Signal Transduction of Paired Inhibitory Receptors of NK Cells and Macrophages

髓样细胞上表达的触发受体表达于多种天然免疫细胞上，包括单核细胞、巨噬细胞、树突状细胞、中性粒细胞和破骨细胞。这些受体通过与信号链DAP12相关联向宿主细胞传递信号。DAP12信号依赖于其胞质尾部是否存在基于免疫受体酪氨酸的激活基序(ITAM)。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，髓系细胞内DAP12信号的生化性质和TREM基因簇的整体免疫学作用一样，在很大程度上还没有确定。因此，我们采取了两部分的方法来了解TREM簇的免疫生物学。首先，我们一直在鉴定我们几年前发现的基因簇中的一个新成员，即Trem-like转录产物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表达的受体。血小板的激活导致TLT-1从血小板的α颗粒移位到细胞表面。我们已经确定了TLT-1的特征，最近培育出了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的血液中有非常高水平的可溶性TLT-1。当在体外产生可溶性TLT-1时，能够增强血小板聚集，这表明患者血液中高水平的sTLT-1可能与这些患者出现的弥漫性血管内凝血有关。我们了解TREM在调节先天免疫和癌症中的作用的第二种方法是剖析髓系细胞中的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位的、发育调节的信号盒。单核细胞表达两种关键的细胞内适配器蛋白，T细胞激活连接物(LAT)和B细胞激活连接物(LAB，也称为非T细胞适配器，NTAL)。我们发现，在单核细胞来源的DC或巨噬细胞体外成熟过程中，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很容易在DAP12信号通路中被磷酸化。此外，对LAB基因缺失的小鼠及其衍生的巨噬细胞的分析表明，ITAM信号过度活跃，MAP激酶级联激活增加。我们已经证明，LAB抑制巨噬细胞中ITAM信号的能力可能来自于它将E3泛素连接酶cCbl招募到TREM受体簇的能力。缺乏实验室介导的调节导致巨噬细胞在内毒素刺激下产生较少的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系部分的信号研究外，我们还研究了自然杀伤细胞的Ly49和KIR的信号传递，自然杀伤细胞是先天免疫系统的淋巴成分。Ly49和KIR家族由抑制性和激活性受体组成，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。我们先前已经剖析了LAT和LAB在该受体通路中的参与。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。这项技术涉及通过内部核糖体进入位点(IRES)将信号基因的mRNA与选择标记的mRNA捆绑在一起。我们选择的选择基因是IL-2受体复合体的公共伽马链(公共伽马链)。这种蛋白是NK细胞发育所必需的，因此当伽马普通零骨髓感染逆转录病毒时，该基因与信号蛋白相连，只有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。我们利用该系统检测了PI3K的表达对NK细胞发育的影响。此外，我们还发现重组NK细胞的伽马链水平较低。这些较低的水平仍然允许NK和T细胞的发展，但这些细胞不能正确地发出信号。IL-2引起的STAT和ERK磷酸化水平降低导致细胞增殖减少。这些数据可能解释了为什么接受伽马普通基因治疗的患者往往不能保留重组的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.