Innate Immunity

天然免疫的细胞介质不表达重排的V区，但在没有抗体的情况下具有识别和反应靶细胞的能力。最近在人类和小鼠中发现了一个古老的病原体识别受体家族，Toll样受体(Toll Like Receptor，TLRs)，其中两个TLRs2和TLRs4被证明能触发对内毒素和其他病原体衍生物质的炎症反应。我们假设IDC对病原体的识别是由Toll样受体(Toll Like Receptor，TLRs)介导的，并询问在单核细胞向MDC的发展过程中，哪些TLRs表达。我们首先通过Northern分析测量了TLR1-5和MD2(一种TLR4功能所需的蛋白质)的mRNA水平。对于大多数TLR来说，随着单核细胞分化为IDC，消息表达减少了几倍，但与此相反，TLR3和MD2在IDC形成过程中显著增加。当用内毒素或肿瘤坏死因子-α诱导IDC成熟时，大多数TLRs的表达一过性增加，然后几乎消失。用内毒素刺激IDC，而不是MDC，可激活TLR信号通路中的早期成分IRAK，强烈建议通过TLR发出该信号。通过单抗结合测定，TLR1和TLR4的表面表达非常低，在单核细胞中相当于每个细胞数千个分子，而在IDC中只有几百个或更少。因此，TLRs在IDC中表达，并参与对至少一种病原体衍生物质--内毒素的反应。人微血管内皮细胞通过TLR4对内毒素产生反应，我们发现TLR1抑制这一反应。当转染293细胞时，TLR1与其他TLR结合，包括TLR2、TLR4和TLR5，使用修改的TLR1的初步结果表明，结合是通过细胞外域发生的。TLR1是最普遍表达的TLR，我们的数据表明，它在负向控制TLR对病原体的反应中发挥作用。以前我们发现，肿瘤诱导的免疫抑制是由一群髓系细胞介导的，这些细胞迁移到淋巴器官，阻断T和B细胞对抗原的反应。这些髓系抑制细胞(MSC)已经永生化，并保留了它们的抑制功能，为研究抑制机制提供了细胞来源。在抗原、同种异体细胞和ConA刺激下，MSC强烈地阻断T细胞的增殖，我们观察到有两种不同的机制用于阻断同种异体和有丝分裂原驱动的反应。对同种异体反应的抑制不是独立的，伴随着应答细胞分泌IL-2的丧失，并且在与MSC接触24小时后不可逆转，与应答人群中的细胞死亡一致。相反，抑制有丝分裂和多肽特异性反应是可逆的，不会导致IL-2产生的损失，需要NO和干扰素-g。MSC系对干扰素-g反应产生高水平NO的独特能力，以及来自响应者群体的接触相关信号，将它们与表型相关的非抑制性同种细胞区分开来。磷酸化STAT5、ERK和Akt的免疫印迹显示，MSC处理直接损害了丝裂原刺激的T细胞中的IL-2信号通路，因为这些分子在与MSC孵育后都不会被IL-2磷酸化。最近的结果表明，抑制也可能作用于其他途径，例如，我们发现除了IL-2外，NO还阻断IL-3和IL-12途径。我们目前正在验证一种假设，即所有的Janus激酶都容易受到NO的抑制，因此，MSC将具有抑制大多数细胞因子诱导的反应的能力。

Cellular mediators of innate immunity do not express rearranged V regions, yet have the capacity to recognize and respond to target cells in the absence of antibody. Recently an ancient family of pathogen recognition receptors, the toll like receptors (TLRs) was discovered in humans and mice and two of these, TLRs2 and 4 were shown to trigger inflammatory responses to LPS and other pathogen derived substances. We hypothesized that pathogen recognition by iDC is mediated by toll like receptors (TLRs), and asked which TLRs are expressed during the progression of monocytes to mDC. We first measured mRNA levels for TLRs 1-5 and MD2 (a protein required for TLR4 function) by Northern analysis. For most TLRs, message expression decreased several fold as monocytes differentiated into iDC, but opposing this trend, TLR3 and MD2 showed marked increases during iDC formation. When iDC were induced to mature with LPS or TNF-a, expression of most TLRs transiently increased and then nearly disappeared. Stimulation of iDC, but not mDC, with LPS resulted in the activation of IRAK, an early component in the TLR signalling pathway, strongly sugesting that LPS signals through a TLR. Surface expression of TLRs 1 and 4, as measured by mAb binding, was very low, corresponding to a few thousand molecules per cell in monocytes, and a few hundred or less in iDC. Thus, TLRs are expressed in iDC and are involved in responses to at least one pathogen derived substance, LPS. Human microvascular endothelial cells respond to LPS through TLR4 and we have found that TLR1 inhibits this response. When transfected into 293 cells, TLR1 binds to other TLRs, including TLRs 2, 4 and 5, and preliminary results using modified TLR1 suggest that binding occurs through the extracellular domain. TLR1 is the most ubiquitously expressed TLR, and our data suggest that it serves a function in negatively controlling TLR responses to pathogens. Previously we showed that tumor induced immunosuppression was mediated by a subset of myeloid cells that migrate to lymphoid organs and block T and B cell responses to antigen. These myeloid suppressor cells (MSC) have been immortalized, retain their suppressive functions, and provide a source of cells for studying mechanisms of suppression. While MSC strongly block T cell proliferation in response to stimulation by antigen, allogeneic cells and Con A, we have observed that two different mechanisms are used to block allogeneic and mitogen driven responses. Suppression of allogeneic responses was NO independent, accompanied by a loss in IL-2 secretion by the responder cells, and was irreversible after 24 hr contact with MSC, consistent with cell death within the responder population. By contrast, inhibition of mitogenic and peptide specific responses was reversible, did not entail a loss in IL-2 production, and required NO and IFN-g. A unique capacity of MSC lines to produce high levels of NO in response to IFN-g and a contact-dependent signal from the responder population distinguished them from phenotypically related non-suppressive counterparts. Western blots for phospho-STAT5, Erk, and Akt demonstrated that MSC treatment directly impaired the IL-2 signaling pathway in mitogen-stimulated T cells, since none of these molecules was phosphorylated in response to IL-2 after incubation with the MSC. Recent results suggest that suppression may operate on other pathways as well, for example, we have found that NO blocks IL-3 and IL-12 pathways in addition to IL-2. We are currently testing the hypothesis that all Janus kinases are susceptible to inhibition by NO, and that as a result, MSC would have the capacity to inhibit most cytokine-induced responses.