Inhibition of Natural Killer Cell Function

人类自然杀伤(NK)细胞活性受一系列针对HLAI类的杀伤细胞Ig样受体(KIR)的调节。KIR和HLA型的组合与疾病易感性有关，包括对病毒感染和妊娠障碍的敏感性。我们研究的目的是确定与人类白细胞抗原-C结合的多肽对抑制性KIR与C1族或C2型HLAC同种异型的特异性结合的影响程度。尽管只有两个氨基酸差异，但内源性HLA-C*05：01相关多肽被鉴定并检测KIR与载多肽的KIR结合，这两个多肽分别是KIR2DL1和KIR2DL3的典型配体。KIR2DL1与C2同种异型的结合在大多数试验中都发生了。相反，KIR2DL3与C1同种异型的结合仅限于部分多肽，这解释了为什么KIR2DL3C1的相互作用似乎弱于KIR2DL1C2。此外，KIR2DL3与C2同种异型的交叉反应和功能结合甚至受到更多的限制，只有几个肽发生。此外，我们还鉴定了两个肽，它们出人意料地促进了C2同种异型特异性KIR2DL1与C1同种异型的结合。我们的数据表明，在多个多肽的背景下，KIR与HLA-C结合是HLA-C同种异型C2的一个强特异性特征，而对多肽的更大选择性与更允许与HLA-C同种异型的反应有关。多肽序列驱动的KIR结合可能导致KIR基因型与特定的HLA等位基因的疾病关联，并被病原体利用来调节NK细胞的反应。 杀伤细胞免疫球蛋白样受体(KIR)结合人类白细胞抗原C并阻断人类自然杀伤(NK)细胞激活的抑制功能依赖于锌。我们已经报道了锌诱导可溶的KIR组装成丝状聚合物，如电子显微镜所检测到的，在锌螯合后解聚。从锌处理的细胞裂解液中分离到类似的KIR丝，扫描电子显微镜和成像质谱仪在整个细胞上检测到富含锌的膜突起。KIR胞外区的两个独立突变，远离HLA-C结合部位，损害了锌驱动的聚合和抑制功能。在NK细胞与HLAC+细胞的功能性抑制性免疫突触处，KIR丝在不加锌的情况下自发形成。除了最近通过高阶分子组装进行信号转导的范例外，锌诱导的抑制性KIR聚合代表了细胞表面受体的一种不寻常的信号传递模式。 IL-15对NK细胞的存活、增殖和激活是必不可少的。IL-15的刺激是通过将IL-15与其他细胞表达的IL-15受体链(IL-15R)结合，反式递呈到NK细胞表达的IL-2受体和c链上。由于IL-15的转压发生在细胞与细胞接触的背景下，我们测试了IL-15向人NK细胞的转压是否对抑制性受体的共同参与敏感。我们发现，IL-15转压诱导的NK细胞增殖受NKG2A或KIR抑制受体共同激活的负向调节。这种负调控选择性地发生在PI3KAktmTORC1S6激酶通路中，因为Akt和核糖体蛋白S6的磷酸化在抑制受体共同作用时被降低，而Stat5的磷酸化和NK细胞的存活没有被抑制。这些结果揭示了抑制IL-15依赖的NK细胞增殖的新机制，并提示抑制性NK细胞受体有助于NK细胞的动态平衡。

Human natural killer (NK) cell activity is regulated by a family of killer-cell Ig-like receptors (KIR) for HLA class I. Combinations of KIR and HLA genotypes are associated with disease susceptibility, including sensitivity to virus infection and disorders of pregnancy. The goal of our study was to determine the extent to which peptides bound to HLA-C influence the specific binding of inhibitory KIR to group C1 or C2 HLA-C allotypes. Endogenous HLA-C*05:01-associated peptides were identified and tested for KIR binding to peptide-loaded HLA-C*05:01 (C2) and HLA-C*08:02 (C1), which are canonical ligands for KIR2DL1 and KIR2DL3, respectively, despite having only two amino acid differences. Specific binding of KIR2DL1 to the C2 allotype occurred with most peptides tested. In contrast, KIR2DL3 binding to the C1 allotype was restricted to a subset of peptides, providing an explanation for why KIR2DL3C1 interactions appear to be weaker than KIR2DL1C2. Furthermore, cross-reactive and functional binding of KIR2DL3 with the C2 allotype was even more restricted, occurring with only a few peptides. Moreover, we identified two peptides that, unexpectedly, promoted binding of the C2 allotype-specific KIR2DL1 to the C1 allotype. Our data show that KIR binding to HLA-C in the context of multiple peptides is a feature of strong specificity for HLA-C allotype C2, whereas greater selectivity for peptides is associated with more permissive reactivity with HLA-C allotypes. Peptide sequence-driven binding of KIR could lead to disease associations of KIR genotypes with specific HLA alleles, and be exploited by pathogens to modulate NK cell responses. The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We have reported that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed spontaneously, without addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C+ cells. Adding to the recent paradigm of signal transduction through higher-order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling by a receptor at the cell surface. IL-15 is essential for NK cell survival, proliferation, and activation. Stimulation by IL-15 occurs through trans-presentation of IL-15, which is bound to the IL-15 receptor chain (IL-15R) expressed on other cells, to the IL-2 receptor and c chains expressed by NK cells. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, we tested if IL-15 transpresentation to human NK cells was sensitive to co-engagement of inhibitory receptors. We found that NK cell proliferation induced by IL-15 transpresentation was negatively regulated by co-engagement of either NKG2A or KIR inhibitory receptors. This negative regulation occurred selectively in the PI3KAktmTORC1S6 kinase pathway, as phosphorylation of Akt and ribosomal protein S6 were reduced upon co-engagement of inhibitory receptor, while phosphorylation of Stat5 and NK cell survival were not inhibited. These results reveal a novel mechanism to attenuate IL-15 dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.