Inhibition of Natural Killer Cell Function

自然杀伤细胞功能的抑制

• 批准号: 7964785
• 负责人: Eric O Long
• 金额: $ 48.72万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964785
• 关键词: ABL1 gene; Abnormal Cell; Actins; Affect; Antigens; Attenuated; Autoimmunity; Binding; Cell Communication; Cell physiology; Cells; Complex; Cytoplasmic Granules; Development; Dissociation; Drosophila genus; Employee Strikes; Family; Goals; Guanine; Guanine Nucleotide Exchange Factors; HLA-C Antigens; Homeostasis; Human; Human Cell Line; Image; Imaging technology; Immune; Inhibitory Synapse; Insecta; Integrins; Interleukin-15; Interleukins; Killer Cells; Life; Ligands; Major Histocompatibility Complex; Malignant Neoplasms; Membrane; Molecular; Mutate; NK Cell Activation; Natural Killer Cells; Normal Cell; Phosphorylation; Physiological; Process; Production; Protein Dephosphorylation; Protein Tyrosine Kinase; Receptor Activation; Receptor Signaling; Regulation; Ribosomal Protein S6; Role; Signal Pathway; Signal Transduction; Site; Small Inducible Cytokine A3; Specificity; Surface; Synapses; T-Lymphocyte; Testing; Tyrosine; Tyrosine Phosphorylation; Virus; base; c-abl Proto-Oncogenes; cancer cell; cytokine; cytotoxic; cytotoxicity; density; interleukin-15 receptor; killings; member; neoplastic cell; novel; pathogen; perforin; prevent; receptor; receptor binding; response

Unlike B and T cells, NK cells do not express antigen-specific receptors, yet they can eliminate virus-infected cells and cancer cells without harming normal cells. An important component in the specific recognition of target cells by NK cells are NK cell inhibitory receptors that recognize surface molecules called major histocompatibility complex (MHC) class I. MHC-specific recognition by inhibitory receptors on NK cells prevents the killing of normal, healthy cells. The major goal of this project is to elucidate the mechanism by which inhibitory receptors block NK cell activation. Natural killer (NK) cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR) family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses. NK cell cytotoxicity is achieved by polarized release of perforin-containing granules towards target cells. As polarization and degranulation can be controlled by separate signals, their respective sensitivity to inhibitory receptors and the requirements for inhibition were evaluated. Expression of HLA-C or HLA-E on the human cell line 221 blocked granule polarization, degranulation, and CD16-dependent MIP-1alpha secretion by NK cell clones with inhibitory receptors of matching HLA specificity. However, HLA-C or HLA-E on Drosophila S2 cells did not fully inhibit CD16-dependent degranulation and MIP-1alpha secretion, suggesting that other receptor-ligand interactions, which occur during contact with 221 cells, are required for complete inhibition. In contrast, HLA-C or HLA-E on S2 cells were sufficient to block granule polarization induced by LFA-1 or by NKG2D. Therefore, engagement of inhibitory receptors by HLA class I on target cells is sufficient to block different signals for granule polarization, but not degranulation. Many cellular responses, such as autoimmunity and cytotoxicity, are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIM). We have shown that binding of inhibitory NK cell receptors to HLA class I on target cells induced tyrosine phosphorylation of the adapter Crk, concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore, Crk dissociated from the guanine exchange factor C3G and bound to tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity, providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex, observed here with two types of inhibitory receptors, expands the signaling potential of the large ITIM-receptor family, and reveals an unsuspected component of the inhibitory mechanism. Interleukin (IL)-15 is presented in trans, bound to the alpha chain of the IL-15 receptor (IL-15Ralpha) on presenting cells, to cells bearing the receptor beta and gamma chains. The confinement of IL-15 stimulation to sites of cell-to-cell contact has potential for regulation by other receptors. We have used primary human NK cells to test the sensitivity of IL-15 transpresentation to NK cell inhibitory receptors. Human target cells expressing ligands for different inhibitory receptor were transfected with IL-15Ralpha. Proliferation of NK cells and phosphorylation of ribosomal protein S6 in response to transpresented IL-15 were reduced by coengagement of inhibitory receptors. Therefore, transpresentation of IL-15 is subject to regulation by MHC class I-specific inhibitory receptors. These conclusions were reached by studying the response of primary, freshly isolated, human NK cells to human cells transfected with IL-15Ralpha, reveal an unsuspected level of regulation in the response of NK cells to the cytokine IL-15, which is essential for their development, survival, and proliferation. These findings demonstrate a novel mechanism to attenuate NK cell responses to IL-15 transpresentation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.

与B和T细胞不同，NK细胞不表达抗原特异性受体，但它们可以在不损害正常细胞的情况下清除病毒感染的细胞和癌细胞。NK细胞对靶细胞的特异性识别的一个重要组成部分是NK细胞抑制受体，它识别称为主要组织相容性复合体(MHC)I类的表面分子。MHC特异性识别NK细胞上的抑制受体可防止对正常、健康细胞的杀伤。本项目的主要目的是阐明抑制性受体阻断NK细胞激活的机制。 自然杀伤(NK)细胞激活受体通过依赖肌动蛋白的过程在细胞毒性免疫突触积聚，在那里它们提供触发NK细胞效应器功能的协同信号。相反，NK细胞抑制性受体，包括MHC-I类特异性杀伤细胞Ig样受体(KIR)家族的成员，聚集在抑制性免疫突触，阻断肌动蛋白动力学，并阻止激活受体的肌动蛋白依赖的磷酸化。因此，人们可以预测，当抑制受体被激活时，依赖肌动蛋白的激活受体的积累将受到抑制。通过与表达NK细胞受体生理配体的靶细胞接触的原代人类NK细胞的共聚焦成像，我们在这里表明这一预测是错误的。靶细胞包括人类细胞系和表达NK细胞激活受体配体和MHC-I类抑制性KIR配体的转基因果蝇昆虫细胞。NK细胞活化受体CD2和2B4与KIR共定位于抑制性免疫突触。事实上，KIR促进了CD2和2B4的聚集，因为CD2和2B4在抑制性突触积累得更有效。相反，KIR和激活受体在抑制性突触的积累与整合素LFA-1密度的降低有关。这些结果表明，抑制性KIR并不是通过阻止CD2和2B4在NK细胞免疫突触上的聚集来阻止它们的信号传递，而是通过阻断它们在抑制性突触内的信号传递能力来阻止它们。 NK细胞的杀伤作用是通过含有穿孔素的颗粒向靶细胞的极化释放来实现的。由于极化和脱颗粒可以由单独的信号控制，因此评估了它们对抑制受体的敏感性和抑制要求。在人细胞系221上表达人类白细胞抗原-C或人类白细胞抗原-E可阻断具有匹配的人类白细胞抗原特异性抑制受体的NK细胞克隆的颗粒极化、脱颗粒和依赖CD16的MIP-1α分泌。然而，果蝇S2细胞上的HLA-C或HLA-E并不能完全抑制CD16依赖的脱颗粒和MIP-1α的分泌，这表明需要完全抑制与221细胞接触时发生的其他受体-配体相互作用。相反，S2细胞表面的HLAC或HLAE足以阻断LFA-1或NKG2D诱导的颗粒极化。因此，HLAI类抑制受体与靶细胞的结合足以阻断颗粒极化的不同信号，但不能阻止脱颗粒。 许多细胞反应，如自身免疫和细胞毒性，是由带有胞浆免疫受体酪氨酸抑制基序(ITIM)的受体控制的。我们已经证明，抑制性NK细胞受体与靶细胞上的HLAI类结合诱导了接头Crk的酪氨酸磷酸化，伴随着鸟嘌呤交换因子Vav1的去磷酸化。此外，在抑制过程中，Crk与鸟嘌呤交换因子C3g解离，并与酪氨酸激酶c-Abl结合。膜靶向的酪氨酸突变形式的Crk可以克服对NK细胞杀伤的抑制，提供了Crk磷酸化有助于抑制的功能证据。CRK的特异性磷酸化及其与两种类型的抑制性受体的信号复合体的解离，扩大了ITIM受体大家族的信号潜力，并揭示了抑制机制中一个意想不到的组成部分。 白介素15以反式形式存在，结合在呈递细胞上的白介素15受体(IL-15Rpha)的阿尔法链上，并结合到带有受体β和伽马链的细胞中。IL-15的刺激局限于细胞与细胞之间的接触部位，有可能被其他受体调节。我们使用原代人NK细胞来检测IL-15转导对NK细胞抑制受体的敏感性。将IL-15Rα导入表达不同抑制性受体配体的人靶细胞。抑制受体的共同作用降低了IL-15对NK细胞的增殖和核糖体蛋白S6的磷酸化水平。因此，IL-15的转压受MHC-I类特异性抑制性受体的调节。这些结论是通过研究原代、新鲜分离的人NK细胞对转IL-15Rpha的人细胞的反应而得出的，揭示了NK细胞对细胞因子IL-15的反应存在意想不到的调节水平，IL-15对其发育、生存和增殖至关重要。这些发现表明了一种新的机制来减弱NK细胞对IL-15转压的反应，并提示抑制性NK细胞受体有助于NK细胞的动态平衡。