ACTIVATION AND TRIGGERING OF EFFECTOR CELLS

天然免疫的细胞介质不表达TCR，但在没有抗体的情况下具有识别和反应靶细胞的能力。在这个项目中，我们使用重定向裂解来证明一些黏附分子，包括CD38，CD44，CD56和CD69，具有在人NK细胞和中性粒细胞中触发细胞毒反应的能力。然而，这些反应依赖于细胞因子，并代表了直接识别致病物质的次要事件。最近，在人和小鼠的EST数据库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Toll样受体TLR2和TLR2被克隆出来，并被证明能触发对内毒素的炎症反应。我们已经开始检测TLR在人类细胞中的表达，并发现在单核细胞、树突状细胞、NK细胞、T细胞和PMN中很容易检测到一些TLR的信息。单核细胞表达最高水平的TLR信息，在单核细胞向树突状细胞分化的过程中，这些水平下降。抗TLR1的单抗对所有单核细胞均呈强染，而对树突状细胞的染色较弱。在293细胞中，TLR1和TLR2共转染可增强TLR2对某些形式的内毒素的反应，但对其他系统的反应具有抑制作用。我们的首要任务是确定TLR的表达模式，然后研究TLR在免疫细胞和周围组织细胞中的信号转导。为此，我们已经克隆了TLR1和5，并正在克隆(或从其他人那里获得)其他TLR。我们的目的是提高针对每一种TLR的抗体，并用它们来研究蛋白质的表达，并进行交联性研究。对病原体的免疫反应始于其固有系统的识别，导致炎症反应的发展。TLRs很可能在这一过程中发挥了重要作用。重定向细胞毒性已被用于临床前小鼠模型以阻止肿瘤生长，但在晚期肿瘤生长往往会导致T和B细胞反应的严重免疫抑制，从而降低任何免疫治疗方法的有效性。我们最初发现，肿瘤生长还导致B和T细胞室中STAT5蛋白表达的选择性丢失，这可能导致对包括IL-2在内的几种细胞因子无反应。为了加深我们对免疫抑制的理解，我们与恩佐·勃朗特博士合作，使用体外系统来研究免疫抑制。Brontes研究小组最初发现，免疫抑制是由一种抑制型巨噬细胞/树突状细胞诱导的，这种细胞在肿瘤生长的后期侵入淋巴组织，他已经使这些细胞永生。我们发现，这些细胞在抗原、同种异体细胞和ConA的刺激下强烈地阻止T细胞的增殖。此外，抑制增殖是由于诱导对IL-2的无反应性。早期事件，如CD69和CD25的上调，以及IL-2的分泌诱导等不会受到损害。无反应状态依赖于一氧化氮的产生。我们已经证明，由脾细胞产生的干扰素-在抑制细胞中诱导一氧化氮的产生，并且干扰素-1的抗体和iNOS(诱导型一氧化氮合酶)的抑制剂都能阻断无反应状态的诱导。无反应性并不伴随着STAT5(A和B)表达的丧失--这发生在较晚的时间，但STAT5的酪氨酸磷酸化显著减少。早期结果尚未检测到JAK3表达或磷酸化的变化。这些结果表明，巨噬细胞相关细胞中iNOS的诱导在决定细胞是刺激还是抑制免疫反应中起着关键作用，我们打算研究iNOS及其产物在遭受各种分化信号的巨噬细胞中的作用。-黏附分子、双特异性抗体、细胞毒性、免疫抑制、重定向细胞溶解、信号分子、先天免疫-人体组织、体液、细胞等

Cellular mediators of innate immunity do not express TcR, yet have the capacity to recognize and respond to target cells in the absence of antibody. In this project we have used redirected lysis to demonstrate that a number of adhesion molecules, including CD38, CD44, CD56, and CD69, have the ability to trigger cytotoxic responses in human NK cells and neutrophils. These responses however are dependent upon cytokines, and represent events secondary to the direct recognition of pathogenic substances. Recently an ancient family of pathogen recognition receptors, the toll like receptors (TLRs) was discovered in human and mouse EST data bases, and two of these, TLR2 and 4 were cloned and shown to trigger inflammatory responses to LPS. We have begun examining TLR expression in human cells, and have found easily detectible message of several of the TLRs in monocytes, dendritic cells, NK cells, T cells and PMN. Monocytes express the highest levels of TLR message, and these levels decrease during differentiation of monocytes to dendritic cells. A mAb to TLR1 stains all monocytes strongly, but gives lower level staining on dentritic cells. Co-transfection of TLR1 with TLR2 in 293 cells potentiates the response of TLR2 to some forms of LPS, but inhibits the response in other systems. Our first priority is to determine the expression patterns of TLRs and then to study TLR signaling in immunocytes and in cells from peripheral tissues. Toward this end we have cloned TLR1 and 5, and are in the process of cloning (or obtaining from others) the other TLRs. Our intention is to raise antibodies to each of the TLRs, and to use them to study protein expression, and to do crosslinking studies. The immune response to a pathogen begins by its recognition by the innate system, resulting in the development of an inflammatory response. It is likely that the TLRs play a major role in this process.Redirected cytotoxicity has been used in preclinical mouse models to block tumor growth, but at late stages tumor growth frequently results in profound immunosuppression of both T and B cell responses, thus reducing the effectiveness of any immunotherapeutic approach. We originally showed that tumor growth also resulted in a selective loss in STAT5 protein expression in B and T cell compartments, which could lead to unresponsiveness to several cytokines including IL-2. To further our understanding of the immune suppression, we have, in collaboration with Dr. Enzo Bronte, used an in vitro system to study immunosuppression. Brontes group originally found that immunosuppression was induced by a suppressor type macrophage/dendritic cell that invades the lymphoid tissue at later stages of tumor growth, and he has immortalized these cells. We have found that these cells strongly block T cell proliferation in response to stimulation by antigen, allogeneic cells and Con A. Moreover, inhibition of proliferation is due to the induction of a non- responsiveness to IL-2. Earlier events, such as up regulation of CD69 and CD25, and induction of IL-2 secretion are not impaired. The non- responsive state is dependent on nitric oxide production. We have shown that IFN- , produced by splenocytes, induces the production of nitric oxide in the suppressor cells, and that antibody to IFN- and an inhibitor of iNOS (inducible nitric oxide synthase) both block the induction of the non-responsive state. Non-responsiveness is not accompanied by a loss in STAT5 (A and B) expression-that occurs later, but there is a marked reduction tyrosine phosphorylation of STAT5. Early results have not yet detected a change in JAK3 expression or phosphorylation. These results suggest that the induction of iNOS in macrophage-related cells plays a pivotal role in determining whether the cells will stimulate or suppress immune responses, and we intend to study iNOS and its product in macrophages that have been subjected to various differentiation signals. - Adhesion Molecules, bispecific antibodies, Cellular cytotoxicity, Immunosuppression, redirected cytolysis, signaling molecules, Innate Immunity, - Human Tissues, Fluids, Cells, etc. & Neither Human Subjects nor Human Tissues