Inhibition of Natural Killer Cell Function

与B和T细胞不同，NK细胞不表达抗原特异性受体，但它们可以消除病毒感染的细胞和癌细胞，而不伤害正常细胞。NK细胞特异性识别靶细胞的一个重要组成部分是NK细胞抑制性受体，其识别称为I类主要组织相容性复合体（MHC）的表面分子。NK细胞上抑制性受体的MHC特异性识别阻止了对正常健康细胞的杀伤。该项目的主要目标是阐明抑制性受体阻断NK细胞活化的机制。 自然杀伤（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处积累，在细胞毒性免疫突触处它们提供触发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，在抑制性免疫突触处积累，阻断肌动蛋白动力学，并防止激活受体的肌动蛋白依赖性磷酸化。因此，当抑制性受体被激活时，可以预测肌动蛋白依赖性激活受体积累的抑制。通过与表达NK细胞受体的生理配体的靶细胞接触的原代人NK细胞的共聚焦成像，我们在这里表明这种预测是不正确的。靶细胞包括人细胞系和表达NK细胞活化受体配体与抑制性KIR的MHC I类配体组合的转染的果蝇昆虫细胞。两种NK细胞活化受体CD 2和2B 4与KIR在抑制性免疫突触处积聚并共定位。事实上，KIR促进了CD 2和2B 4的聚集，因为CD 2和2B 4在抑制性突触中的积累更有效。相反，KIR和激活受体在抑制性突触的积累与整合素LFA-1的密度降低相关。这些结果表明，抑制性KIR不阻止CD 2和2B 4信号通过阻断其在NK细胞免疫突触的积累，但通过阻断其在抑制性突触内的信号能力。 NK细胞的细胞毒性通过含穿孔素的颗粒朝向靶细胞的极化释放来实现。由于极化和脱粒可以通过单独的信号控制，因此评价了它们各自对抑制性受体的敏感性和抑制要求。人类细胞系221上HLA-C或HLA-E的表达阻断了具有匹配HLA特异性抑制性受体的NK细胞克隆的颗粒极化、脱颗粒和CD 16依赖性MIP-1 α分泌。然而，果蝇S2细胞上的HLA-C或HLA-E不能完全抑制CD 16依赖性脱粒和MIP-1 α分泌，这表明在与221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必需的。相反，S2细胞上的HLA-C或HLA-E足以阻断LFA-1或NKG 2D诱导的颗粒极化。因此，靶细胞上的抑制性受体与HLA I类的结合足以阻断颗粒极化的不同信号，但不阻断脱粒。 许多细胞反应，如自身免疫和细胞毒性，是由具有细胞质免疫受体酪氨酸基序（ITIM）的受体控制的。我们已经表明，抑制性NK细胞受体与靶细胞上的HLA I类的结合诱导了接头Crk的酪氨酸磷酸化，伴随着鸟嘌呤交换因子Vav 1的去磷酸化。此外，在抑制过程中，Crk从鸟嘌呤交换因子C3 G解离并与酪氨酸激酶c-Abl结合。酪氨酸突变形式的Crk的膜靶向可以克服NK细胞毒性的抑制，提供了Crk磷酸化有助于抑制的功能证据。特异性磷酸化的Crk和它的解离信号复合物，观察到这里与两种类型的抑制性受体，扩大了大ITIM受体家族的信号转导潜力，并揭示了一个未知的组成部分的抑制机制。 白细胞介素（IL）-15以反式形式呈递给携带受体β和γ链的细胞，与呈递细胞上IL-15受体（IL-15 R α）的α链结合。将IL-15刺激限制在细胞与细胞接触的位点具有通过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转表达对NK细胞抑制性受体的敏感性。用IL-15 Ra转染表达不同抑制性受体的配体的人靶细胞。NK细胞的增殖和核糖体蛋白S6的磷酸化反应transpresented IL-15的抑制性受体的共同参与减少。因此，IL-15的转表达受到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IL-15 R α转染的人细胞的反应而得出的，揭示了NK细胞对细胞因子IL-15的反应中的未知调节水平，这对于它们的发育、存活和增殖是必不可少的。这些发现证明了减弱NK细胞对IL-15转表达的应答的新机制，并表明抑制性NK细胞受体有助于NK细胞稳态。

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.