The inhibitory NK cell receptor CD94/NKG2A and the activating receptor CD94/NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues

The human non-classical MHC class I molecule HLA-E is a ligand for both an inhibitory NK cell receptor (CD94/NKG2A) and an activating receptor (CD94/NKG2C). To identify HLA-E surface recognized by both receptors, especially to determine if both receptors recognize the same epitope, we made a series of individually Ala-substituted HLA-E proteins and analyzed their binding to CD94/NKG2A orCD94/NKG2C. Eight HLA-E mutations that significantly impaired HLA-E binding to CD94/NKG2A are all found in the top of alpha1/alpha2 domain of HLA-E. These results suggest that CD94/NKG2A binds a HLA-E surface equivalent to a NKG2D binding site on MICA. Of the eight mutations that impaired HLA-E binding to CD94/NKG2A, six significantly impaired HLA-E binding to CD94/NKG2C suggesting that CD94/NKG2C also binds a similar surface of HLA-E. Unexpectedly, the two HLA-E mutations (D69A and H155A) selectively abrogated HLA-E binding to CD94/NKG2A, not largely affected CD94/NKG2C. These results indicate that a mostly shared, but partly distinct set of HLA-E residues is discriminated by the two receptors.     

Inhibitory NKG2A and activating NKG2D and NKG2C natural killer cell receptor genes: susceptibility for rheumatoid arthritis

The inhibitory (NKG2A) and activating (NKG2D and NKG2C) natural killer (NK) cell receptors are expressed on a subset of NK and T cells. They regulate the innate and adaptive immune systems related to cytotoxicity and cytokine production that are involved in the pathogenesis of rheumatoid arthritis (RA). The role of inhibitory and activating NK cell receptor genes might contribute to chronic inflammation and destruction of bone and cartilage in RA. Therefore, we investigated the association of the NKG2A, NKG2C, and NKG2D genotypes with RA. NKG2A (KLRC1) NKG2C (KLRC2), and NKG2D (KLRK1, D12S249E) genes were genotyped in 210 unrelated patients with RA and 298 controls using a polymerase chain reaction-restriction fragment length polymorphism. We further investigated the relationships between the genotypes of each single nucleotide polymorphism and the presence of rheumatoid factor (RF), antinuclear antibody (ANA), and bony erosions in RA patients. The major NKG2A c.338-90*A/*A, NKG2C102*Ser/*Ser, and NKG2D72*Ala/*Ala genotypes in RA were significantly associated compared with controls [P = 0.013, odds ratio (OR) = 0.6, 95% confidence interval (CI) = 0.44-0.91; P < 0.0001, OR = 2.1, 95% CI = 1.44-2.96; and P = 0.019, OR = 0.6, 95% CI = 0.45-0.93, respectively]. The minor NKG2A c.338-90*G/*G, NKG2C102*Phe/*Phe, and NKG2D72*Thr/*Thr genotypes showed a risk of RA (P = 0.010, OR = 2.0, 95% CI = 1.17-3.54; P < 0.0001, OR = 0.2, 95% CI = 0.12-0.48; and P = 0.032, OR = 2.3, 95% CI = 1.05-5.01, respectively) compared with controls. No significance was observed between the inhibitory (NKG2A) or activating (NKG2C and NKG2D) receptor genotypes and the presence of RF, ANA, or bony erosions in RA.     

Clinical Significance of the HLA-E and CD94/NKG2 Interaction

HLA-E belongs to the non-classical HLA (class Ib family) broadly defined by a limited polymorphism and a restricted pattern of cellular expression. So far, only two functional alleles differing at only one amino acid position (non-synonymous mutation) in the α2 heavy chain domain, where an arginine in position 107 in HLA-E*0101 is replaced by a glycine in HLA-E*0103, have been reported. The interaction between non-classical HLA-E molecule and CD94/NKG2A receptor plays a crucial role in the immunological response involving natural killer (NK) cells and cytotoxic T lymphocytes. All proteins forming CD94/NKG2 receptors are encoded by genes situated in the same cluster on chromosome 12, allowing tight control over the order of their expression. The inhibitory members of the NKG2 receptor family are available on the cell surface before activating the members to prevent autoimmune incidents during immune cells’ ontogenesis. In the present review, the potential role of this interaction in viral infection, pregnancy and transplantation of allogeneic hematopoietic stem cells (HSC) is presented and discussed. The review will also include the effect of HLA-E polymorphism on the outcome of HSC transplants in humans.     

HLA-E*0101 and HLA-G*010101 reduce the risk of Behcet's disease

The nonclassical human leukocyte antigen (HLA)-E and -G molecules have previously been shown to inhibit natural killer- and cytotoxic T-lymphocyte-mediated cell lysis and have also been shown to prevent the proliferation of CD4 T cells and secrete cytokines that appear to be important in the modulation of the Behcet's disease (BD) immune systems. Polymorphisms in the HLA-E and HLA-G genes have been associated with differential expression and function. Thus, we conducted an analysis of the HLA-E and HLA-G alleles using Amplification Refractory Mutation System-polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism techniques in a study comprising 312 patients with BD and 486 controls. The HLA-E*0101 and HLA-G*010101 alleles were associated with a reduced risk of BD (P = 0.0002, odds ratio (OR) = 0.7 and P = 0.002, OR = 0.7, respectively). By way of contrast, the variants HLA-E*010302, HLA-G*010102, G*0105N alleles and 3741_3754ins14bp were all associated with an increased risk of BD (P < 0.0001, OR = 1.6; P = 0.002, OR = 1.8; P = 0.024, OR = 2.0 and P = 0.003, OR = 1.4, respectively). Individuals carrying both the HLA-E*0101 and the HLA-G*010101 alleles evidenced significantly lower frequency in the patients than in the controls (35.6% vs 49.6%; P < 0.0001, OR = 0.6). These results indicate that variant HLA-E and HLA-G molecules appear to function independently and synergistically, increasing the risk of BD, and may result in an imbalance of lymphocytic functions, which may culminate in the development of BD.     

The CD94/NKG2C killer lectin-like receptor constitutes an alternative activation pathway for a subset of CD8+ T cells

The CD94/NKG2C killer lectin-like receptor (KLR) specific for HLA-E is coupled to the KARAP/DAP12 adapter in a subset of NK cells, triggering their effector functions. We have studied the distribution and function of this KLR in T lymphocytes. Like other NK cell receptors (NKR), CD94/NKG2C was predominantly expressed by a CD8(+) T cell subset, though TCRgammadelta(+) NKG2C(+) and rare CD4(+) NKG2C(+) cells were also detected in some individuals. Coculture with the 721.221 HLA class I-deficient lymphoma cell line transfected with HLA-E (.221-AEH) induced IL-2Ralpha expression in CD94/NKG2C+ NK cells and a minor subset of CD94/NKG2C(+) T cells, promoting their proliferation; moreover, a similar response was triggered upon selective engagement of CD94/NKG2C with a specific mAb. CD8(+) TCRalphabeta CD94/NKG2C(+) T cell clones, that displayed different combinations of KIR and CD85j receptors, expressed KARAP/DAP12 which was co-precipitated by an anti-CD94 mAb. Specific engagement of the KLR triggered cytotoxicity and cytokine production in CD94/NKG2C(+) T cell clones, inducing as well IL-2Ralpha expression and a proliferative response. Altogether these results support that CD94/NKG2C may constitute an alternative T cell activation pathway capable of driving the expansion and triggering the effector functions of a CTL subset.     

HLA-E regulates NKG2C+ natural killer cell function through presentation of a restricted peptide repertoire

NK cells interact with the HLA-E molecule via the inhibitory receptor NKG2A and the activating receptor NKG2C. Hence, HLA-E can have a dual role in the immune response. In the present study, we aim to investigate the functional consequences of HLA-E for NKG2A and NKG2C expressing NK cell subsets by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. PBMC derived from healthy subjects were used as targets for isolated NK cells and NK cell activation was examined by analysis of the expression of the degranulation marker CD107a. Peptide induced HLA-E expression inhibited degranulation of NKG2A+ NK cell subsets with almost all peptides, whereas NKG2A− NKG2C+ NK cell responses were enhanced only after incubation with four peptides; 1.3-fold with CMV(I), A80 and B13 and 3.2-fold with HLA-G derived peptide. In addition, the HLA-E:G peptide complex triggered NKG2C receptor internalization, as evidenced by reduction in the percentage of NKG2C+ NK cells when incubated with the peptide, which could be restored by addition of Bafilomycin. In conclusion: in contrast to NKG2A, NKG2C is regulated by HLA-E only when HLA-E is in complex with a restricted peptide repertoire, especially in combination with the HLA-G leader peptide.     

MS患者CD94/NKG2A-bright和CD94/NKG2A-dim NK细胞亚群对IFN-β的差异性反应

目的： 分析多发性硬化（MS）进展型患者不同表型NK细胞亚群对临床主要治疗方法的反应性差异.方法： 分离患者外周血中的NK细胞, 以流式细胞术根据表面抑制性受体CD94/NKG2A表达情况分为两个亚群CD94/NKG2A-bright和CD94/NKG2A-dim.分别加入IFN-β, 测定两个亚群表面CD94/NKG2A变化及细胞增殖, 同时检测两种亚群分泌IL-10和TGF-β情况.结果： CD94/NKG2A阳性表达的NK细胞占25.5%, 其中CD94/NKG2A-bright和CD94/NKG2A-dim分别占其中的23.6%和76.4%.加入IFN-β, CD94/NKG2A-bright组增殖率明显低于CD94/NKG2A-dim组, CD94/NKG2A表达峰度变化不大.CD94/NKG2A-dim组中CD94/NKG2A表达显著增加.两个亚群分泌的IL-10和TGF-β与未刺激组相比, 均有明显差异.CD94/NKG2A-bright和CD94/NKG2A-dim组间亦有明显差异.结论： IFN-β通过诱导NK细胞CD94/NKG2A表达在非特异免疫系统中抑制NK细胞; 同时刺激IL-10 和TGF-β分泌进一步发挥对免疫系统的抑制.CD94/NKG2A-bright和CD94/NKG2A-dim对IFN-β反应有差异性.     

Specific engagement of the CD94/NKG2-A killer inhibitory receptor by the HLA-E class Ib molecule induces SHP-1 phosphatase recruitment to tyrosine-phosphorylated NKG2-A: evidence for receptor function in heterologous transfectants

It has been recently demonstrated that the CD94/NKG2-A killer inhibitory receptor (KIR) specifically recognizes the HLA-E class Ib molecule. Moreover, the apparent CD94-mediated specific recognition of different HLA class Ia allotypes, transfected into the HLA-defective cell line 721.221, indeed depends on their selective ability to concomitantly stabilize the surface expression of endogenous HLA-E molecules, which confer protection against CD94/NKG2-A⁺ effector cells. In the present study, we show that a selective engagement of the CD94/NKG2-A inhibitory receptor with a specific monoclonal antibody (mAb) (Z199) was sufficient to induce tyrosine phosphorylation of the NKG2-A subunit and SHP-1 recruitment. These early biochemical events, commonly related to negative signaling pathways, were also detected upon the specific interaction of NK cells with an HLA-E⁺ 721.221 transfectant (.221-AEH), and were prevented by pre-incubation of .221-AEH with an anti-HLA class I mAb. Furthermore, mAb cross-linking of the CD94/NKG2-A receptor, segregated from other NK-associated molecules by transfection into a rat basophilic leukemia cell line (RBL-2H3), promoted tyrosine phosphorylation of NKG2-A and co-precipitation of SHP-1, together with an inhibition of secretory events triggered via FcϵRI. Remarkably, interaction of CD94/NKG2-A⁺ RBL cells with the HLA-E⁺ .221-AEH transfectant specifically induced a detectable association of SHP-1 with NKG2-A, constituting a more formal evidence for the receptor-HLA class I interaction.     

HLA-E-bound peptides influence recognition by inhibitory and triggering CD94/NKG2 receptors: preferential response to an HLA-G-derived nonamer

The HLA-E class Ib molecule constitutes a major ligand for the lectin-like CD94/NKG2 natural killer (NK) cell receptors. Specific HLA class I leader sequence-derived nonapeptides bind to endogenous HLA-E molecules in the HLA-defective cell line 721.221, inducing HLA-E surface expression, and promote CD94/NKG2A-mediated recognition. We compared the ability of NK clones which expressed either inhibitory or activating CD94/NKG2 receptors to recognize HLA-E molecules on the surface of 721.221 cells loaded with a panel of synthetic nonamers derived from the leader sequences of most HLA class I molecules. Our results support the notion that the primary structure of the HLA-E-bound peptides influences CD94/NKG2-mediated recognition, beyond their ability to stabilize surface HLA-E. Further, CD94/NKG2A⁺ NK clones appeared more sensitive to the interaction with most HLA-E-peptide complexes than did effector cells expressing the activating CD94/NKG2C receptor. However, a significant exception to this pattern was HLA-E loaded with the HLA-G-derived nonamer. This complex triggered cytotoxicity very efficiently over a wide range of peptide concentrations, suggesting that the HLA-E/G-nonamer complex interacts with the CD94/NKG2 triggering receptor with a significantly higher affinity. These results raise the possibility that CD94/NKG2-mediated recognition of HLA-E expressed on extravillous cytotrophoblasts plays an important role in maternal-fetal cellular interactions.     

Thermal-exchange HLA-E multimers reveal specificity in HLA-E and NKG2A/CD94 complex interactions

There is growing interest in HLA-E-restricted T-cell responses as a possible novel, highly conserved, vaccination targets in the context of infectious and malignant diseases. The developing field of HLA multimers for the detection and study of peptide-specific T cells has allowed the in-depth study of TCR repertoires and molecular requirements for efficient antigen presentation and T-cell activation. In this study, we developed a method for efficient peptide thermal exchange on HLA-E monomers and multimers allowing the high-throughput production of HLA-E multimers. We optimized the thermal-mediated peptide exchange, and flow cytometry staining conditions for the detection of TCR and NKG2A/CD94 receptors, showing that this novel approach can be used for high-throughput identification and analysis of HLA-E-binding peptides which could be involved in T-cell and NK cell-mediated immune responses. Importantly, our analysis of NKG2A/CD94 interaction in the presence of modified peptides led to new molecular insights governing the interaction of HLA-E with this receptor. In particular, our results reveal that interactions of HLA-E with NKG2A/CD94 and the TCR involve different residues. Altogether, we present a novel HLA-E multimer technology based on thermal-mediated peptide exchange allowing us to investigate the molecular requirements for HLA-E/peptide interaction with its receptors.     

Activating CD94:NKG2C and inhibitory CD94:NKG2A receptors are expressed by distinct subsets of committed CD8+ TCR alphabeta lymphocytes

A subset of CD8(+) T cells express the natural killer cell receptors CD94:NKG2A or CD94:NKG2C. We found that although many CD8(+) T cells transcribe CD94 and NKG2C, expression of a functional CD94:NKG2C receptor is restricted to highly differentiated effector cells. CD94:NKG2A is expressed by a different subset consisting of CCR7(+) memory cells and CCR7(-) effector cells. Since NKG2A can only be induced on naive CD8(+) T cells while CD94(-) memory cells are refractory, it is likely that commitment to the CD94:NKG2A(+) subset occurs during the first encounter with antigen. CCR7(+)CD94:NKG2A(+) T cells recirculate through lymph nodes where upon activation, they produce large quantities of IFN-gamma. These cells occur as a separate CD94:NKG2A(+) T cell lineage with a distinct TCR repertoire that differs from that of the other CD8(+)CD94(-) T cells activated in situ.     

Structure of CD94 reveals a novel C-type lectin fold: implications for the NK cell-associated CD94/NKG2 receptors

The crystal structure of the extracellular domain of CD94, a component of the CD94/NKG2 NK cell receptor, has been determined to 2.6 A resolution, revealing a unique variation of the C-type lectin fold. In this variation, the second alpha helix, corresponding to residues 102-112, is replaced by a loop, the putative carbohydrate-binding site is significantly altered, and the Ca2+-binding site appears nonfunctional. This structure may serve as a prototype for other NK cell receptors such as Ly-49, NKR-P1, and CD69. The CD94 dimer observed in the crystal has an extensive hydrophobic interface that stabilizes the loop conformation of residues 102-112. The formation of this dimer reveals a putative ligand-binding region for HLA-E and suggests how NKG2 interacts with CD94.     

The role of CD94/NKG2 in innate and adaptive immunity

CD94/NKG2 is a heterodimer expressed on natural killer (NK) and a small subset of T cells. This receptor varies in function as an inhibitor or activator depending on which isoform of NKG2 is expressed. The ligand for CD94/NKG2 is HLA-E in human and its homolog, Qa1 in mouse, which are both nonclassical class I molecules that bind leader peptides from other class I molecules. Although <5% of CD8 T cells express the receptor in a naïve mouse, its expression is upregulated upon specific recognition of antigen. Similar to NK cells, most CD8 T cells that express high levels of CD94 co-express NKG2A, the inhibitory isoform. The engagement of this receptor can lead to a blocking of cytotoxicity. However, these receptors have also been implicated in the cell survival of both NK and CD8T cells. The level of CD94 expression is inversely correlated with the level of apoptosis in culture. Thus, CD94/NKG2 receptors may regulate effector functions and cell survival of NK cells and CD8 T cells, thereby playing a crucial role in the innate and adaptive immune response to a pathogen.     

HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells

Non-classical MHC class I molecule HLA-E is the ligand for CD94/NKG2 NK cell receptors. Surface expression of HLA-E requires binding of specific HLA class I leader sequences. The uterine mucosa in early pregnancy (decidua) is infiltrated by large numbers of NK cells, which are closely associated with placental trophoblast cells. In this study we demonstrate that trophoblast cells express HLA-E on their cell surface in addition to the previously reported expression of HLA-G and HLA-C. Furthermore, we show that the vast majority of decidual NK cells bind to HLA-E tetrameric complexes and this binding is inhibited by mAb to CD94. Thus, recognition of fetal HLA-E by decidual NK cells may play a key role in regulation of placentation. The functional consequences of decidual NK cell interaction were investigated in cytotoxicity assays using polyclonal decidual NK cells. The overall effect of CD94/NKG2 interaction with HLA-E is inhibition of cytotoxicity by decidual NK cells. However, since decidual NK cells are unable to kill trophoblast even in the presence of mAb to MHC class I molecules and NK cell receptors, HLA-E interaction with CD94/NKG2 receptors may regulate other functions besides cytolysis during implantation.     

The cell biology of the human natural killer cell CD94/NKG2A inhibitory receptor

To avoid destruction of normal bystander cells, natural killer (NK) cells must provide a continuous supply of functional inhibitory receptors to their cell surface. After interaction with its ligand HLA-E, which is expressed on normal cells, the C-type lectin inhibitory receptor CD94/NKG2A suppresses activation signaling processes. CD94/NKG2A receptors continuously recycle from the cell surface through endosomal compartments and back again in a process that requires energy and the cytoskeleton. This steady state process appears to be largely unaffected by exposure to ligand. CD94/NKG2A receptors move freely within the plasma membrane and accumulate at the site of contact with the ligand bearing target cells (or monoclonal antibodies (mAb) coated beads). As expected, ligated CD94/NKG2A receptors are less mobile than the nonligated receptors, and the lipid raft marker cholera toxin B is excluded from the CD94/NKG2A enriched target cell contact sites. Also, methylcyclodextrin does not interfere with CD94/NKG2A accumulation at these contact sites. The constant renewal of CD94/NKG2A receptors at the cell surface and their free mobility within the plasma membrane likely facilitates and insures inhibitory capacity.     

NKG2C zygosity influences CD94/NKG2C receptor function and the NK‐cell compartment redistribution in response to human cytomegalovirus

Human cytomegalovirus (HCMV) infection promotes a persistent expansion of a functionally competent NK‐cell subset expressing the activating CD94/NKG2C receptor. Factors underlying the wide variability of this effect observed in HCMV‐seropositive healthy individuals and exacerbated in immunocompromized patients are uncertain. A deletion of the NKG2C gene has been reported, and an apparent relation of NKG2C genotype with circulating NKG2C⁺ NK‐cell numbers was observed in HCMV⁺ children. We have assessed the influence of NKG2C gene dose on the NK‐cell repertoire in a cohort of young healthy adults (N = 130, median age 19 years). Our results revealed a relation of NKG2C copy number with surface receptor levels and with NKG2C⁺ NK‐cell numbers in HCMV⁺ subjects, independently of HLA‐E dimorphism. Functional studies showed quantitative differences in signaling (i.e. iCa²⁺ influx), degranulation, and IL‐15‐dependent proliferation, in response to NKG2C engagement, between NK cells from NKG2C^+/+ and hemizygous subjects. These observations provide a mechanistic interpretation on the way the NKG2C genotype influences steady‐state NKG2C⁺ NK‐cell numbers, further supporting an active involvement of the receptor in the HCMV‐induced reconfiguration of the NK‐cell compartment. The putative implications of NKG2C zygosity over viral control and other clinical variables deserve attention.     

The natural killer cell-mediated killing of autologous dendritic cells is confined to a cell subset expressing CD94/NKG2A,but lacking inhibitory killer Ig-like receptors

The cognate NK-DC interaction in inflamed tissues results in NK cell activation and acquisition of cytotoxicity against immature DC (iDC). This may represent a mechanism of DC selection required for the control of downstream adaptive immune responses. Here we show that killing of monocyte-derived iDC is confined to the NK cell subset that expresses CD94/NKG2A, but not killer Ig-like receptors (KIR). Consistent with these data, the expression of HLA-E (i.e. the cellular ligand of CD94/NKG2A) was down-regulated in iDC. On the other hand, HLA-B and HLA-C down-regulation in iDC was not sufficient to induce cytotoxicity in NK cells expressing KIR3DL1 or KIR2DL. Remarkably, CD94/NKG2A(+)KIR(-) NK cells were heterogeneous in their ability to kill iDC and an inverse correlation existed between their CD94/NKG2A surface density and the magnitude of their cytolytic activity. It is conceivable that the reduced CD94/NKG2A surface density enables these cells to efficiently sense the decrease of HLA-E surface expression in iDC. Finally, most NK cells that lysed iDC did not kill mature DC that express higher amounts of HLA class I molecules (including HLA-E)as compared with iDC. However, a small NK cell subset was capable of killing not only iDC but also mature DC.     

Deletion of the NKG2C receptor encoding KLRC2 gene and HLA-E variants are risk factors for severe COVID-19

PurposeHost genetic variants may contribute to severity of COVID-19. NKG2C⁺ NK cells are potent antiviral effector cells, potentially limiting the extent of SARS-CoV-2 infections. NKG2C is an activating NK cell receptor encoded by the KLRC2 gene, which binds to HLA-E on infected cells leading to NK cell activation. Heterozygous or homozygous KLRC2 deletion (KLRC2^del) may naturally occur and is associated with a significantly lower or absent NKG2C expression level. In addition, HLA-E*0101/0103 genetic variants occur, caused by a single-nucleotide polymorphism. We therefore investigated whether the severity of COVID-19 is associated with these genetic variants.MethodsWe investigated the distribution of KLRC2 deletion and HLA-E*0101/0103 allelic variants in a study cohort of 361 patients with either mild (N = 92) or severe (N = 269) COVID-19.ResultsEspecially the KLRC2^del, and at a lower degree the HLA-E*0101, allele were significantly overrepresented in hospitalized patients (p = 0.0006 and p = 0.01), particularly in patients requiring intensive care (p < 0.0001 and p = 0.01), compared with patients with mild symptoms. Both genetic variants were independent risk factors for severe COVID-19.ConclusionOur data show that these genetic variants in the NKG2C/HLA-E axis have a significant impact on the development of severe SARS-CoV-2 infections, and may help to identify patients at high-risk for severe COVID-19.