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，但它们可以消除感染病毒的细胞和癌细胞而不会损害正常细胞。 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，它们识别称为主要的组织相容性复合物（MHC）I级的表面分子I。NK细胞上抑制受体的MHC特异性识别可预防正常健康细胞的杀死。该项目的主要目标是阐明抑制受体阻止NK细胞激活的机制。 天然杀伤（NK）细胞活化受体通过肌动蛋白依赖性过程在细胞毒性免疫突触中积累，它们提供了触发NK细胞效应函数的协同信号。相反，NK细胞抑制受体，包括MHC I类特异性杀伤细胞Ig样受体（KIR）家族的成员，在抑制性免疫突触中积累，阻断肌动蛋白动力学并防止肌动蛋白依赖性活化受体的磷酸化。因此，当抑制受体参与时，将预测抑制肌动蛋白依赖性受体的积累。通过与表达NK细胞受体生理配体的靶细胞接触的原代人NK细胞的共聚焦成像，我们在这里表明该预测是不正确的。靶细胞包括人类细胞系和转染的果蝇昆虫细胞，该细胞表达NK细胞活化受体的配体与MHC I类抑制性KIR配体结合使用。两个NK细胞活化受体CD2和2B4在抑制性免疫突触下与KIR累积并共定位。实际上，KIR促进了CD2和2B4聚类，因为CD2和2B4在抑制突触下积累了更有效的积累。相反，KIR和激活受体在抑制性突触上的积累与整联蛋白LFA-1密度降低相关。这些结果表明，抑制性KIR不会通过阻止其在NK细胞免疫突触中的积累而导致其在抑制突触中信号的能力来阻止其积累来阻止CD2和2B4信号传导。 NK细胞细胞毒性是通过极化对靶细胞的含状颗粒释放来实现的。由于可以通过单独的信号控制极化和脱粒，因此评估了它们对抑制受体的敏感性以及抑制的要求。 HLA-C或HLA-E在人类细胞系221上的表达阻断了NK细胞克隆具有抑制性HLA特异性的抑制受体的NK细胞克隆的颗粒极化，脱粒和CD16依赖的MIP-1Alpha分泌。但是，果蝇S2细胞上的HLA-C或HLA-E并未完全抑制CD16依赖性脱粒和MIP-1Alpha分泌，这表明在与221个细胞接触期间发生的其他受体配体相互作用是完全抑制所需的。相反，S2细胞上的HLA-C或HLA-E足以阻止LFA-1或NKG2D诱导的颗粒极化。因此，HLA I类在靶细胞上对抑制受体的接合足以阻止不同的信号以进行颗粒极化，但不能阻止脱粒。 许多细胞反应，例如自身免疫性和细胞毒性，都由具有细胞质免疫受体酪氨酸抑制基序（ITIM）的受体控制。我们已经表明，抑制性NK细胞受体与靶细胞I类HLA I类的结合诱导了适配器CRK的酪氨酸磷酸化，这与鸟嘌呤交换因子VAV1的脱磷酸化。此外，在抑制过程中，CRK与鸟嘌呤交换因子C3G分离，并与酪氨酸激酶C-ABL结合。将酪氨酸突变形式的CRK靶向膜可以克服NK细胞细胞毒性的抑制作用，提供功能证据表明CRK磷酸化有助于抑制。 CRK的特定磷酸化及其与信号传导复合物的解离，在此观察到了两种类型的抑制受体，扩大了大型ITIM受体受体家族的信号传导潜力，并揭示了抑制性机制的未经引起的组件。 白介素（IL）-15以反式形式呈现，与呈现细胞的IL-15受体（IL-15ralpha）的α链结合到带有受体β和伽马链的细胞。将IL-15刺激限制在细胞对细胞接触部位具有其他受体调节的潜力。我们已经使用原代人NK细胞来测试IL-15超代理对NK细胞抑制受体的敏感性。用IL-15ralpha转染表达不同抑制受体的配体的人靶细胞。 NK细胞的增殖和核糖体蛋白S6响应于超代理IL-15的磷酸化降低，通过抑制受体的促进。因此，IL-15的跨代理受到MHC I类抑制受体的调节。通过研究原代，新鲜分离的人类NK细胞对用IL-15Ralpha转染的人类细胞的反应得出的结论，揭示了NK细胞对细胞因子IL-15的反应的不受欢迎的调节水平，这对于它们的发育，存活和增殖至关重要。这些发现证明了一种减弱NK细胞对IL-15先生反应的新机制，并表明抑制性NK细胞受体有助于NK细胞稳态。