Inhibition of Natural Killer Cell Function

NK细胞具有几组激活和抑制受体，其控制细胞毒性淋巴细胞介导的靶细胞杀伤中的不同步骤，包括NK细胞与靶细胞的缀合、裂解颗粒朝向靶细胞的极化和脱粒。通过抑制性受体将NK细胞与MHC I类分子预先接合，可以通过称为NK细胞许可（又名教育）的过程对随后的激活刺激产生更大的内在反应。与MHC结合的抑制性受体的数量和MHC与抑制性受体结合的强度校准了每个NK细胞对细胞毒性和细胞因子分泌的潜在响应性。 我们测试了结合HLA-C组1等位基因的较弱抑制性受体KIR 2DL 3是否比结合HLA-C组2等位基因的较强KIR 2DL 1赋予较弱的许可。我们分析了NK细胞亚群表达单一和多种HLA I类受体的脱粒。结合HLA-E的NKG 2A具有最强的许可影响，而结合HLA-B的KIR 2DL 3，KIR 2DL 1和KIR 3DL 1较弱，但彼此相似。因此，KIR 2DL 3和KIR 2DL 1具有类似的许可NK细胞的能力，表明抑制性信号强度和可用HLA-C配体的量与NKC许可无关。因此，与HLA-C和KIR 2DL的特定组合相关的疾病的基础可能包括许可证以外的因素。 KIR 2DL 1、KIR 2DL 2和KIR 2DL 3结合定义为C1和C2组的HLA-C等位基因的二态形式。KIR 2DL 2和KIR 2DL 3结合含有Ser 77和Asn 80的C1组等位基因，而KIR 2DL 1结合含有Asn 77和Lys 80的C2组等位基因。然而，这种二元系统受到挑战，因为一些HLA-C2等位基因显示与KIR 2DL 2和KIR 2DL 3结合。特别地，C2组等位基因HLA-C*05：01以同样高的强度结合KIR 2DL 1、KIR 2DL 2和KIR 2DL 3。由于已知KIR与HLA-C的结合是肽选择性的，我们假设与HLA-C*05：01结合的肽可以赋予与KIR 2DL 1、KIR 2DL 2和KIR 2DL 3的结合。为此，我们洗脱并测序了与HLA-C*05：01结合的肽。我们通过表达病毒TAP抑制剂ICP 47产生TAP缺陷型HLA-C*05：01细胞。我们监测了单个合成HLA-C*05：01肽赋予可溶性KIR 2DL 1、KIR 2DL 2和KIR 2DL 3分子的直接结合以及KIR 2DL 1+和KIR 2DL 3 + NK细胞的功能抑制的能力。我们表明，在测试的20种肽中，当在.221-C*05：01-ICP 47上呈递时，16种肽赋予与KIR 2DL 1的结合，而只有两种赋予与KIR 2DL 2和KIR 2DL 3的结合。结合KIR 2DL 2和KIR 2DL 3的能力依赖于位置7和8处的肽侧链，并且能够抑制KIR 2DL 3 + NK细胞。这两种肽也能够与KIR 2DL 1结合并抑制KIR 2DL 1 + NK细胞，这表明这两种肽可以超越由C1和C2组定义的KIR特异性。 免疫受体的信号传导由胞质酪氨酸的磷酸化启动，然后招募效应分子。在MHC I类特异性抑制性受体的情况下，基于免疫受体酪氨酸的抑制基序（ITIM）内的胞质酪氨酸残基的磷酸化导致阻断激活信号的蛋白酪氨酸磷酸酶的募集。最近的研究表明，HLA-C特异性杀伤细胞Ig样受体（KIR）的信号传导与活化受体的信号传导无关。目前尚不清楚ITIM磷酸化是如何启动和调节的。我们已经证明，在KIR 2DL 1的第一个IG结构域中的组氨酸36（His-36）被丙氨酸（H36 A）取代导致组成性KIR 2DL 1自缔合和磷酸化，以及酪氨酸磷酸酶SHP-1的募集。KIR 2DL 1和KIR 2DL 1-H36 A在通过过钒酸盐抑制酪氨酸磷酸酶后同样强的磷酸化表明KIR 2DL 1-H36 A被选择性地保护免于去磷酸化。我们认为KIR磷酸化是由ITIM对酪氨酸磷酸酶的可及性控制的，并且KIR与HLA-C的结合必须克服His-36对KIR 2DL 1自结合的阻碍。NK细胞上KIR 2DL 1-H36 A的表达导致比野生型KIR 2DL 1的表达更强的对HLA-C+靶细胞裂解的抑制。这些结果表明，ITIM磷酸化是由KIR的自缔合控制的，并且His-36作为看门人来防止通过KIR 2DL 1的不受调节的信号传导。 IL-15对NK细胞增殖、活化和存活至关重要。与IL-15受体α链结合的IL-15（IL-15 R α）反式呈递给携带IL-2受体β和共同γ链的细胞。由于IL-15转表达发生在细胞与细胞接触的背景下，因此它具有通过其他受体-配体相互作用进行调节和调节其他受体-配体相互作用的潜力。我们测试了人NK细胞的IL-15转表达对抑制性受体的敏感性。用IL-15 Ra转染表达抑制性受体KIR 2DL 1、KIR 2DL 2/3或CD 94-NKG 2A的HLA I类配体的人细胞。通过同源HLA-C配体与KIR 2DL 1或KIR 2DL 2/3的接合，降低了原代NK细胞响应于转表达的IL-15的增殖。抑制性KIRHLA-C相互作用不降低可溶性IL-15诱导的增殖。因此，IL-15的转表达受到MHC I类特异性抑制性受体的下调。类似地，由IL-15转表达诱导的NKG 2A+细胞系NKL的增殖被HLA-E抑制。抑制性受体KIR 2DL 1或CD 94-NKG 2A的共结合不抑制Stat 5的磷酸化，但选择性抑制Akt和S6激酶的磷酸化。这些结果证明了减弱IL-15依赖性NK细胞增殖的新机制，并表明抑制性NK细胞受体有助于NK细胞稳态。

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.