Role of LAIR-1 (CD305), FcmuR and CD300 Receptors in Regulating Inflammation

为了研究LAIR-1的体内功能，我们在C57 B1/6背景下产生了LAIR-1-/-和LAIR-1flox/flox小鼠。虽然LAIR-1-/-小鼠是健康的和可生育的，并且在无病原体的环境中具有正常的寿命，但是它们确实显示出与野生型（wt）小鼠不同的某些表型特征。它们产生脾B细胞和树突状细胞的百分比增加，沿着T细胞水平降低，其中较高频率是CD 4 + CD 25+。随着LAIR-1-/-小鼠年龄的增长，脾T细胞群显示出更高频率的活化T细胞和更高百分比的效应/记忆T细胞。由于LAIR-1+/+和LAIR-1-/- T细胞以相同的效率运输到外周淋巴器官，这可能不是异常T淋巴细胞运输的结果。功能实验显示抗原（Ag）特异性LAIR-1-/-CD 4 T细胞增殖显著低于Ag特异性LAIR-1+/+ CD 4 T细胞。虽然小鼠B细胞先前已报道为LAIR-1表达阴性，但我们发现边缘区B细胞表达LAIR-1。LAIR-1-/-小鼠在血清中具有较低水平的IgG 3和IgG 1，并且响应于TNP-0 VA的T依赖性免疫，LAIR-1-/-转换为IgG 2a和IgG 2b产生的效率较低，而转换为IgG 1不受影响。利用几种小鼠疾病模型，包括实验性自身免疫性脑脊髓炎（EAE）和实验性结肠炎的那些，来检查LAIR-1缺陷在体内的作用，并且没有观察到LAIR-1-/-和LAIR-1+/+小鼠的应答的差异。总之，这些观察结果表明，LAIR-1在形成免疫应答中起作用，该免疫应答可以通过其他抑制性受体在体内进行补偿（手稿在准备中）。 免疫调节受体CD 300 a在人B细胞上表达。裸B细胞表达非常低水平的该受体，而记忆B细胞和血浆母细胞/细胞表达可变水平的CD 300 a。老年中心B细胞CD 300 a表达阴性。通过BCR和TLR 9刺激幼稚B细胞，沿着T细胞的帮助，尽管记忆标记物CD 27的表达增加和CD 305的下调，但不能上调CD 300 a细胞表面表达。TLR 9单独刺激显著增加记忆B细胞上的CD 300 a表达，而IL-4和TGF-β 1作为记忆B细胞上的CD 300 a表达的负调节剂。BCR和CD 300 a的共连接抑制单独BCR连接引起的Ca 2+动员和NFAT转录活性。用siRNA抑制原代B细胞的CD 300 a表达导致BCR介导的增殖增加，从而证实了CD 300 a的抑制能力。最后，我们发现，CD 300 a的表达水平下调，在循环B细胞的HIV感染患者。总之，这些数据证明了一种抑制B细胞活性的新机制，并表明CD 300 a在HIV诱导的免疫缺陷中观察到的B细胞功能障碍中的潜在作用（提交的手稿）。 人原始CD 4 T细胞表达低水平的免疫调节受体CD 300 a，而效应/记忆CD 4细胞可以是CD 300 a+或CD 300 a-。这表明，CD 300 a表达可以定义效应/记忆CD 4 T细胞亚群内的特定亚群。事实上，产生IFN-γ的CD 4 T细胞的离体分析显示它们富集在CD 300 α +亚群中。此外，除了IFN-γ外，产生TNF-β和IL-2的刺激的CD 4 T细胞（多功能的）主要是CD 300 α +。除了产生明显更高水平的Th 1相关细胞因子外，受刺激的CD 300 a + CD 4 T细胞的特征在于T盒转录因子eomesodermin（Eomes）的显著上调，而T-bet在CD 300 a+和CD 300活化的CD 4 T细胞中上调至相似水平。多效性细胞因子TGF-β 1在决定该Th 1亚群的发展中具有决定性作用，因为其存在抑制CD 300 α的表达并下调Eomes和IFN-γ的表达。我们得出结论，CD 300 a+人Th 1细胞往往是多功能的，刺激后上调Eomes。

To study the in vivo function of LAIR-1, we generated LAIR-1-/- and LAIR-1flox/flox mice on a C57Bl/6 background. Although LAIR-1-/- mice are healthy and fertile, and have normal longevity in pathogen free environment, they do show certain phenotypic characteristics distinct from wild-type (wt) mice. They develop an increased percentage of splenic B cells and dendritic cells, along with decreased levels of T cells, of which a higher frequency are CD4+CD25+. As the LAIR-1-/- mice age, the splenic T cell population shows a higher frequency of activated T cells and a higher percentage of effector/memory T cells. Since LAIR-1+/+ and LAIR-1-/- T cells traffic with equal proficency to peripheral lymphoid organs, this is probably not the result of abnormal T lymphocyte trafficking. Functional experiments showed that antigen (Ag) specific LAIR-1-/- CD4 T cells proliferate significantly less than Ag specific LAIR-1+/+ CD4 T cells. Although mouse B cells have previously been reported to be negative for LAIR-1 expression, we find that marginal zone B cells express LAIR-1. LAIR-1-/- mice have lower levels of IgG3 and IgG1 in serum and, in response to T-dependent immunization with TNP-OVA, LAIR-1-/- switch less efficiently to IgG2a and IgG2b production, while switching to IgG1 is not affected. Several mouse disease models, including those for experimental autoimmune encephalomyelitis (EAE) and experimental colitis, were utilized to examine the effect of LAIR-1 deficiency in vivo, and no difference in the response of LAIR-1-/- and LAIR-1+/+ mice were observed. Taken together, these observations indicate that LAIR-1 plays a role in shaping the immune response that may be compensated for in vivo by other inhibitory receptors(manuscript in preparation). The immunomodulatory receptor CD300a is expressed on human B cells. Nave B cells express very low levels of this receptor, while memory B cells and plasma blasts/cells express variable levels of CD300a. Germinal center B cells are negative for CD300a expression. Stimulation of naive B cells via BCR and TLR9, along with T cell help, failed to up-regulate CD300a cell surface expression despite the increased expression of the memory marker CD27 and the down-regulation of CD305. TLR9 stimulation alone significantly increased CD300a expression on memory B cells, whereas IL-4 and TGF-beta1 act as negative regulators of CD300a expression on memory B cells. Co-ligation of BCR and CD300a inhibits Ca2+ mobilization and NFAT transcriptional activity evoked by BCR ligation alone. Suppression of CD300a expression by primary B cells with siRNA resulted in increased BCR-mediated proliferation, thereby confirming the inhibitory capacity of CD300a. Finally, we show that of CD300a expression levels are down-regulated in the circulating B cells of HIV-infected patients. Altogether, these data demonstrate a novel mechanism for suppressing the activity of B cells and suggest a potential role for CD300a in the B cell dysfunction observed in HIV induced immunodeficiency (manuscript submitted). Human nave CD4 T cells express low levels of the immunomodulatory receptor CD300a, whereas effector/memory CD4 cells can be either CD300a+ or CD300a-. This suggested that CD300a expression could define a specific subset within the effector/memory CD4 T cell subpopulations. In fact, ex vivo analysis of the IFN-gamma; producing CD4 T cells showed that they are enriched in the CD300a+ subset. Moreover, stimulated CD4 T cells producing TNF-beta; and IL-2 besides IFN-gamma; (polyfunctional) are predominantly CD300a+. In addition to producing markedly higher levels of Th1-associated cytokines, the stimulated CD300a+ CD4 T cells are distinguished by a striking up-regulation of the T-box transcription factor eomesodermin (Eomes), whereas T-bet is up-regulated in both CD300a+ and CD300- activated CD4 T cells to similar levels. The pleiotropic cytokine TGF-beta1 has a determinant role in dictating the development of this Th1 subset, as its presence inhibits the expression of CD300a and down-regulates the expression of Eomes and IFN-gamma. We conclude that CD300a+ human Th1 cells tend to be polyfunctional and after stimulation up-regulate Eomes.