| Chronic lymphocytic leukemia(CLL) is the most prevalent adult leukemia in western countries. Given improved understanding of the etiology and prognosis in CLL biology, the development of therapeutic approaches shifts to targeting critical molecules relied on by CLL cells. Overall, the mechanism and the effect of these drugs on immnune system need investigate deeply. Fortheremore, The leukemia micro-environment is associated with heterogeneous cell populations predominantly resembling tumor-associated macrophages. Nurse-like cells(NLCs) are M2-like macrophages that interact with B-CLL cells to promote their survival, and to protect them from spontaneous and antibody-induced apoptosis. Therefore, how to switch the M2 phenotype of NLCs towards the M1 thereby enhancing antibody-mediated killing of CLL-B cells become more and more important in CLL antibody thrapy.The irreversible Bruton’s tyrosine kinase(Btk) inhibitor, Ibrutinib, has shown efficacy against B-cell tumors such as Chronic Lymphocytic Leukemia(CLL) and B-cell Non-Hodgkin lymphoma. Fcγ receptors(FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses but many such responses involve Btk. In chapter 2, we tested the effects of Ibrutinib on FcγR-mediated activities in monocytes. We found that Ibrutinib did not affect monocyte FcγR-mediated phagocytosis even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice, in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. Results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that Ibrutinib did not inhibit Rac activation, nor did the calcium chelator BAPTA-AM. Collectively, these results suggest that the Btk inhibitor ibrutinib blocks FcγR-mediated cytokine production, likely by its effect on calcium signaling, but this has little to no effect on Rac activation. Hence, ibrutinib shows minimal effects on monocyte-mediated phagocytosis. Fortheremore, we detected how Btk phospharylation happened induced by FcγR, data shows that FcγRIIa(CD32)plays a most important role in Btk phospharylation. As controls for the efficacy and relative specificity of ibrutinib, we also tested the effects of ibrutinib on TLR4-and TLR8-mediated cytokine production. Results showed that Btk is closely involved with TLR8 activation but not for TLR4 activation.Monoclonal antibody immunotherapy has been widely used on the B-CLL clinical treatment. Fcγ receptors are critical for antibody-mediated responses, as they provide for initial contact of the effector cell to the opsonized target cell and subsequently activate effector signaling pathways that lead to target destruction. Activation of FcγR also elicits cytokine production by monocytes, which serves to activate other immune effectors such as natural killer(NK) cells. In chapter 3, We next asked whether the effect of Ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFN-γ priming, as NK cells produce IFN-γ in response to antibody therapy. Pretreatment of monocytes with IFN-γ abrogated the effects of Ibrutinib on FcγR-mediated cytokine production, suggesting that IFN-γ priming could overcome this Btk inhibition. We then proceeded to test the effects of ibrutinib on downstream mediators of FcγR activation in order to determine which were affected and which might be rescued by IFNγ, the results suggest that IFN-γ can rescue Erk pathway activation downstream of FcγR. Furthermore, in monocyte-NK-cell co-cultures, Ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining Ibrutinib with monoclonal antibody therapy could enhance B-CLL killing without affecting macrophage effector function.Over the past decade, the essential role of the tumor microenvironment in the survival and progression of CLL has become increasingly clear. The CLL tumor microenvironment describes an admixture of malignant cells with host immune cells, stromal elements, and vascular cells that create a niche wherein signals can be transmitted through cell-cell interactions, antigen presentation, and paracrine signaling. The CLL tumor microenvironment contains cells and cytokines that support CLL survival. CD14+ monocytes when co-cultured with chronic lymphocytic leukemia B cells can differentiate in vitro into large, round, adherent cells termed “nurse-like cells”(NLCs) that can support the survival of CLL cells survival. Interferon-γ(IFN-γ) has been long known to induce M1 polarization of macrophages. In chapter 4, we illuminated that IFN-γ can switch the M2 phenotype of NLCs towards the M1 thereby enhancing cytokine production, phagocytosis and antibody-mediated killing of CLL-B cells. Specifically, experiments revealed that treatment of NLCs with IFN-γ resulted in enhanced expression of M1-associated genes CD80 and CD86, and down-regulated expression of the M2-associated gene CD206. Our results were further supported by flow cytometry analysis of surface expression of these markers. Additionally, IFN-γ treated NLCs showed enhanced expression of FcγR1 and phagocytosis. NLCs also showed enhanced antibodymediated B cell depletion upon stimulation with IFN-γ. Finally, IFN-γ treatment of NLCs resulted in increased cytotoxicity and reduced B-CLL survival as measured by MTS. Monocytes to NLCs generation and their TAM like function is solely determined by the CLL microenvironment. Thus, we propose that IFN-γ can re-educate Nurse-like cells and skew them towards anti-cancerous, effector type for proper immune response against CLL and can be used as potential anti-CLL therapy in combination with antibody treatment for a maximal treatment success.This dissertation focused the effects of the Btk inhibitor Ibrutinib on monocyte FcγR function, and illuminated the feasiblity and potentiality of Ibrutinib in combination with antibody therapy in CLL. Furthermore, we discussed and examined that IFN-γ is capable of polarizing NLCs to interrupt B-CLL survival. Importantly, a more complete understanding for further enhancement of the efficacy of therapeutic antibodies have unlimited potential. It will be further important to explore cellular sources and induce immune mechanisms in both innate and adaptive immune response to generate spontaneous IFN-γ for sustained anti-CLL environment. |
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