Inhibition of Natural Killer Cell Function

NK细胞具有多组激活和抑制受体，控制着细胞毒性淋巴细胞介导的靶细胞杀伤的不同步骤，包括NK细胞与靶细胞的结合，溶解颗粒对靶细胞的极化，以及脱颗粒。抑制性受体事先使NK细胞与MHC-I类分子结合，通过一个称为NK细胞许可(又名教育)的过程，使NK细胞对随后的激活刺激具有更强的内在反应性。与MHC结合的抑制性受体的数量和MHC与抑制性受体的结合强度决定了每个NK细胞对细胞毒作用和细胞因子分泌的潜在反应性。 我们测试了与人类白细胞抗原-C组1等位基因结合的较弱的抑制受体KIR2DL3是否比与人类白细胞抗原-C组2等位基因结合的较强的KIR2DL1授予较弱的许可。我们分析了表达单个和多个人类白细胞抗原I受体的NK细胞亚群中的脱颗粒现象。与人类白细胞抗原E结合的NKG2A具有最强的许可效应，而与人类白细胞抗原B结合的KIR2DL3、KIR2DL1和KIR3DL1较弱，但彼此相似。因此，KIR2DL3和KIR2DL1具有相似的许可NK细胞的能力，这表明抑制信号强度和可用的HLA-C配体的数量与NKC许可无关。因此，与特定组合的人类白细胞抗原-C和KIR2DL相关的疾病的基础可能包括许可以外的因素。 KIR2DL1、KIR2DL2和KIR2DL3结合定义为组1和组C2的二态形式的HLA-C等位基因。KIR2DL2和KIR2DL3结合了含有Ser77和Asn80的C1组等位基因，而KIR2DL1结合了含有Asn77和Lys80的C2组等位基因。然而，这个二元系统受到了挑战，因为一些HLA-C2等位基因显示出与KIR2DL2和KIR2DL3结合。尤其是C2组等位基因HLAC*05：01与KIR2DL1、KIR2DL2和KIR2DL3的结合强度相当高。由于已知KIR与人类白细胞抗原C的结合具有多肽选择性，我们推测与人类白细胞抗原C*05：01结合的多肽可能与KIR2DL1、KIR2DL2和KIR2DL3结合。为此，我们对与人类白细胞抗原C*05：01结合的多肽进行了洗脱和测序。我们通过表达病毒TAP抑制因子ICP47，获得了TAP缺陷的人类白细胞抗原-C*05：01细胞。我们监测了单个人工合成的HLA-C*05：01多肽与可溶性KIR2DL1、KIR2DL2和KIR2DL3分子直接结合的能力，以及对KIR2DL1+和KIR2DL3+NK细胞的功能抑制。我们发现，在所测试的20个多肽中，有16个多肽与KIR2DL1结合，而只有两个多肽与KIR2DL2和KIR2DL3结合。KIR2DL2和KIR2DL3的结合能力依赖于第7位和第8位的多肽侧链，能够抑制KIR2DL3+NK细胞。这两个多肽还具有KIR2DL1结合和抑制KIR2DL1+NK细胞的能力，表明这两个多肽可以取代C1群和C2群定义的KIR特异性。 免疫受体的信号是通过胞浆酪氨酸的磷酸化来启动的，然后胞浆酪氨酸招募效应分子。在MHC I类特异性抑制性受体中，免疫受体酪氨酸抑制基序(ITIM)中胞浆酪氨酸残基的磷酸化导致蛋白酪氨酸磷酸酶的募集，从而阻断激活信号。最近的研究表明，由人类白细胞抗原C特异性杀伤细胞Ig样受体(KIR)发出的信号不依赖于激活受体发出的信号。目前尚不清楚ITIM的磷酸化是如何启动和调节的。我们发现，KIR2DL1的第一个Ig结构域的组氨酸36(His-36)被丙氨酸(H36A)取代，导致KIR2DL1的结构性自结合和磷酸化，以及酪氨酸磷酸酶SHP-1的募集。过钒酸抑制酪氨酸磷酸酶后，KIR2DL1和KIR2DL1-H36A的磷酸化程度相当强，这表明KIR2DL1-H36A被选择性地保护而不被去磷酸化。我们认为KIR的磷酸化是由ITIM对酪氨酸磷酸酶的可及性控制的，并且KIR与人类白细胞抗原C的结合必须推翻His-36对KIR2DL1自结合的阻碍。KIR2DL1-H36A在NK细胞上的表达比野生型KIR2DL1对HLA-C+靶细胞裂解的抑制作用更强。这些结果表明，ITIM的磷酸化是由KIR的自结合控制的，His-36是一个守门人，通过KIR2DL1阻止未调控的信号传递。 IL-15对NK细胞的增殖、激活和存活至关重要。与IL-15受体α链(IL-15Rpha)结合的IL-15存在于携带IL-2受体β链和常见伽马链的反式TO细胞中。由于IL-15的转压发生在细胞与细胞之间的接触中，它有可能被其他受体和配体的相互作用所调节。我们测试了人NK细胞IL-15转压对抑制性受体的敏感性。将IL-15Rpha导入表达抑制受体KIR2DL1、KIR2DL2/3或CD94-NKG2A的HLAI类配体的人细胞。KIR2DL1或KIR2DL2/3被同源的HLA-C配体结合，抑制了原代NK细胞对IL-15的反应。抑制KIRHLA-C相互作用并不能减少可溶性IL-15诱导的细胞增殖。因此，IL-15的转压受到MHC-I类特异性抑制性受体的下调。同样，人类白细胞抗原E可抑制IL-15转导诱导的NKG2A+细胞株NKL增殖。抑制受体KIR2DL1或CD94-NKG2A的共同作用不能抑制Stat5的磷酸化，但选择性地抑制Akt和S6激酶的磷酸化。这些结果证明了一种新的抑制IL-15依赖的NK细胞增殖的机制，提示抑制性NK细胞受体有助于NK细胞的动态平衡。

NK cells possess several sets of activating and inhibitory receptors that control different steps in cytotoxic lymphocyte-mediated killing of target cells, including conjugation of NK cells to target cells, polarization of lytic granules towards target cells, and degranulation. Prior engagement of NK cells with MHC class I molecules by inhibitory receptors allows for greater intrinsic responsiveness to subsequent activation stimuli through a process called NK cell licensing (aka education). The number of inhibitory receptors engaged with MHC and the strength of MHC binding to inhibitory receptors calibrate the potential responsiveness of each NK cell for cytotoxicity and cytokine secretion. We tested whether the weaker inhibitory receptor KIR2DL3, which binds HLA-C group 1 alleles, conferred weaker licensing than the stronger KIR2DL1, which binds HLA-C group 2 alleles. We analyzed degranulation in NK cell subsets expressing single and multiple receptors for HLA class I. NKG2A, which binds HLA-E, had the strongest licensing impact, while KIR2DL3, KIR2DL1, and KIR3DL1, which binds HLA-B, were weaker but similar to each other. Thus, KIR2DL3 and KIR2DL1 have similar capacity to license NK cells, suggesting that inhibitory signal strength and amount of available HLA-C ligands do not correlate with NKC licensing. Therefore, the basis for disease associations with specific combinations of HLA-C and KIR2DL likely encompasses factors other than licensing. KIR2DL1, KIR2DL2 and KIR2DL3 bind dimorphic forms of HLA-C alleles defined as group C1 and C2. KIR2DL2 and KIR2DL3 bind group C1 alleles that contain Ser77 and Asn80 while KIR2DL1 binds group C2 alleles that contain Asn77 and Lys80. However, this binary system has been challenged as some HLA-C2 alleles showed binding to KIR2DL2 and KIR2DL3. In particular, the group C2 allele HLA-C*05:01 bound KIR2DL1, KIR2DL2 and KIR2DL3 with equally high intensity. As it is known that KIR binding to HLA-C is peptide selective, we hypothesized that peptides bound to HLA-C*05:01 may confer binding to KIR2DL1, KIR2DL2 and KIR2DL3. To this end, we eluted and sequenced peptides bound to HLA-C*05:01. We generated TAP deficient HLA-C*05:01 cells through expression of the viral TAP inhibitor ICP47. We monitored the capacity of individual synthetic HLA-C*05:01 peptides to confer direct binding of soluble KIR2DL1, KIR2DL2 and KIR2DL3 molecules, and functional inhibition of KIR2DL1+ and KIR2DL3+ NK cells. We showed that of twenty peptides tested, 16 peptides conferred binding to KIR2DL1 when presented on .221-C*05:01-ICP47, while only two conferred binding to KIR2DL2 and KIR2DL3. The capacity to bind KIR2DL2 and KIR2DL3 was dependent on peptide side chains at positions 7 and 8 and was competent for inhibition of KIR2DL3+ NK cells. These two peptides were also competent for KIR2DL1 binding and inhibition of KIR2DL1+ NK cells showing that these two peptides can override KIR specificity defined by group C1 and C2. Signaling by immunoreceptors is initiated by phosphorylation of cytosolic tyrosines, which then recruit effector molecules. In the case of MHC class I-specific inhibitory receptors, phosphorylation of cytosolic tyrosine residues within immunoreceptor tyrosine-based inhibition motifs (ITIM) results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work has shown that signaling by an HLA-C-specific killer cell Ig-like receptor (KIR) is independent of signaling by activation receptors. It is not known how ITIM phosphorylation is initiated and regulated. We have shown that substitution of histidine 36 (His-36) in the first Ig domain of KIR2DL1 with alanine (H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, and recruitment of tyrosine phosphatase SHP-1. The equally strong phosphorylation of KIR2DL1 and KIR2DL1-H36A after inhibition of tyrosine phosphatase by pervanadate suggested that KIR2DL1-H36A is selectively protected from dephosphorylation. We propose that KIR phosphorylation is controlled by the accessibility of ITIM to tyrosine phosphatases, and that KIR binding to HLA-C must override the hindrance His-36 puts on KIR2DL1 self-association. Expression of KIR2DL1-H36A on NK cells led to stronger inhibition of lysis of HLA-C+ target cells than expression of wild type KIR2DL1. These results have revealed that ITIM phosphorylation is controlled by self-association of KIR and that His-36 serves as a gatekeeper to prevent unregulated signaling through KIR2DL1. IL-15 is essential for NK cell proliferation, activation and survival. IL-15 bound to the IL-15 receptor alpha chain (IL-15Ralpha) is presented in trans to cells bearing the IL-2 receptor beta and common gamma chains. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptorligand interactions. We tested human NK cells for the sensitivity of IL-15 transpresentation to inhibitory receptors. Human cells expressing HLA class I ligands for inhibitory receptors KIR2DL1, KIR2DL2/3, or CD94-NKG2A were transfected with IL-15Ralpha. Proliferation of primary NK cells in response to transpresented IL-15 was reduced by engagement of either KIR2DL1 or KIR2DL2/3 by cognate HLA-C ligands. Inhibitory KIRHLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to down-regulation by MHC class I-specific inhibitory receptors. Similarly, proliferation of the NKG2A+ cell line NKL induced by IL-15 transpresentation was inhibited by HLA-E. Co-engagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 kinase. These results demonstrate a novel mechanism to attenuate IL-15 dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.