Structure of the murine CD94-NKG2A receptor in complex with Qa-1b presenting an MHC-I leader peptide.

The heterodimeric natural killer cells antigen CD94 (CD94)-NKG2-A/NKG2-B type II integral membrane protein (NKG2A) receptor family expressed on human and mouse natural killer (NK) cells monitors global major histocompatibility complex (MHC) class I cell surface expression levels through binding to MHC class Ia-derived leader sequence peptides presented by HLA class I histocompatibility antigen, alpha chain E (HLA-E; in humans) or H-2 class I histocompatibility antigen, D-37 (Qa-1b; in mice). Although the molecular basis underpinning human CD94-NKG2A recognition of HLA-E is known, the equivalent interaction in the murine setting is not. By determining the high-resolution crystal structure of murine CD94-NKG2A in complex with Qa-1b presenting the Qa-1 determinant modifier peptide (QDM), we resolved the mode of binding. Compared to the human homologue, the murine CD94-NKG2A-Qa-1b-QDM displayed alterations in the distribution of interactions across CD94 and NKG2A subunits that coincide with differences in electrostatic complementarity of the ternary complex and the lack of cross-species reactivity. Nevertheless, we show that Qa-1b could be modified through W65R + N73I mutations to mimic HLA-E, facilitating binding with both human and murine CD94-NKG2A. These data underscore human and murine CD94-NKG2A cross-species heterogeneity and provide a foundation for humanising Qa-1b in immune system models.

Transcriptomic Remodelling of Fetal Endothelial Cells During Establishment of Inflammatory Memory.

Inflammatory memory involves the molecular and cellular 'reprogramming' of innate immune cells following exogenous stimuli, leading to non-specific protection against subsequent pathogen exposure. This phenomenon has now also been described in non-hematopoietic cells, such as human fetal and adult endothelial cells. In this study we mapped the cell-specific DNA methylation profile and the transcriptomic remodelling during the establishment of inflammatory memory in two distinct fetal endothelial cell types - a progenitor cell (ECFC) and a differentiated cell (HUVEC) population. We show that both cell types have a core transcriptional response to an initial exposure to a viral-like ligand, Poly(I:C), characterised by interferon responsive genes. There was also an ECFC specific response, marked by the transcription factor ELF1, suggesting a non-canonical viral response pathway in progenitor endothelial cells. Next, we show that both ECFCs and HUVECs establish memory in response to an initial viral exposure, resulting in an altered subsequent response to lipopolysaccharide. While the capacity to train or tolerize the induction of specific sets of genes was similar between the two cell types, the progenitor ECFCs show a higher capacity to establish memory. Among tolerized cellular pathways are those involved in endothelial barrier establishment and leukocyte migration, both important for regulating systemic immune-endothelial cell interactions. These findings suggest that the capacity for inflammatory memory may be a common trait across different endothelial cell types but also indicate that the specific downstream targets may vary by developmental stage.

Features of HLA class I expression and its clinical relevance in SARS-CoV-2: What do we know so far?

Modifications in HLA-I expression are found in many viral diseases. They represent one of the immune evasion strategies most widely used by viruses to block antigen presentation and NK cell response, and SARS-CoV-2 is no exception. These alterations result from a combination of virus-specific factors, genetically encoded mechanisms, and the status of host defences and range from loss or upregulation of HLA-I molecules to selective increases of HLA-I alleles. In this review, I will first analyse characteristic features of altered HLA-I expression found in SARS-CoV-2. I will then discuss the potential factors underlying these defects, focussing on HLA-E and class-I-related (like) molecules and their receptors, the most documented HLA-I alterations. I will also draw attention to potential differences between cells transfected to express viral proteins and those presented as part of authentic infection. Consideration of these factors and others affecting HLA-I expression may provide us with improved possibilities for research into cellular immunity against viral variants.

Viral infection modulates Qa-1b in infected and bystander cells to properly direct NK cell killing.

Natural killer (NK) cell activation depends on the signaling balance of activating and inhibitory receptors. CD94 forms inhibitory receptors with NKG2A and activating receptors with NKG2E or NKG2C. We previously demonstrated that CD94-NKG2 on NK cells and its ligand Qa-1b are important for the resistance of C57BL/6 mice to lethal ectromelia virus (ECTV) infection. We now show that NKG2C or NKG2E deficiency does not increase susceptibility to lethal ECTV infection, but overexpression of Qa-1b in infected cells does. We also demonstrate that Qa-1b is down-regulated in infected and up-regulated in bystander inflammatory monocytes and B cells. Moreover, NK cells activated by ECTV infection kill Qa-1b-deficient cells in vitro and in vivo. Thus, during viral infection, recognition of Qa-1b by activating CD94/NKG2 receptors is not critical. Instead, the levels of Qa-1b expression are down-regulated in infected cells but increased in some bystander immune cells to respectively promote or inhibit their killing by activated NK cells.

Host genetic control of natural killer cell diversity revealed in the Collaborative Cross.

Natural killer (NK) cells are innate effectors armed with cytotoxic and cytokine-secreting capacities whose spontaneous antitumor activity is key to numerous immunotherapeutic strategies. However, current mouse models fail to mirror the extensive immune system variation that exists in the human population which may impact on NK cell-based therapies. We performed a comprehensive profiling of NK cells in the Collaborative Cross (CC), a collection of novel recombinant inbred mouse strains whose genetic diversity matches that of humans, thereby providing a unique and highly diverse small animal model for the study of immune variation. We demonstrate that NK cells from CC strains displayed a breadth of phenotypic and functional variation reminiscent of that reported for humans with regards to cell numbers, key marker expression, and functional capacities. We took advantage of the vast genetic diversity of the CC and identified nine genomic loci through quantitative trait locus mapping driving these phenotypic variations. SNP haplotype patterns and variant effect analyses identified candidate genes associated with lung NK cell numbers, frequencies of CD94+ NK cells, and expression levels of NKp46. Thus, we demonstrate that the CC represents an outstanding resource to study NK cell diversity and its regulation by host genetics.

Identification of human cytotoxic ILC3s.

Human ILCs are classically categorized into five subsets; cytotoxic CD127- CD94+ NK cells and non-cytotoxic CD127+ CD94- , ILC1s, ILC2s, ILC3s, and LTi cells. Here, we identify a previously unrecognized subset within the CD127+ ILC population, characterized by the expression of the cytotoxic marker CD94. These CD94+ ILCs resemble conventional ILC3s in terms of phenotype, transcriptome, and cytokine production, but are highly cytotoxic. IL-15 was unable to induce differentiation of CD94+ ILCs toward mature NK cells. Instead, CD94+ ILCs retained RORγt, CD127 and CD200R1 expression and produced IL-22 in response to IL-15. Culturing non-cytotoxic ILC3s with IL-12 induced upregulation of CD94 and cytotoxic activity, effects that were not observed with IL-15 stimulation. Thus, human helper ILCs can acquire a cytotoxic program without differentiating into NK cells.

NKG2A/CD94 Is a New Immune Receptor for HLA-G and Distinguishes Amino Acid Differences in the HLA-G Heavy Chain.

Natural killer (NK) cell therapies are a tool to antagonize a dysfunctional immune system. NK cells recognize malignant cells, traffic to a tumor location, and infiltrate the solid tumor. The immune checkpoint molecule human leukocyte antigen (HLA)-G is upregulated on malignant cells but not on healthy surrounding cells, the requirement of understanding the basis of receptor mediated events at the HLA-G/NK cell interface becomes obvious. The NK cell receptors ILT2 and KIR2DL4 have been described to bind to HLA-G; however, their differential function and expression levels on NK cell subsets suggest the existence of an unreported receptor. Here, we performed a ligand-based receptor capture on living cells utilizing sHLA-G*01:01 molecules coupled to TriCEPS and bound to NK cells followed by mass spectrometric analyses. We could define NKG2A/CD94 as a cognate receptor of HLA-G. To verify the results, we used the reciprocal method by expressing recombinant soluble heterodimeric NKG2A/CD94 molecules and used them to target HLA-G*01:01 expressing cells. NKG2A/CD94 could be confirmed as an immune receptor of HLA-G*01:01. Despite HLA-G is marginal polymorphic, we could previously demonstrate that the most common allelic subtypes HLA-G*01:01/01:03 and 01:04 differ in peptide repertoire, their engagement to NK cells, their catalyzation of dNK cell proliferation and their impact on NK cell development. Continuing these studies with regard to NKG2A/CD94 engagement we engineered recombinant single antigen presenting K562 cells and targeted the surface expressed HLA-G*01:01, 01:03 or 01:04 molecules with NKG2A/CD94. Specificity and sensitivity of HLA-G*01:04/NKG2A/CD94 engagement could be significantly verified. The binding affinity decreases when using K562-G*01:03 or K562-G*01:01 cells as targets. These results demonstrate that the ligand-receptor assignment between HLA-G and NKG2A/CD94 is dependent of the amino acid composition in the HLA-G heavy chain. Understanding the biophysical basis of receptor-mediated events that lead to NK cell inhibition would help to remove non-tumor reactive cells and support personalized mild autologous NK cell therapies.

CD93 hematopoietic stem cells improve diabetic wound healing by VEGF activation and downregulation of DAPK-1.

Diabetes is associated with numerous complications, such as diabetic skin wounds or ulcerations. The aim of this study was to evaluate experimentally the effectiveness of applying polycaprolactone (PCL)-gelatin scaffold, with or without rat CD93 hematopoietic stem cells (HSCs), in diabetic wound healing in a rat model. CD93 HSCs were aseptically isolated from rat bone marrow using fluorescent activated cell sorting (FACS) method and FACS-SORTER. A total of 25 Wistar rats were divided into five groups including Group I (sham, nondiabetic, and wound covered only with sterile dressing), II (control, diabetic rat), III (CD93 HSCs alone), IV (PCL-gelatin scaffold), and V (CD93 HSCs+PCL-gelatin scaffold). Animals were killed on Days 7, 14, or 28 posttreatment and histological sections were blindly evaluated by two expert pathologists. Death-associated protein kinase 1 (DAPK-1) gene and vesicular endothelial growth factors (VEGF) protein expression were evaluated using reverse transcription-polymerase chain reaction and western blot, respectively. The thickest and the thinnest epidermises microscopically were belonged to CD93+HSCs+scaffold and the control group, respectively. The growth rate of the epidermis and adnexal epithelia was the highest in both the cell and cell+scaffold groups. Evaluation of the protein expression level of VEGF indicated that the expression levels of this growth factor were the most on Day 7 posttreatment in sham, HSCs alone, and HSCs cell+scaffold groups. While the lowest expression levels of this growth factor was detected in the control and scaffold groups. The gene expression level of DAPK-1 on Day 7 posttreatment was higher than that of the Day 14 posttreatment in all groups. The highest and lowest gene expression levels of DAPK-1 belonged to control and sham groups, respectively. According to our findings, CD93 HSCs offer new prospects for the treatment of diabetic ulcers and concomitant application of these cells with PCL-gelatin nanofiber scaffold significantly improves diabetic wound treatment.

Evidence of functional Cd94 polymorphism in a free-living house mouse population.

The CD94 receptor, expressed on natural killer (NK) and CD8+ T cells, is known as a relatively non-polymorphic receptor with orthologues in humans, other primates, cattle, and rodents. In the house mouse (Mus musculus), a single allele is highly conserved among laboratory strains, and reports of allelic variation in lab- or wild-living mice are lacking, except for deficiency in one lab strain (DBA/2J). The non-classical MHC-I molecule Qa-1b is the ligand for mouse CD94/NKG2A, presenting alternative non-americ fragment of leader peptides (Qa-1 determinant modifier (Qdm)) from classical MHC-I molecules. Here, we report a novel allele identified in free-living house mice captured in Norway, living among individuals carrying the canonical Cd94 allele. The novel Cd94LocA allele encodes 12 amino acid substitutions in the extracellular lectin-like domain. Flow cytometric analysis of primary NK cells and transfected cells indicates that the substitutions prevent binding of CD94 mAb and Qa-1b/Qdm tetramers. Our data further indicate correlation of Cd94 polymorphism with the two major subspecies of house mice in Europe. Together, these findings suggest that the Cd94LocA/NKG2A heterodimeric receptor is widely expressed among M. musculus subspecies musculus, with ligand-binding properties different from mice of subspecies domesticus, such as the C57BL/6 strain.

A herpesvirus encoded Qa-1 mimic inhibits natural killer cell cytotoxicity through CD94/NKG2A receptor engagement.

A recurrent theme in viral immune evasion is the sabotage of MHC-I antigen presentation, which brings virus the concomitant issue of 'missing-self' recognition by NK cells that use inhibitory receptors to detect surface MHC-I proteins. Here, we report that rodent herpesvirus Peru (RHVP) encodes a Qa-1 like protein (pQa-1) via RNA splicing to counteract NK activation. While pQa-1 surface expression is stabilized by the same canonical peptides presented by murine Qa-1, pQa-1 is GPI-anchored and resistant to the activity of RHVP pK3, a ubiquitin ligase that targets MHC-I for degradation. pQa-1 tetramer staining indicates that it recognizes CD94/NKG2A receptors. Consistently, pQa-1 selectively inhibits NKG2A+ NK cells and expression of pQa-1 can protect tumor cells from NK control in vivo. Collectively, these findings reveal an innovative NK evasion strategy wherein RHVP encodes a modified Qa-1 mimic refractory to MHC-I sabotage and capable of specifically engaging inhibitory receptors to circumvent NK activation.

Effect of the in vivo application of granulocyte colony-stimulating factor on NK cells in bone marrow and peripheral blood.

Granulocyte colony-stimulating factor (G-CSF) has been widely used in the field of allogeneic haematopoietic stem cell transplantation (allo-HSCT) for priming donor stem cells from the bone marrow (BM) to peripheral blood (PB) to collect stem cells more conveniently. Donor-derived natural killer (NK) cells have important antitumour functions and immune regulatory roles post-allo-HSCT. The aim of this study was to evaluate the effect of G-CSF on donors' NK cells in BM and PB. The percentage of NK cells among nuclear cells and lymphocyte was significantly decreased and led to increased ratio of T and NK cells in BM and PB post-G-CSF in vivo application. Relative expansion of CD56bri NK cells led to a decreased ratio of CD56dim and CD56bri NK subsets in BM and PB post-G-CSF in vivo application. The expression of CD62L, CD54, CD94, NKP30 and CXCR4 on NK cells was significantly increased in PB after G-CSF treatment. G-CSF treatment decreased the IFN-γ-secreting NK population (NK1) dramatically in BM and PB, but increased the IL-13-secreting NK (NK2), TGF-ß-secreting NK (NK3) and IL-10-secreting NK (NKr) populations significantly in BM. Clinical data demonstrated that higher doses of NK1 infused into the allograft correlated with an increased incidence of chronic graft-vs-host disease post-transplantation. Taken together, our results show that the in vivo application of G-CSF can modulate NK subpopulations, leading to an increased ratio of T and NK cells and decreased ratio of CD56dim and CD56bri NK cells as well as decreased NK1 populations in both PB and BM.

A 3'UTR polymorphism marks differential KLRG1 mRNA levels through disruption of a miR-584-5p binding site and associates with pemphigus foliaceus susceptibility.

Genetic variations mapping to 3' untranslated regions (3'UTRs) may overlap with microRNA (miRNA) binding sites, therefore potentially interfering with translation inhibition or messenger RNA (mRNA) degradation. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) located within the 3'UTRs of six candidate genes and predicted to interfere with miRNA ligation could account for disease-relevant differential mRNA levels. Focusing on pemphigus foliaceus (PF) - an autoimmune blistering skin condition with unique endemic patterns - we investigated whether nine 3'UTR SNPs from the CD1D, CTLA4, KLRD1, KLRG1, NKG7, and TNFSF13B genes differentially expressed in PF were disease-associated. The heterozygous genotype of the KLRG1 rs1805672 polymorphism was associated with increased predisposition to PF (A/G vs. A/A: P=0.038; OR=1.60), and a trend for augmented susceptibility was observed for carriers of the G allele (P=0.094; OR=1.44). In silico analyses suggested that rs1805672 G allele could disrupt binding of miR-584-5p, and indicated rs1805672 as an expression Quantitative Trait Locus (eQTL), with an effect on KLRG1 gene expression. Dual-luciferase assay showed that miR-584-5p mediated approximately 50% downregulation of the reporter gene's activity through the 3'UTR of KLRG1 harboring rs1805672 A allele (vs. miRNA-negative condition, P=0.006). This silencing relationship was lost after site-directed mutation to G allele (vs. miRNA-negative condition, P=0.391; vs. rs1805672 A allele, P=0.005). Collectively, these results suggest that a disease-associated SNP located within the 3'UTR of KLRG1 directly interferes with miR-584-5p binding, allowing for KLRG1 mRNA differential accumulation, which in turn may contribute to pathogenesis of autoimmune diseases, such as pemphigus.

Spontaneous and natural cytotoxicity receptor-mediated cytotoxicity are effector functions of distinct natural killer subsets in hepatitis C virus-infected chimpanzees.

In humans, CD16 and CD56 are used to identify functionally distinct natural killer (NK) subsets. Due to ubiquitous CD56 expression, this marker cannot be used to distinguish between NK cell subsets in chimpanzees. Therefore, functional analysis of distinct NK subsets during hepatitis C virus (HCV) infection has never been performed in these animals. In the present study an alternative strategy was used to identify four distinct NK subsets on the basis of the expression of CD16 and CD94. The expression of activating and inhibiting surface receptors showed that these subsets resemble human NK subsets. CD107 expression was used to determine degranulation of the different subsets in naive and HCV-infected chimpanzees. In HCV-infected chimpanzees increased spontaneous cytotoxicity was observed in CD94(high/dim) CD16(pos) and CD94(low) CD16(pos) subsets. By contrast, increased natural cytotoxicity receptor (NCR)- mediated degranulation after NKp30 and NKp44 triggering was demonstrated in the CD94(dim) CD16(neg) subset. Our findings suggest that spontaneous and NCR-mediated cytotoxicity are effector functions of distinct NK subsets in HCV-infected chimpanzees.

Influence of CD94 and NKG2A variants on susceptibility to rheumatoid arthritis and efficacy of anti-TNF treatment.

OBJECTIVES: The present study aimed to investigate relationships between the CD94 and NKG2A gene polymorphisms and the risk of rheumatoid arthritis (RA) development as well as their association with the response to anti-TNF therapy. METHODS: A total of 284 patients with RA receiving anti-TNF therapy and 124 healthy subjects were enrolled to the study. Genotypings for CD94 (rs2302489) and NKG2A (rs7301582, rs2734440, rs2734414) polymorphisms were performed using a polymerase chain reaction (PCR) amplification employing LightSNiP assays (TIB-MolBiol, Berlin, Germany). Clinical response was evaluated at 12th and 24th week after initiation of the therapy according to the EULAR response criteria. RESULTS: The frequency of the CD94 AA genotype was significantly decreased in RA patients compared to controls (OR=0.44; P=0.016). The CD94 AA homozygotes were also more common among patients negative to anti-cyclic citrullinated peptide (anti-CCP) antibodies (OR=11.28; P=0.001) as compared to anti-CCP-positive patients and the presence of the CD94 allele A was associated with lack of anti-CCP antibodies (OR=5.00; P=0.0005). The CD94 rs2302489 TT genotype was over-represented in patients exhibiting worse EULAR response at 12th week (OR=3.33; P=0.017). Furthermore, the lack of response after 12 weeks was more frequent among patients carrying the NKG2A rs7301582C allele (OR=3.68; P=0.019) or the CC genotype (OR=3.58; P=0.035) in comparison to allele T or CT/TT genotypes, respectively. CONCLUSIONS: These results indicate that CD94 and NKG2A polymorphisms may contribute to genetic susceptibility to RA or affect the response to anti-TNF therapy in patients of Caucasian origin.

The Presence of HLA-E-Restricted, CMV-Specific CD8+ T Cells in the Blood of Lung Transplant Recipients Correlates with Chronic Allograft Rejection.

The human cytomegalovirus (CMV) immune evasion protein, UL40, shares an identical peptide sequence with that found in the leader sequence of many human leukocyte antigen (HLA)-C alleles and when complexed with HLA-E, can modulate NK cell functions via interactions with the CD94-NKG2 receptors. However the UL40-derived sequence can also be immunogenic, eliciting robust CD8+ T cell responses. In the setting of solid organ transplantation these T cells may not only be involved in antiviral immunity but also can potentially contribute to allograft rejection when the UL40 epitope is also present in allograft-encoded HLA. Here we assessed 15 bilateral lung transplant recipients for the presence of HLA-E-restricted UL40 specific T cells by tetramer staining of peripheral blood mononuclear cells (PBMC). UL40-specific T cells were observed in 7 patients post-transplant however the magnitude of the response varied significantly between patients. Moreover, unlike healthy CMV seropositive individuals, longitudinal analyses revealed that proportions of such T cells fluctuated markedly. Nine patients experienced low-grade acute cellular rejection, of which 6 also demonstrated UL40-specific T cells. Furthermore, the presence of UL40-specific CD8+ T cells in the blood was significantly associated with allograft dysfunction, which manifested as Bronchiolitis Obliterans Syndrome (BOS). Therefore, this study suggests that minor histocompatibility antigens presented by HLA-E can represent an additional risk factor following lung transplantation.

Polymorphisms in inflammatory genes are associated with term small for gestational age and preeclampsia.

PROBLEM: Inflammatory biomarkers are associated with preeclampsia (PE) and poor fetal growth; however, genetic epidemiologic studies have been limited by reduced gene coverage and the exclusion of African American mothers. METHOD OF STUDY: Cases and controls (N = 1646) from a pregnancy cohort were genotyped for 503 tagSNPs in 40 genes related to inflammation. Gene-set analyses were stratified by race and were followed by a single SNP analysis within significant gene sets. RESULTS: Gene-level associations were found for IL6 and KLRD1 for term small for gestational age (SGA) among African Americans. LTA/TNF and TBX21 were associated with PE among European Americans. The strongest association was for PE among European Americans for an upstream regulator of TNF with RR = 1.8 (95% CI 1.1-2.7). CONCLUSION: Although previous studies have suggested null associations, increased tagging and stratification by genetic ancestry suggests important associations between IL6 and term SGA for African Americans, and a TNF regulator and PE among European Americans (N = 149).

Adaptive reconfiguration of the human NK-cell compartment in response to cytomegalovirus: a different perspective of the host-pathogen interaction.

As discussed in this review, human cytomegalovirus (HCMV) infection in healthy individuals is associated with a variable and persistent increase of NK cells expressing the CD94/NKG2C activating receptor. The expansion of NKG2C(+) NK cells reported in other infectious diseases is systematically associated with HCMV co-infection. The functionally mature NKG2C(bright) NK-cell subset expanding in HCMV(+) individuals displays inhibitory Ig-like receptors (KIR and LILRB1) specific for self HLA class I, and low levels of NKp46 and NKp30 activating receptors. Such reconfiguration of the NK-cell compartment appears particularly marked in immunocompromised patients and in children with symptomatic congenital infection, thus suggesting that its magnitude may be inversely related with the efficiency of the T-cell-mediated response. This effect of HCMV infection is reminiscent of the pattern of response of murine Ly49H(+) NK cells against murine CMV (MCMV), and it has been hypothesized that a cognate interaction of the CD94/NKG2C receptor with HCMV-infected cells may drive the expansion of the corresponding NK-cell subset. Yet, the precise role of NKG2C(+) cells in the control of HCMV infection, the molecular mechanisms underlying the NK-cell compartment redistribution, as well as its putative influence in the response to other pathogens and tumors remain open issues.

Impaired natural killer cell self-education and "missing-self" responses in Ly49-deficient mice.

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.

A structural basis for antigen presentation by the MHC class Ib molecule, Qa-1b.

The primary function of the monomorphic MHC class Ib molecule Qa-1(b) is to present peptides derived from the leader sequences of other MHC class I molecules for recognition by the CD94-NKG2 receptors expressed by NK and T cells. Whereas the mode of peptide presentation by its ortholog HLA-E, and subsequent recognition by CD94-NKG2A, is known, the molecular basis of Qa-1(b) function is unclear. We have assessed the interaction between Qa-1(b) and CD94-NKG2A and shown that they interact with an affinity of 17 µM. Furthermore, we have determined the structure of Qa-1(b) bound to the leader sequence peptide, Qdm (AMAPRTLLL), to a resolution of 1.9 Å and compared it with that of HLA-E. The crystal structure provided a basis for understanding the restricted peptide repertoire of Qa-1(b). Whereas the Qa-1(b-AMAPRTLLL) complex was similar to that of HLA-E, significant sequence and structural differences were observed between the respective Ag-binding clefts. However, the conformation of the Qdm peptide bound by Qa-1(b) was very similar to that of peptide bound to HLA-E. Although a number of conserved innate receptors can recognize heterologous ligands from other species, the structural differences between Qa-1(b) and HLA-E manifested in CD94-NKG2A ligand recognition being species specific despite similarities in peptide sequence and conformation. Collectively, our data illustrate the structural homology between Qa-1(b) and HLA-E and provide a structural basis for understanding peptide repertoire selection and the specificity of the interaction of Qa-1(b) with CD94-NKG2 receptors.

Rat and mouse CD94 associate directly with the activating transmembrane adaptor proteins DAP12 and DAP10 and activate NK cell cytotoxicity.

Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family.