macrophages from residual lymphoma tissue in mice were down-regulated in ibrutinib group (P=0.011and P=0.006) and CAR-T+ ibrutinib group (P=0.009 and P=0.004) compared with CAR-T group.

Conclusion: 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.

Key words: Bruton's tyrosine kinase inhibitor, Diffuse large B-cell lymphoma cells, Macrophages, Polarization, Chimeric antigen receptor