Myeloid cell-derived Granzyme B as an inducible enhancer of cancer immunotherapy

项目总结 我们广泛而长期的目标是了解Fcγ受体(FcγR)功能的分子细节， 其目标是改进癌症的单抗治疗。抗体疗法的抗肿瘤作用 在很大程度上是由FcγR介导的，当结合到Fc部分时，Fc Fc R就会聚集并激活 抗体。单核细胞和巨噬细胞是抗体治疗有效的关键，FcγR 这些细胞的激活可以导致多效性反应。这些包括吞噬作用，抗体依赖 细胞毒性(ADCC)，炎性细胞因子的产生，或它们的组合。其中包括 单核细胞/巨噬细胞进行ADCC的机制仍然知之甚少。 我们最近发现，FcγR聚集性可以诱导人单核细胞产生颗粒酶B，并且 Toll样受体(TLR)4和8的配体可以显著增强这种反应，并诱导 颗粒酶B本身。颗粒酶B是一种丝氨酸蛋白酶，主要由自然杀伤(NK)细胞表达 和CD8+T细胞，是其细胞毒功能所必需的。鉴于颗粒酶B在NK细胞中的核心作用- 我们发现单核细胞也能产生颗粒酶B，这是值得注意的，因为它提供了一种AS- 尚未发现的抗肿瘤功能的单抗。最值得注意的是，我们还发现了那个护士- 慢性淋巴细胞性白血病患者外周血中产生的类细胞在Fc-γ-R活化后也产生颗粒酶B。 TLR8配体处理后，可参与CLL细胞颗粒酶依赖性ADCC。后者 观察特别有趣，因为NLCS通常促进CLL细胞的存活和增殖。因此，我们 假设单核细胞和非淋巴细胞产生颗粒酶B代表效应器的一个关键方面 对包被抗体的靶细胞的反应。我们建议具体探讨这些发现的意义。 与CLL免疫治疗相关的3个具体目的：1)阐明颗粒酶B的作用机制 Fc-γ-R对单核/巨噬细胞和非淋巴细胞的诱导作用 FcγR诱导免疫调节剂产生颗粒酶B 3)表达及功能分析 在CLL患者样本和小鼠模型中单核细胞和单核细胞系细胞表达颗粒酶B。 这项研究完成后，我们将全面探索一种全新的抗体介导机制。 单核/巨噬细胞和非淋巴细胞对肿瘤的破坏。这些机械论研究将显著地 增强我们对这些细胞介导的ADCC的理解，并可能提供可能导致 以进一步加强抗体治疗。

PROJECT SUMMARY Our broad, long term objective is to understand the molecular details of Fcγ receptor (FcγR) function, with the goal of improving monoclonal antibody therapy for cancer. The antitumor effects of antibody therapy are largely mediated by FcγR, which become clustered and activated upon binding to the Fc portion of antibodies. Monocytes and macrophages are essential for antibody therapy to be effective, and FcγR activation in these cells can lead to pleiotropic responses. These include phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), production of inflammatory cytokines, or a combination of these. Amongst these activities, the mechanisms by which monocytes / macrophages carry out ADCC remain the least understood. We have recently found that FcγR clustering can induce Granzyme B production by human monocytes and that ligands for Toll-like receptors (TLR) 4 and 8 can significantly enhance this response, as well as induce Granzyme B themselves. Granzyme B is a serine protease expressed predominantly by natural killer (NK) cells and CD8+ T cells, and is required for their cytotoxic functions. Given the central role of Granzyme B in NK cell- mediated ADCC, our finding that monocytes can also produce Granzyme B is remarkable as it provides an as- yet undiscovered anti-tumor function of monoclonal antibodies. Most notably we have also found that nurse- like cells (NLCs) generated from CLL patient blood also produce Granzyme B following FcγR activation and TLR8 ligand treatment, and they can engage in Granzyme-dependent ADCC of CLL cells. This latter observation is of particular interest, as NLCs typically promote CLL-cell survival and proliferation. We therefore hypothesize that Granzyme B production by monocytes and NLCs represents a critical aspect of the effector response to antibody-coated target cells. We propose to explore the significance of these findings specifically related to CLL immunotherapy in the following 3 specific aims: 1) Elucidate the mechanisms of Granzyme B induction by FcγR in monocytes/macrophages and NLCs, 2) Determine the mechanism of augmentation of FcγR-induced Granzyme B production by immune modulators and 3) Analyze the expression and function of Granzyme B by monocytes and monocyte-lineage cells in patient samples and mouse models of CLL. Upon completion of this study we will have fully explored an entirely new mechanism of antibody-mediated destruction of tumors by monocytes/macrophages and NLCs. These mechanistic studies will significantly enhance our understanding of ADCC mediated by these cells, and will likely provide information that can lead to the further enhancement of antibody therapy.