Signal Transduction of Paired Inhibitory Receptors of NK Cells and Macrophages

髓样细胞上表达的触发受体（TREM）在多种先天性免疫细胞上表达，包括单核细胞、巨噬细胞、树突状细胞（DC）、嗜中性粒细胞和破骨细胞。这些受体通过与信号链DAP 12结合将信号传递到其宿主细胞。DAP 12信号传导依赖于其胞质尾内基于免疫受体酪氨酸的激活基序（ITAM）的存在。在刺激DAP 12偶联受体后，DAP 12被磷酸化并募集对下游信号传播至关重要的蛋白质。最近的研究表明，TREM家族的成员通过DAP 12可以向单核细胞和巨噬细胞传递激活或抑制信号。然而，骨髓细胞内DAP 12信号传导的生物化学性质在很大程度上未被表征，TREM基因簇的整体免疫学作用也是如此。因此，我们已经采取了一个双向的方法来了解TREM集群的免疫生物学。首先，我们一直在表征我们几年前发现的基因簇的一个新成员，TREM样转录本1（TLT-1）。该TREM样基因编码巨核细胞和外周血血小板中特异性表达的受体。血小板的活化导致TLT-1从血小板的α颗粒易位到细胞表面。我们已经对TLT-1进行了表征，并且最近生产了具有TLT-1特定突变的小鼠。此外，我们还研究了该基因在人类疾病中的作用。我们发现脓毒症患者血液中可溶性TLT-1水平很高。当在体外产生时，可溶性TLT-1能够增强血小板聚集，这表明患者血液中高水平的sTLT-1可能有助于在这些患者中观察到的弥散性血管内凝血。我们理解TREM在先天免疫和癌症调节中的作用的第二种方法是解剖骨髓细胞中的DAP 12信号通路。我们的研究已经在巨噬细胞和单核细胞中确定了一个变化的、发育调节的信号盒。单核细胞表达两种关键的细胞内衔接子蛋白，T细胞活化接头（LAT）和B细胞活化接头（LAB，也称为非T细胞衔接子，NTAL）。我们发现，在体外从单核细胞成熟DC或巨噬细胞的过程中，LAT的水平下降，而LAB的水平增加。结果是DAP 12利用的信号通路发生了变化。因此，我们已经发现LAB响应于DAP 12信号传导而容易磷酸化。此外，对LAB缺失小鼠和源自它们的巨噬细胞的分析证明了ITAM信号传导过度活跃和MAP激酶级联的活化增加。我们已经证明LAB抑制巨噬细胞中ITAM信号传导的能力可能来源于其将E3泛素连接酶cCbl募集到TREM受体簇的能力。缺乏LAB介导的调节导致巨噬细胞在用LPS刺激时产生较少的IL-12/23和较高水平的IL-10。目前的研究正在解决这些受体在调节与癌症相关的白细胞中的作用。除了我们在先天免疫系统的髓样区室中的信号传导研究之外，我们还研究了天然杀伤细胞的Ly 49和KIR的信号传导，天然杀伤细胞是先天免疫系统的淋巴成分。Ly 49和KIR家族由抑制性和激活性受体组成;后者通过DAP 12相互作用和信号传导。因此，我们一直在解剖NK细胞的ITAM信号传导，重点是DAP 12信号传导。我们以前已经解剖了LAT和LAB在该受体通路中的参与。为了进一步研究，我们最近开发了一种在原代小鼠NK细胞中表达基因或shRNA的方法。该技术涉及通过内部核糖体进入位点（IRES）将信号传导基因的mRNA与选择标记的mRNA拴系。我们选择的选择基因是IL-2受体复合物的共同γ链（γ共同）。这种蛋白质是NK细胞发育所需的，因此当γ共同无效骨髓被含有与信号蛋白连接的这种基因的逆转录病毒感染时，只有转导的骨髓细胞应该能够产生NK细胞，并且所有这些NK细胞都应该携带感兴趣的信号蛋白。我们已经使用该系统来测试PI 3 K的表达对NK细胞发育的影响。此外，我们还发现重组NK细胞的γ链水平略低。这些较低的水平仍然允许NK细胞和T细胞的发育，但这些细胞不能正确地发出信号。响应于IL-2的STAT和ERK磷酸化减少导致增殖减少。这些数据可以解释为什么接受γ共同基因治疗的患者通常不能保留重建的NK细胞群。

The Triggering Receptors Expressed on Myeloid Cells (TREM) are expressed on a variety of innate immune cells including monocytes, macrophages, dendritic cells (DC), neutrophils, and osteoclasts. These receptors deliver signals to their host cells via association with the signaling chain, DAP12. DAP12 signaling is dependent on the presence of an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic tail. Upon stimulation of a DAP12-coupled receptor, DAP12 is phosphorylated and recruits proteins critical to the propagation of downstream signals. Recent work has demonstrated that members of the TREM family, via DAP12, can deliver either activation or inhibitory signals to monocytes and macrophages. However, the biochemical nature of DAP12 signaling within myeloid cells is largely uncharacterized as is the overall immunological role of the TREM gene cluster. Thus, we have taken a bipartite approach to understand the immunobiology of the TREM cluster. First of all we have been characterizing a novel member of the gene cluster we discovered several years ago, TREM-Like transcript 1 (TLT-1). This TREM-like gene encodes a receptor specifically expressed in megakaryocytes and peripheral blood platelets. Activation of the platelets results in a translocation of TLT-1 from the alpha granules of platelets to the cell surface. We have characterized TLT-1, and recently produced mice with a specific mutation in TLT-1. In addition, we have investigated the role of this gene in human disease. We find the patients with sepsis have very high levels of soluble TLT-1 in their blood. When produced in vitro soluble TLT-1 is capable of enhancing platelet aggregation suggesting that the high levels of sTLT-1 in patients blood may contribute to the disseminated intravascular coagulation seen in these patients. Our second approach to understanding the role of TREM in regulation of innate immunity and cancer is dissection of the DAP12 signaling pathway in myeloid cells. Our studies have identified a shifting, developmentally regulated signaling cassette in macrophages and monocytes. Monocytes express two key intracellular adaptor proteins, the Linker for Activation of T cells (LAT) and the Linker for Activation of B cells (LAB, also known as the Non-T cell Adaptor, NTAL). We find that during maturation of DC or macrophages from monocytes in vitro, the levels of LAT fall whereas the levels of LAB increase. The result is that the signaling pathway utilized by DAP12 changes. Accordingly we have found that LAB is readily phosphorylated in response to DAP12 signaling. Moreover, analysis of LAB null mice, and macrophages derived from them, demonstrated hyperactive ITAM signaling and increased activation of the MAP kinase cascade. We have demonstrated that the ability of LAB to suppress ITAM signaling in macrophages may derive from its ability to recruit the E3 ubiquitin ligase cCbl to the TREM receptor cluster. The lack of LAB-mediated regulation results in macrophages that produce less IL-12/23 and higher levels of IL-10 upon stimulation with LPS. Current studies are addressing the role of these receptors in the regulation of leukocytes associated with cancers. In addition to our signaling studies in the myeloid compartment of the innate immune system we study the signaling of the Ly49 and KIR of natural killer cells, the lymphoid component of the innate immune system. The Ly49 and KIR families are comprised of both inhibitory and activating receptors; the latter interacting and signaling through DAP12. Thus we have been dissecting the ITAM signaling of NK cells with emphasis on on DAP12 signaling. We have previously dissected the involvement of LAT and LAB in this receptor pathway. In order to further these studies we have recently developed a method to express genes or shRNA in primary murine NK cells. This technique involves tethering the mRNA of a signaling gene to that of a selection marker via an internal ridosome entry site (IRES). The selection gene we selected is the common gamma chain of the IL-2 receptor complex (gamma common). This protein is required for the development of NK cells so when gamma common null bone marrow is infected with a retrovirus containing this gene tethered to a signaling protein, only transduced bone marrow cells should be able to give rise to NK cells and all those NK cells should carry the signaling protein of interest. We have used this system to test the effects of the expression of PI3K on NK cell development. In addition, we have found that reconstituted NK cells have somewhat lower levels of gamma chain. These lower levels still permit the development of NK and T cells but these cells do not signal properly. Reduced STAT and ERK phosphorylation in response to IL-2 results in reduced proliferation. These data may explain why patients treated with gamma common gene therapy often do not retain reconstituted NK cell populations.