Regulation of human gamma/delta T cell activation by neutrophils: mechanisms and translational implications

VGamm9Vdelta2(Vg9Vd2)T细胞是人类外周血中主要的γ/Delta T细胞亚群。与传统的CD4和CD8T细胞不同，Vg9Vd2T细胞不识别抗原肽，而识别来自微生物或真核生物合成异戊二烯的焦磷酸分子(磷酸抗原，PAg)。这种PAg的呈现独立于MHC I类或II类，但需要表达丁咯菲林成员3A1(BTN3A1/CD277)。唑来膦(ZOL)等氨基二膦酸盐可显著增加肿瘤细胞和单核细胞内源性PAg的产生，临床上用于治疗骨质疏松症和各种癌症的骨转移。ZOL处理的单核细胞刺激Vg9Vd2T细胞的选择性激活和扩增，而ZOL处理的肿瘤细胞被Vg9Vd2T细胞更有效地杀伤。因此，ZOL加小剂量IL-2已被用于治疗癌症患者，试图在体内激活肿瘤反应性Vg9Vd2T细胞，尽管效果有限。我们最近报道，中性粒细胞有效地摄取ZOL，并随后强烈抑制Vg9Vd2 T细胞的激活，这可能是在ZOL治疗过程中观察到的Vg9Vd2 T细胞无能和数量下降的原因之一。我们发现ROS的产生是一个促成因素，然而，这并不能完全解释抑制作用。在这个项目中，我们计划在细胞和分子水平上详细研究中性粒细胞对gd T细胞激活的调节作用。作为第一步，我们将比较纯化的中性粒细胞和单核细胞向Vg9Vd2 T细胞递送PAg的能力(重要分子BTN3A1/CD277和法尼基焦磷酸合成酶的表达/调节，内源性PAg对ZOL刺激的反应)。此外，我们将研究对照和ZOL激活的粒细胞和单核细胞的ROS产生、NADPH氧化酶活性、细胞骨架重组和差异基因表达模式。新接受ZOL治疗的癌症患者的血液中也将检测到Vg9Vd2激活和中性粒细胞的抑制/调节活性。重要的是，我们还将制定策略，防止或抵消中性粒细胞的抑制活性。我们项目的长期目标是通过防止中性粒细胞的抑制作用来改进基于GDT细胞的免疫治疗方法

Vgamm9Vdelta2 (Vg9Vd2) T cells comprise the major subset of gamma/delta T cells in human peripheral blood. In contrast to conventional CD4+ and CD8+ T cells, Vg9Vd2 T cells do not recognize antigenic peptides but rather pyrophosphate molecules (phosphoantigens, pAg) derived from the microbial or eukaryotic biosynthesis of isoprenoids. The presentation of such pAg is independent of MHC class I or class II but requires the expression of the butyrophilin member 3A1 (BTN3A1/CD277). The endogenous production of pAg in tumor cells and monocytes can be drastically increased by aminobisphosphonates such as zoledronate (ZOL) which are in clinical use for the treatment of osteoporosis and bone metastasis in various types of cancer. ZOL-treated monocytes stimulate the selective activation and expansion of Vg9Vd2 T cells, and ZOL-treated tumor cells are much more efficiently killed by Vg9Vd2 T cells. Therefore, ZOL plus low-dose IL-2 has been used to treat cancer patients in an attempt to activate tunmor-reactive Vg9Vd2 T cells in vivo, though with limited success. We recently reported that neutrophils take up ZOL efficiently and subsequently strongly suppress Vg9Vd2 T cell activation in response to ZOL which might contribute to the observed anergy and numerical decline of Vg9Vd2 T cells in the course of ZOL therapy. We identified production of reactive oxygen species (ROS) as a contributing factor which, however, could not fully explain the inhibitory effect. In this project we plan to study in detail the regulatory impact of neutrophils on gd T cell activation at the cellular and molecular level. As a first step, we will compare purified neutrophils and monocytes with respect to their capacity to present pAg to Vg9Vd2 T cells (expression/regulation of important molecules such as BTN3A1/CD277 and farnesyl pyrophosphate synthase, endogenous production of pAg in response to ZOL stimulation). Furthermore, we will investigate ROS production, NADPH oxidase activity, cytoskeleton reorganization, and differential gene expression patterns in both control and ZOL-activated granulocytes and monocytes. Vg9Vd2 activation and suppressive/regulatory activiy of neutrophils will be also determined in the blood of cancer patients who are newly put on intravenous ZOL therapy. Importantly, we will also devise strategies to prevent or counteract the inhibitory activity of neutrophils. The longterm goal of our project is to improve gdT cell-based immunotherapeutic approaches by preventing the suppressive role of neutrophils