Signal Transduction of Paired Inhibitory Receptors of NK Cells and Macrophages

髓样细胞（TREM）上表达的触发受体在多种先天免疫细胞上表达，包括单核细胞、巨噬细胞、树突状细胞（DC）、中性粒细胞和破骨细胞。这些受体通过与信号链DAP12的关联将信号传递给宿主细胞。DAP12信号通路依赖于其细胞质尾部免疫受体酪氨酸激活基序（ITAM）的存在。在DAP12偶联受体的刺激下，DAP12被磷酸化并招募对下游信号传播至关重要的蛋白质。最近的研究表明，TREM家族的成员可以通过DAP12向单核细胞和巨噬细胞传递激活或抑制信号。然而，髓细胞内DAP12信号的生化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未被表征。因此，我们采取了两方面的方法来了解TREM簇的免疫生物学。首先，我们描述了几年前发现的基因簇的一个新成员，TREM-Like transcript 1 （TLT-1）。这种类似trem的基因编码一种在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α颗粒转运到细胞表面。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的可溶性TLT-1含量非常高。体外产生的可溶性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可能有助于这些患者所见的弥散性血管内凝血。我们了解TREM在先天免疫和癌症调控中的作用的第二种方法是解剖骨髓细胞中的DAP12信号通路。我们的研究已经在巨噬细胞和单核细胞中发现了一个移动的、发育调节的信号盒。单核细胞表达两个关键的细胞内接头蛋白，T细胞活化接头（LAT）和B细胞活化接头（LAB，也称为非T细胞接头，NTAL）。我们发现，在体外单核细胞的DC或巨噬细胞成熟过程中，LAT水平下降，而LAB水平上升。结果是DAP12利用的信号通路发生了变化。因此，我们发现LAB在响应DAP12信号时很容易磷酸化。此外，对LAB缺失小鼠及其衍生的巨噬细胞的分析表明，ITAM信号异常活跃，MAP激酶级联的激活增加。我们已经证明，LAB抑制巨噬细胞ITAM信号的能力可能源于其将E3泛素连接酶cCbl募集到TREM受体簇的能力。cCbl募集的缺乏导致近端ITAM信号的增加，导致巨噬细胞发育转变为免疫抑制状态，其特征是在LPS刺激下IL-12/23的产生较低，IL-10的产生较高。最后，当在Shwartzman反应中测试时，LAB-/-比野生型小鼠有更少的炎症，这表明巨噬细胞极性的改变与生理相关。最近，我们发现DC也使用LAB作为它们的信号，并且与野生型DC相比，无LAB DC产生更高水平的IL-10和更低水平的IL-12。除了我们在先天免疫系统髓系室的信号研究外，我们还研究了自然杀伤细胞（先天免疫系统的淋巴成分）的Ly49和KIR信号。Ly49和KIR家族由抑制受体和激活受体组成；后者通过DAP12相互作用并发出信号。因此，我们一直在剖析NK细胞的ITAM信号，重点是DAP12信号。我们之前已经剖析了LAT和LAB在这一受体通路中的作用。为了进一步进行这些研究，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。该技术涉及通过核糖核酸内进入位点（IRES）将信号基因的mRNA与选择标记的mRNA连接在一起。我们选择的选择基因是IL-2受体复合物的共同γ链（gamma common）。这种蛋白质对NK细胞的发育是必需的所以当γ - common null骨髓被含有这种与信号蛋白相连的基因的逆转录病毒感染时，只有被转导的骨髓细胞才能产生NK细胞所有这些NK细胞都应该携带感兴趣的信号蛋白。我们通过将绿色荧光蛋白与原理验证实验中常见的伽马联系起来来测试这一原理。这些实验表明，我们的病毒确实重组了NK细胞，所有这些细胞都是绿色的，这证实了病毒有能力将信号货物蛋白传递给所有NK细胞。我们现在用这个系统来表达信号蛋白和shrna。

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 likely derives from its ability to recruit the E3 ubiquitin ligase cCbl to the TREM receptor cluster. The lack of cCbl recruitment results in increased proximal ITAM signaling resulting in a developmental shift in the macrophages to a more immunosuppressive state characterized by lower production of IL-12/23 and higher production of IL-10 upon stimulation with LPS. Lastly, when tested in the Shwartzman reaction, LAB-/- have less inflammation than wild type mice suggesting the shift in macrophage polarity is physiologically relevant. Most recently we have found that DC also use LAB for their signalingf and that LAB null DC make higher levels of IL-10 and lower levels of IL-12 than wild type DC. 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 tested this principle by linking green fluorescent protein to gamma common in proof of principle experiments. These experiments showed that our virus did reconstitute NK cells and that all those cells were green confirming the ability of the virus to deliver the signaling cargo protein to all NK cells. We are now using this system to express signaling proteins and shRNAs.