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。这些TLT-1缺失小鼠表现出血小板聚集减少，因此与出血性血管炎的Shwartzman模型相关的出血更为明显。此外，我们已经确定了TLT-1的推定配体，并与Valance Washington博士合作继续对TLT-1和TLT-1无效小鼠进行表征。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。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。我们在NK细胞中发现了LAT和LAB的平行作用。通过研究缺乏LAT、LAB或两种接头的小鼠，我们已经证明ITAM相关受体NK1.1可以利用其中一种接头引发干扰素γ (IFNgamma)的产生。肥大细胞也可以直接通过LAB发送一些信号，但其机制尚不清楚，因此我们正在解剖LAT无效小鼠中与LAB相关的蛋白质复合物，以进一步了解LAB传播信号的性质。为此，我们开发了一种将信号蛋白或shrna传递到NK细胞体内的新方法

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. These TLT-1 null mice exhibit decreased platelet aggregation and as a consequence have more pronounced hemorrhage associated with the Shwartzman model of hemorrhagic vasculitis. In addition, we have identified putative ligands for TLT-1 and are continuing our characterization of TLT-1 and TLT-1 null mice in collaboration with Dr. Valance Washington. 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. 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 discovered the parallel use of LAT and LAB in NK cells. Through the study mice lacking LAT, LAB, or both adaptors, we have demonstrated that the ITAM associated receptor NK1.1 can utilized either adaptor to elicit Interferon gamma (IFNgamma) production. Mast cells can also send some signals directly via LAB but the mechanism is unknown so we are dissecting the protein complexes associated with LAB in LAT null mice to further understand the nature of a LAB propagated signal. Toward this end we have developed a novel method for the delivery of signaling proteins or shRNAs to NK cells in vivo