Innate Immunity

先天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响应靶细胞的能力。 最近，在人类和小鼠中发现了病原体识别受体的古老家族，Toll样受体（TLR），并且其中两个TLR 2和4显示出触发对LPS和其他病原体衍生物质的炎症反应。我们假设iDC的病原体识别是由Toll样受体（TLR）介导的，并询问在单核细胞向mDC的进展过程中表达哪些TLR。 我们首先通过北方分析测量TLR 1-5和MD 2（TLR 4功能所需的蛋白质）的mRNA水平。对于大多数TLR，随着单核细胞分化成iDC，信使表达降低数倍，但与此趋势相反，TLR 3和MD 2在iDC形成期间显示出显著增加。 当用LPS或TNF-α诱导iDC成熟时，大多数TLR的表达短暂增加，然后几乎消失。 用LPS刺激iDC而不是mDC导致IRAK（TLR信号传导途径中的早期组分）的活化，强烈表明LPS通过TLR进行信号传导。 如通过mAb结合测量的，TLR 1和4的表面表达非常低，对应于单核细胞中每个细胞几千个分子，而iDC中几百个或更少。 因此，TLR在iDC中表达并且参与对至少一种病原体衍生物质LPS的应答。 人微血管内皮细胞通过TLR 4应答LPS，我们发现TLR 1抑制这种应答。 当转染到293细胞中时，TLR 1与其他TLR结合，包括TLR 2、4和5，并且使用修饰的TLR 1的初步结果表明结合通过细胞外结构域发生。 TLR 1是最普遍表达的TLR，我们的数据表明，它在负控制TLR对病原体的反应中起作用。以前我们表明，肿瘤诱导的免疫抑制是由髓样细胞亚群介导的，这些细胞迁移到淋巴器官并阻断T和B细胞对抗原的反应。这些骨髓抑制细胞（MSC）已经永生化，保留其抑制功能，并为研究抑制机制提供细胞来源。虽然MSC强烈地阻断T细胞增殖以响应抗原、同种异体细胞和Con A的刺激，但是我们已经观察到两种不同的机制用于阻断同种异体和有丝分裂原驱动的应答。 同种异体反应的抑制是NO非依赖性的，伴随着应答细胞IL-2分泌的损失，并且在与MSC接触24小时后是不可逆的，与应答群体内的细胞死亡一致。 相比之下，有丝分裂和肽特异性反应的抑制是可逆的，不需要IL-2产生的损失，并需要NO和IFN-g。 MSC系响应于IFN-g产生高水平NO的独特能力和来自应答者群体的接触依赖性信号将它们与表型相关的非抑制性对应物区分开。 磷酸化STAT 5、Erk和Akt的蛋白质印迹证明MSC处理直接损害了促分裂原刺激的T细胞中的IL-2信号通路，因为这些分子在与MSC孵育后均未响应于IL-2而磷酸化。 最近的研究结果表明，抑制也可能对其他途径起作用，例如，我们已经发现，除了IL-2外，NO还阻断IL-3和IL-12途径。 我们目前正在测试的假设是，所有的Janus激酶都容易受到NO的抑制，因此，MSC将有能力抑制大多数精氨酸诱导的反应。

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.