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C57BL/6背景下建立了LAIR-1-/-和LAIR-1FLOX/FLOX小鼠。虽然LAIR-1-/-小鼠是健康的、多产的，在无病原体的环境中也有正常的寿命，但它们确实表现出不同于野生型(Wt)小鼠的某些表型特征。他们的脾B细胞和树突状细胞比例增加，而T细胞水平降低，其中更高比例的是CD4+CD25+。随着LAIR-1-/-小鼠年龄的增长，脾T细胞群表现出更高的激活T细胞频率和更高比例的效应/记忆T细胞。由于LAIR-1+/+和LAIR-1-/-T细胞对外周淋巴器官的转运能力相同，这可能不是T淋巴细胞异常转运的结果。功能实验表明，抗原特异性LAIR-1-/-CD4T细胞的增殖明显低于抗原特异性LAIR-1+/+CD4T细胞。尽管先前有报道称小鼠B细胞表达LAIR-1，但我们发现边缘带B细胞表达LAIR-1。LAIR-1-/-小鼠血清中的IgG3和IgG1水平较低，在TNP-OVA的T依赖免疫反应中，LAIR-1-/-小鼠转换为IgG2a和IgG2b的效率较低，而转换为IgG1的效果不受影响。采用实验性自身免疫性脑脊髓炎(EAE)和实验性结肠炎等多种疾病模型，观察了LAIR-1缺陷对小鼠体内免疫功能的影响，结果表明，LAIR-1-/-和LAIR-1+/+小鼠的免疫应答无明显差异。综上所述，这些观察表明，LAIR-1在塑造免疫反应方面发挥了作用，这种免疫反应可能在体内被其他抑制性受体补偿(手稿正在准备中)。 免疫调节受体CD300a在人B细胞上表达。幼稚B细胞表达这种受体的水平非常低，而记忆B细胞和浆母细胞/细胞表达不同水平的CD300a。生发中心B细胞CD300a表达阴性。通过BCR和TLR9刺激幼稚B细胞，以及T细胞的帮助，未能上调CD300a细胞表面的表达，尽管记忆标记CD27的表达增加，CD305的表达下调。TLR9单独刺激显著增加记忆B细胞CD300a的表达，而IL-4和转化生长因子-β1对记忆B细胞CD300a的表达起负调控作用。Bcr和CD300a共连接可抑制单独bcr连接引起的钙动员和NFAT转录活性。用siRNA抑制原代B细胞CD300a的表达，可促进BCR介导的增殖，从而证实CD300a的抑制能力。最后，我们发现HIV感染者循环B细胞中CD300a的表达水平下调。总之，这些数据显示了一种抑制B细胞活性的新机制，并表明CD300a在艾滋病毒诱导的免疫缺陷中观察到的B细胞功能障碍中可能起到作用(提交的手稿)。 人类幼稚的CD4T细胞表达低水平的免疫调节受体CD300a，而效应/记忆的CD4细胞可以是CD300a+或CD300a-。这表明CD300a的表达可以在效应/记忆的CD4T细胞亚群中定义一个特定的亚群。事实上，对产生干扰素的CD4T细胞的体外分析表明，它们富含CD300a+亚群。此外，受刺激的CD4T细胞除产生干扰素-γ外，还产生肿瘤坏死因子-β和IL-2(多功能)，主要是CD300a+。除了产生明显更高水平的Th1相关细胞因子外，受刺激的CD300a+CD4T细胞的显著特点是T-box转录因子eomesodermin(EOMES)显著上调，而T-bet在CD300a+和CD300激活的CD4T细胞中上调至相似水平。多效性细胞因子转化生长因子-β1在Th1亚群的发育中起决定性作用，因为它的存在抑制了CD300a的表达，并下调了eome和干扰素-γ的表达。我们得出结论：CD300a+的人Th1细胞趋向于多功能，并且在刺激后上调eome。

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.