In our clinical trial of CD19 CAR-T cell therapy (ChiCTR-ONN-16009862) for R/R B-cell lymphoma, we found that after the patients with R/R B-cell lymphoma failed to the first time CD19 CAR-T cell therapy, resistance of CAR-T cell was reversed after 7–16 months of Ibrutinib salvage therapy. They obtained very satisfactory efficacy in the same second CD19 CAR-T cell therapy [17]. In our further exploration work, we found that in BALB/c mouse of subcutaneous tumor formation of B lymphoma cell lines, Ibrutinib reversed the resistance of lymphoma cells to CAR-T cells and improved the efficacy. Moreover, we observed significant expansion of CAR-T cells in the combined group of lymphoma BALB/c mouse [11]. However, what is the mechanism of Ibrutinib to reverse the resistance of CAR-T cells and improve efficacy? It was considered that the tumor tissue of subcutaneous tumorigenic mice formed a local mass with local TME. TME has been shown to play an important role in the drug resistance and progression of DLBCL [8, 9]. It promotes drug resistance and progression by inhibiting effector T cell activity and recruiting immunosuppressive cells such as macrophages [18–20]. In this study, we have made a preliminary exploration on the improvement of TME and regulation of macrophage M2 polarization by BTK inhibitors.
First, we analyzed the effects of different doses of BTK inhibitors on DLBCL cells (HBL-1/U2932 cells), and the sensitivity of DLBCL cells to CAR-T cells after pretreatment with BTK inhibitors. We found that HBL-1/U2932 cells were more sensitive to CAR-T cells after pretreatment with ibrutinib/orelabrutinib. Moreover, HBL-1/U2932 cells pretreated with ibrutinib/orelabrutinib for 48 hours were more sensitive to CAR-T cells than that of 24 hours. However, in our previous studies, we did not find a synergistic effect of simultaneous co-culture of CAR-T cells and BTK inhibitors in vitro [11]. Based on these results, we further studied the effect of pretreatment with BTK inhibitors on substitute activated M2 macrophages.
We successfully constructed substitute activated M2 macrophages using human THP-1 macrophage cells. After co-culture with HBL-1 cells, the expression of CD206 and IL-10 in activated M2 macrophages was up-regulated. The expression of CD206 and IL-10 were down-regulated by ibrutinib/orelabrutinib. It is interesting that the up-regulation of the expression of CD206 and IL-10 by Notch1 agonists could be reversed by pretreatment with ibrutinib/orelabrutinib. Moreover, the sensitivity of HBL-1 cells co-cultured with substitute activated M2 macrophages of each group to CAR-T cells changed accordingly. We further compared the sensitivity of HBL-1 cells which were co-cultured with or without substitute activated M2 macrophages to CAR-T cells.HBL-1 cells co-cultured with substitute activated M2 macrophages showed significantly down-regulated sensitivity to CAR-T cells, but it could be reversed by the pretreatment with BTK inhibitors to M2 macrophages. We conclude that pretreatment with BTK inhibitors down-regulate polarization of M2 macrophages and reverse the resistance of HBL-1 cells to CAR-T cells induced by substitute activated M2 macrophages.
In the mechanism exploration, we also found that Notch1 agonists up-regulated polarization of M2 macrophage and further induced the resistance of HBL-1 cells in the co-culture system to CAR-T cells. Activation of Notch1 receptor in malignancies was first reported in T cell acute lymphoblastic leukemia [21]. Subsequently, many studies have found that Notch1 receptor activation occurs in a variety of tumors, including CLL [22], mantle cell lymphoma [23], and DLBCL [24]. Patients with Notch1 mutations are often associated with higher risk of disease progression, drug resistance and poorer clinical outcomes [25, 26]. However, whether the Notch1 pathway regulates polarization of M2 macrophages remains unclear. It has been reported that Notch1 signaling could affect macrophage subtypes and function [27, 28]. When Notch1 signaling is blocked, macrophages are inhibited to differentiate into M2 macrophage type [29, 30]. It has also been reported that activation of Notch-RBP-J pathway promotes polarization of M2-type macrophages [31]. The Notch signaling pathway regulates cell differentiation and proliferation. Then Notch intracellular domain (NICD) translocates to the nucleus and binds to the DNA-binding protein RBP-J [31]. The most definite function of Notch signaling is to regulate the function of lymphocytes [32], and it has been reported that Notch pathway could regulate myeloid cell function [33–35]. However, the mechanism of effect of Notch-RBP-J pathway in polarization of macrophages remains unclear.
In our study, we found that pretreatment with ibrutinib/orelabrutinib down-regulated the polarization of M2 macrophages in each group of co-culture systems. Expression of macrophage was detected in macrophages extracted from co-culture system with HBL-1 cells. The polarization of M2 macrophages was down-regulated by pretreatment with ibrutinib/orelabrutinib, while was up-regulated by Notch1 agonists. The up-regulation polarization of M2 macrophages by Notch1 agonists could be reversed by pretreatment with ibrutinib/orelabrutinib. Interestingly, drug resistance of M2 macrophage-induced HBL-1 cells to CAR-T cells could be reversed by the effect of ibrutinib/orelabrutinib pretreatment on M2 macrophages.
In our further exploration of the mechanism by which pretreatment with BTK inhibitors down-regulate the polarization of M2 macrophages, we found that the expression of RBP-J protein was consistent with that of the expression of Notch1 protein. We verified the consistency of Notch-1 and RBP-J expression in activated M2 macrophages by siRNA transfection of Notch-1.
Therefore, we found that HBL-1/U2932 cells were more sensitive to CAR-T cells after pretreatment with BTK inhibitors. Further, we found that the activation of Notch-RBP-J pathway promotes the polarization of M2 macrophages. We demonstrated that pretreatment with BTK inhibitors down-regulate the Notch-RBP-J pathway of M2 macrophages and subsequently reverse the resistance of DLBCL cells which were co-cultured with substitute activated M2 macrophages to CAR-T cells. We also demonstrated our results in lymphoma mouse model.