KIR2DS1 and KIR2DL1-C245 Dominantly Repress NK Cell Degranulation Triggered by Monoclonal or Bispecific Antibodies, whereas Education by Uptuning Inhibitory Killer Ig-related Receptors Exerts No Advantage in Ab-dependent Cellular Cytotoxicity.

NK cell responsiveness to target cells is tuned by interactions between inhibitory NK cell receptors and their cognate HLA class I ligands in a process termed "NK cell education." Previous studies addressing the role for NK cell education in Ab-dependent cellular cytotoxicity (ADCC) show ambiguous results and do not encompass full educational resolution. In this study, we systematically characterized human NK cell CD16-triggered degranulation toward defined human tumor cell lines in the presence of either the mAb rituximab or a recently developed CD34xCD16 bispecific killer engager. Despite positive correlation between killer Ig-related receptor (KIR)-mediated education and CD16 expression, NK cells educated by one or even two inhibitory KIRs did not perform better in terms of ADCC than uneducated NK cells in either missing-self or KIR-ligand matched settings at saturating Ab concentrations. Instead, NKG2A+ NK cells consistently showed more potent ADCC in the missing-self context despite lower levels of CD16 expression. KIR2DS1+ NK cells demonstrated dampened ADCC in both the missing-self and KIR-ligand matched settings, even in the presence of its ligand HLA C2. The lower response by KIR2DS1+ NK cells was also observed when stimulated with a bispecific killer engager. Surprisingly, repression of ADCC was also observed by NKG2A+ NK cells coexpressing the inhibitory KIR2DL1-C245 receptor that confers weak education. In conclusion, our study suggests that NK cell education by inhibitory KIRs does not augment ADCC per se, whereas expression of KIR2DS1 and KIR2DL1-C245 dominantly represses ADCC. These insights add to the fundamental understanding of NK cells and may have implications for their therapeutic use.

The KIR2DL1 intermediate upstream element participates in gene activation.

The human KIR genes encode a family of class I MHC receptors that are expressed on subsets of NK cells. The expression of KIR proteins is controlled by a stochastic process, and competition between sense and antisense promoter elements has been suggested to program the variegated expression of these genes. Previous studies have demonstrated distinct roles of distal, intermediate, and proximal sense promoter/enhancer elements in gene activation and expression. Conversely, proximal and intronic antisense promoter transcripts have been associated with gene silencing at different stages of NK cell development. In the current study, we examine the effect of intermediate promoter deletion on KIR2DL1 expression in the YTS cell line. Homozygous deletion of the KIR2DL1 intermediate element did not affect proximal promoter activity but resulted in increased detection of upstream transcripts. No significant changes in alternative mRNA splicing or expression levels of KIR2DL1 protein were observed. However, intermediate element deletion was associated with a reduced frequency of gene activation by 5-azacytidine. Taken together, these results indicate that the intermediate element is not an enhancer required for KIR expression; however, it is required for the efficient activation of the gene.

The combinatorial diversity of KIR and HLA class I allotypes in Peninsular Malaysia.

Killer cell immunoglobulin-like receptors (KIRs) interact with polymorphic human leucocyte antigen (HLA) class I molecules, modulating natural killer (NK) cell functions and affecting both the susceptibility and outcome of immune-mediated diseases. The KIR locus is highly diverse in gene content, copy number and allelic polymorphism within individuals and across geographical populations. To analyse currently under-represented Asian and Pacific populations, we investigated the combinatorial diversity of KIR and HLA class I in 92 unrelated Malay and 75 Malaysian Chinese individuals from the Malay Peninsula. We identified substantial allelic and structural diversity of the KIR locus in both populations and characterized novel variations at each analysis level. The Malay population is more diverse than Malay Chinese, likely representing a unique history including admixture with immigrating populations spanning several thousand years. Characterizing the Malay population are KIR haplotypes with large structural variants present in 10% individuals, and KIR and HLA alleles previously identified in Austronesian populations. Despite the differences in ancestries, the proportion of HLA allotypes that serve as KIR ligands is similar in each population. The exception is a significantly reduced frequency of interactions of KIR2DL1 with C2+ HLA-C in the Malaysian Chinese group, caused by the low frequency of C2+ HLA. One likely implication is a greater protection from preeclampsia, a pregnancy disorder associated with KIR2DL1, which shows higher incidence in the Malay than in the Malaysian Chinese. This first complete, high-resolution, characterization of combinatorial diversity of KIR and HLA in Malaysians will form a valuable reference for future clinical and population studies.

Influence of HLA-C environment on the spontaneous clearance of hepatitis C in European HIV-HCV co-infected individuals.

Natural killer (NK) cell functions are regulated by diverse inhibitory and activating receptors, including killer cell immunoglobulin-like receptors (KIR), which interact with human leukocyte antigen (HLA) class I molecules. Some KIR/HLA genetic combinations were reported associated with spontaneous clearance (SC) of hepatitis C virus (HCV) but with discordant results, possibly reflecting KIR and/or HLA gene polymorphism according to populations. KIR/HLA genetic combinations associated with both an exhaustive NK and T cell repertoire were investigated in a cohort of HIV-HCV co-infected individuals with either SC (n = 68) or chronic infection (CI, n = 163) compared to uninfected blood donors [controls (Ctrl), n = 100]. Multivariate analysis showed that the HLA C2C2 environment was associated with SC only in European HIV-HCV co-infected individuals [odds ratio (OR) = 4·30, 95% confidence interval = 1·57-12·25, P = 0·005]. KIR2D+ NK cell repertoire and potential of degranulation of KIR2DL1/S1+ NK cells were similar in the SC European cohort compared to uninfected individuals. In contrast, decreased frequencies of KIR2DS1+ and KIR2DL2+ NK cells were detected in the CI group of Europeans compared to SC and a decreased frequency of KIR2DL1/S1+ NK cells compared to controls. Regarding T cells, higher frequencies of DNAX accessory molecule-1 (DNAM-1)+ and CD57+ T cells were observed in SC in comparison to controls. Interestingly, SC subjects emphasized increased frequencies of KIR2DL2/L3/S2+ T cells compared to CI subjects. Our study underlines that the C2 environment may activate efficient KIR2DL1+ NK cells in a viral context and maintain a KIR2DL2/L3/S2+ mature T cell response in the absence of KIR2DL2 engagement with its cognate ligands in SC group of HCV-HIV co-infected European patients.

KIR Polymorphism Modulates the Size of the Adaptive NK Cell Pool in Human Cytomegalovirus-Infected Individuals.

Acute infection with human CMV (HCMV) induces the development of adaptive NKG2C+ NK cells. In some cases, large expansions of this subset, characterized by coexpression of HLA-C-specific KIR, are stably maintained during the life-long latent phase of infection. The factors that control these unusual expansions in vivo are currently unknown. In this study, the role of KIR polymorphism and expression in this process was analyzed. It is shown that strong NKG2C+ NK cell expansions are dominated by single KIR clones, whereas moderate expansions are frequently polyclonal (p < 0.0001). Importantly, the choice of KIR was not arbitrary but biased toward usage of HLA-C-specific KIR encoded by the centromeric part of group A (cenA) haplotypes. Consideration of KIR allelic variation and gene copy number revealed that the cenA effect was predominantly due to the HLA-C2-specific KIR2DL1 receptor; presence of KIR2DL1 on NKG2C+ NK cells led to significantly larger clonal expansions than the cenB-encoded KIR2DL2 (p = 0.002). Expansion of NKG2C+KIR2DL1+ NK cells was always accompanied by the cognate ligand HLA-C2. Moreover, in these donors the frequency of NKG2C+ NK cells correlated with the concentration of anti-HCMV IgG (r = 0.62, p = 0.008), suggesting direct relevance of NKG2C+KIR2DL1+ NK cells for virus control. Altogether, the study suggests that the homeostasis of NKG2C+ NK cells in HCMV infection is at least partly controlled by coexpression of cognate inhibitory KIR. In particular, the strong interaction of KIR2DL1 and HLA-C2 ligands seems to promote large and stable expansion of adaptive NK cells in HCMV infection.

NK Response Correlates with HIV Decrease in Pegylated IFN-α2a-Treated Antiretroviral Therapy-Suppressed Subjects.

We previously reported that pegylated IFN-α2a (Peg-IFN-α2a) added to antiretroviral therapy (ART)-suppressed, HIV-infected subjects resulted in plasma HIV control and integrated HIV DNA decrease. We now evaluated whether innate NK cell activity or PBMC transcriptional profiles were associated with decreases in HIV measures. Human peripheral blood was analyzed prior to Peg-IFN-α2a administration (ART, baseline), after 5 wk of ART+Peg-IFN-α2a, and after 12 wk of Peg-IFN-α2a monotherapy (primary endpoint). After 5 wk of ART+Peg-IFN-α2a, immune subset frequencies were preserved, and induction of IFN-stimulated genes was noted in all subjects except for a subset in which the lack of IFN-stimulated gene induction was associated with increased expression of microRNAs. Viral control during Peg-IFN-α2a monotherapy was associated with 1) higher levels of NK cell activity and IFN-γ-induced protein 10 (IP-10) on ART (preimmunotherapy) and 2) downmodulation of NK cell KIR2DL1 and KIR2DL2/DL3 expression, transcriptional enrichment of expression of genes associated with NK cells in HIV controller subjects, and higher ex vivo IFN-α-induced NK cytotoxicity after 5 wk of ART+Peg-IFN-α2a. Integrated HIV DNA decline after immunotherapy was also associated with gene expression patterns indicative of cell-mediated activation and NK cytotoxicity. Overall, an increase in innate activity and NK cell cytotoxicity were identified as correlates of Peg-IFN-α2a-mediated HIV control.

Tuning of human NK cells by endogenous HLA-C expression.

NK cells are primarily responsible for detecting malignant or pathogen-infected cells, and their function is influenced both by stress-associated activating signals and opposing inhibitory signals from receptors that recognize self MHC. The receptors that produce this inhibitory signal shift from the NKG2A:HLA-E system to that of KIR:HLA as the NK cells mature. This maturation is associated with an increase in lytic activity, as well as an increase in HLA-C protein levels controlled by the NK-specific HLA-C promoter, NK-Pro. We propose that modulation of the translatability of HLA-C transcripts in NK cells constitutes an evolutionary mechanism to control cis inhibitory signaling by HLA-C, which fine tunes NK cell activity. Furthermore, the high degree of variability in KIR receptor affinity for HLA alleles, as well as the variable expression levels of both KIR and HLA, suggest an evolutionary requirement for the tuning of NK lytic activity. Various data have demonstrated that mature NK cells may gain or lose lytic activity when placed in different environments. This indicates that NK cell activity may be more a function of constant tuning by inhibitory signals, rather than a static, irreversible "license to kill" granted to mature NK cells. Inhibitory signaling controls the filling of the cytolytic granule reservoir, which becomes depleted if there are insufficient inhibitory signals, leading to a hyporesponsive NK cell. We propose a novel model for the tuning of human NK cell activity via cis interactions in the context of recent findings on the mechanism of NK education.

Study of KIR gene expression at the mRNA level in specific donor-derived NK cells after allogeneic HSCT.

The function of natural killer (NK) cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is regulated by the balance between inhibitory KIRs (iKIRs) and activating KIRs (aKIRs). However, few studies have examined the subsequent expression of KIR genes unique to the donor. We defined the set of KIR genes expressed only in the donor and designed a method for measuring the expression of these KIR genes by quantitative real-time polymerase chain reaction (RT-qPCR) based on genetic cloning techniques. In this study, we evaluated the recovery pattern of KIR genes in 252 donor-recipient pairs. The expression of each KIR unique to the donor was in line with that of KIR genes shared by the donor and recipient, such as KIR2DS1, KIR3DS1, KIR2DS4, or KIR2DS3. The timing of the peak mRNA expression of aKIRs unique to the donor was inconsistent but occurred within the first 3 months posttransplantation, whereas the peak mRNA expression of iKIRs was consistently observed in the third month after transplantation. The expression of KIR2DL2 in the third month posttransplantation was significantly higher in the transplant recipients than in the donors (p = 0.01). The KIR2DL1 and KIR3DL1 levels in the transplant recipients in the second and third months posttransplantation were also obviously higher than the donor levels (p < 0.0001). Thus, these observations should be considered when attempting to predict the correlation between mRNA expression and prognosis after allo-HSCT.

The Education of NK Cells Determines Their Responsiveness to Autologous HIV-Infected CD4 T Cells.

Several studies support a role for specific killer immunoglobulin-like receptor (KIR)-HLA combinations in protection from HIV infection and slower progression to AIDS. Natural killer (NK) cells acquire effector functions through education, a process that requires the interaction of inhibitory NK cell receptors with their major histocompatibility complex (MHC) class I (or HLA class I [HLA-I]) ligands. HLA-C allotypes are ligands for the inhibitory KIRs (iKIRs) KIR2DL1, KIR2DL2, and KIR2DL3, whereas the ligand for KIR3DL1 is HLA-Bw4. HIV infection reduces the expression of HLA-A, -B, and -C on the surfaces of infected CD4 (iCD4) T cells. Here we investigated whether education through iKIR-HLA interactions influenced NK cell responses to autologous iCD4 cells. Enriched NK cells were stimulated with autologous iCD4 cells or with uninfected CD4 cells as controls. The capacities of single-positive (sp) KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1 NK cells to produce CCL4, gamma interferon (IFN-γ), and/or CD107a were assessed by flow cytometry. Overall, we observed that the potency of NK cell education was directly related to the frequency of each spiKIR+ NK cell's ability to respond to the reduction of its cognate HLA ligand on autologous iCD4 cells, as measured by the frequency of production by spiKIR+ NK cells of CCL4, IFN-γ, and/or CD107a. Both NK cell education and HIV-mediated changes in HLA expression influenced NK cell responses to iCD4 cells.IMPORTANCE Epidemiological studies show that natural killer (NK) cells have anti-HIV activity: they are able to reduce the risk of HIV infection and/or slow HIV disease progression. How NK cells contribute to these outcomes is not fully characterized. We used primary NK cells and autologous HIV-infected cells to examine the role of education through four inhibitory killer immunoglobulin-like receptors (iKIRs) from persons with HLA types that are able to educate NK cells bearing one of these iKIRs. HIV-infected cells activated NK cells through missing-self mechanisms due to the downmodulation of cell surface HLA expression mediated by HIV Nef and Vpu. A higher frequency of educated than uneducated NK cells expressing each of these iKIRs responded to autologous HIV-infected cells by producing CCL4, IFN-γ, and CD107a. Since NK cells were from non-HIV-infected individuals, they model the consequences of healthy NK cell-HIV-infected cell interactions occurring in the HIV eclipse phase, when new infections are susceptible to extinction.

High-Resolution Genetic and Phenotypic Analysis of KIR2DL1 Alleles and Their Association with Pre-Eclampsia.

Killer-cell Ig-like receptor (KIR) genes are inherited as haplotypes. They are expressed by NK cells and linked to outcomes of infectious diseases and pregnancy in humans. Understanding how genotype relates to phenotype is difficult because of the extensive diversity of the KIR family. Indeed, high-resolution KIR genotyping and phenotyping in single NK cells in the context of disease association is lacking. In this article, we describe a new method to separate NK cells expressing allotypes of the KIR2DL1 gene carried by the KIR A haplotype (KIR2DL1A) from those expressing KIR2DL1 alleles carried by the KIR B haplotype (KIR2DL1B). We find that in KIR AB heterozygous individuals, different KIR2DL1 allotypes can be detected in both peripheral blood and uterine NK cells. Using this new method, we demonstrate that both blood and uterine NK cells codominantly express KIR2DL1A and KIR2DL1B allotypes but with a predominance of KIR2DL1A variants, which associate with enhanced NK cell function. In a case-control study of pre-eclampsia, we show that KIR2DL1A, not KIR2DL1B, associates with increased disease risk. This method will facilitate our understanding of how individual KIR2DL1 allelic variants affect NK cell function and contribute to disease risk.

Different Selected Mechanisms Attenuated the Inhibitory Interaction of KIR2DL1 with C2+ HLA-C in Two Indigenous Human Populations in Southern Africa.

The functions of human NK cells in defense against pathogens and placental development during reproduction are modulated by interactions of killer cell Ig-like receptors (KIRs) with HLA-A, -B and -C class I ligands. Both receptors and ligands are highly polymorphic and exhibit extensive differences between human populations. Indigenous to southern Africa are the KhoeSan, the most ancient group of modern human populations, who have highest genomic diversity worldwide. We studied two KhoeSan populations, the Nama pastoralists and the ≠Khomani San hunter-gatherers. Comprehensive next-generation sequence analysis of HLA-A, -B, and -C and all KIR genes identified 248 different KIR and 137 HLA class I, which assort into ∼200 haplotypes for each gene family. All 74 Nama and 78 ≠Khomani San studied have different genotypes. Numerous novel KIR alleles were identified, including three arising by intergenic recombination. On average, KhoeSan individuals have seven to eight pairs of interacting KIR and HLA class I ligands, the highest diversity and divergence of polymorphic NK cell receptors and ligands observed to date. In this context of high genetic diversity, both the Nama and the ≠Khomani San have an unusually conserved, centromeric KIR haplotype that has arisen to high frequency and is different in the two KhoeSan populations. Distinguishing these haplotypes are independent mutations in KIR2DL1, which both prevent KIR2DL1 from functioning as an inhibitory receptor for C2+ HLA-C. The relatively high frequency of C2+ HLA-C in the Nama and the ≠Khomani San appears to have led to natural selection against strong inhibitory C2-specific KIR.

Association of killer-cell immunoglobulin-like receptor genes with acute myelogenous leukaemia.

Killer-cell immunoglobulin-like receptors (KIRs) are important because of their key roles in NK cell development and function. Some KIR genes have been associated with the incidence of haematological malignancies. This study was designed to determine whether the inheritance of specific KIR genes is associated with susceptibility to acute myelogenous leukaemia (AML) in Persians living in south-western Iran. KIR genes and KIR2DS4 variants were typed by polymerase chain reaction-sequence-specific primer (PCR-SSP) in 167 patients with AML and 169 healthy controls. Our results showed 10% of patients-mostly females-were classified as M3. Flt3 mutations were detected in 26% of patients, most of whom had internal tandem duplication (ITD). The frequency of activating KIRs (aKIRs)-mainly KIR3DS1-was higher in patients, whereas inhibitory KIRs (iKIRs)-particularly KIR3DL1 and KIR2DL1-were more common among controls. The incidence of the KIR2DS4fl allele was higher among patients with non-M3 AML than controls. We also found a higher frequency of 4 or more iKIR genes in the controls and a higher frequency of 4 or more aKIR genes in the patients. Individuals with more iKIR than aKIR belonged predominantly to the control group. Individuals with the telomeric AA genotype who had inherited the KIR2DS4fl allele were more frequent in the patient group. According to our results, increased frequency of aKIRs in patients with AML may lead to the hyperactivation of NK cells against malignant cells with reduced or lack of HLA class I molecules followed by NK cell exhaustion which allow malignant cells to progress.

Impact of HLA Allele-KIR Pairs on HIV Clinical Outcome in South Africa.

BACKGROUND: HLA class I contributes to HIV immune control through antigen presentation to cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. In contrast to investigations of CTL, studies of NK cells in HIV control through HLA-killer immunoglobulin-like receptor (KIR) interactions remain sparse in African cohorts. METHODS: Treatment-naive, chronically HIV-infected adults (N = 312) were recruited from South Africa, and the effects of HLA-KIR pairs on clinical outcome were analyzed. RESULTS: There was no significant difference in viral load among all subjects with HLA alleles from the HLA-C1 group (P = .1). However, differences in HLA-C type significantly influenced viremia among 247 KIR2DL3 positives (P = .04), suggesting that specific HLA-KIR interactions contribute to immune control. Higher viral load (P = .02) and lower CD4+ T-cell counts (P = .008) were observed in subjects with HLA-C*16:01+KIR2DL3+. Longitudinal analysis showed more rapid progression to AIDS among HLA-C*16:01+KIR2DL3+ subjects (adjusted hazard ratio 1.9, P = .03) than those without this genotype, independent of CD4+ T-cell count and viral load. CONCLUSIONS: These results highlight the existence of unique anti-HIV innate immunity within distinct populations and the contribution of KIR on NK cells and some CTLs to the well-described HLA-mediated impact on HIV disease progression.

Resurrecting KIR2DP1: A Key Intermediate in the Evolution of Human Inhibitory NK Cell Receptors That Recognize HLA-C.

KIR2DP1 is an inactive member of the human lineage III KIR family, which includes all HLA-C-specific receptor genes. The lethal, and only, defect in KIR2DP1 is a nucleotide deletion in codon 88. Fixed in modern humans, the deletion is also in archaic human genomes. KIR2DP1 is polymorphic, with dimorphism at specificity-determining position 44. By repairing the deletion, we resurrected 11 alleles of KIR2DP1F , the functional antecedent of KIR2DP1 We demonstrate how K44-KIR2DP1F with lysine 44 recognized C1+HLA-C, whereas T44-KIR2DP1F recognized C2+HLA-C. Dimorphisms at 12 other KIR2DP1F residues modulate receptor avidity or signaling. KIR2DP1 and KIR2DL1 are neighbors in the centromeric KIR region and are in tight linkage disequilibrium. Like KIR2DL1, KIR2DP1 contributed to CenA and CenB KIR haplotype differences. Encoded on CenA, C1-specific K44-KIR2DP1F were stronger receptors than the attenuated C2-specific T44-KIR2DP1F encoded on CenB The last common ancestor of humans and chimpanzees had diverse lineage III KIR that passed on to chimpanzees but not to humans. Early humans inherited activating KIR2DS4 and an inhibitory lineage III KIR, likely encoding a C1-specific receptor. The latter spawned the modern family of HLA-C receptors. KIR2DP1F has properties consistent with KIR2DP1F having been the founder gene. The first KIR2DP1F alleles encoded K44-C1 receptors; subsequently KIR2DP1F alleles encoding T44-C2 receptors evolved. The emergence of dedicated KIR2DL2/3 and KIR2DL1 genes encoding C1 and C2 receptors, respectively, could have led to obsolescence of KIR2DP1F Alternatively, pathogen subversion caused its demise. Preservation of KIR2DP1F functional polymorphism was a side effect of fixation of the deletion in KIR2DP1F by micro gene conversion.

The unique evolution of the pig LRC, a single KIR but expansion of LILR and a novel Ig receptor family.

The leukocyte receptor complex (LRC) encodes numerous immunoglobulin (Ig)-like receptors involved in innate immunity. These include the killer-cell Ig-like receptors (KIR) and the leukocyte Ig-like receptors (LILR) which can be polymorphic and vary greatly in number between species. Using the recent long-read genome assembly, Sscrofa11.1, we have characterized the porcine LRC on chromosome 6. We identified a ~ 197-kb region containing numerous LILR genes that were missing in previous assemblies. Out of 17 such LILR genes and fragments, six encode functional proteins, of which three are inhibitory and three are activating, while the majority of pseudogenes had the potential to encode activating receptors. Elsewhere in the LRC, between FCAR and GP6, we identified a novel gene that encodes two Ig-like domains and a long inhibitory intracellular tail. Comparison with two other porcine assemblies revealed a second, nearly identical, non-functional gene encoding a short intracellular tail with ambiguous function. These novel genes were found in a diverse range of mammalian species, including a pseudogene in humans, and typically consist of a single long-tailed receptor and a variable number of short-tailed receptors. Using porcine transcriptome data, both the novel inhibitory gene and the LILR were highly expressed in peripheral blood, while the single KIR gene, KIR2DL1, was either very poorly expressed or not at all. These observations are a prerequisite for improved understanding of immune cell functions in the pig and other species.

Maternal obesity alters uterine NK activity through a functional KIR2DL1/S1 imbalance.

In pregnancy, uterine natural killer cells (uNK) play essential roles in coordinating uterine angiogenesis, blood vessel remodeling and promoting maternal tolerance to fetal tissue. Deviances from a normal uterine microenvironment are thought to modify uNK function(s) by limiting their ability to establish a healthy pregnancy. While maternal obesity has become a major health concern due to associations with adverse effects on fetal and maternal health, our understanding into how obesity contributes to poor pregnancy disorders is unknown. Given the importance of uNK in pregnancy, this study examines the impact of obesity on uNK function in women in early pregnancy. We identify that uNK from obese women show a greater propensity for cellular activation, but this difference does not translate into increased effector killing potential. Instead, uNK from obese women express an altered repertoire of natural killer receptors, including an imbalance in inhibitory KIR2DL1 and activating KIR2DS1 receptors that favors HLA-C2-directed uNK activation. Notably, we show that obesity-related KIR2DS1 skewing potentiates TNFα production upon receptor crosslinking. Together, these findings suggest that maternal obesity modifies uNK activity by altering the response toward HLA-C2 antigen and KIR2DL1/2DS1-controlled TNFα release. Furthermore, this work identifies alterations in uNK function resulting from maternal obesity that may impact early developmental processes important in pregnancy health.

Loss and Gain of Natural Killer Cell Receptor Function in an African Hunter-Gatherer Population.

Modulating natural killer cell functions in human immunity and reproduction are diverse interactions between the killer cell immunoglobulin-like receptors (KIR) of Natural Killer (NK) cells and HLA class I ligands on the surface of tissue cells. Dominant interactions are between KIR2DL1 and the C2 epitope of HLA-C and between KIR2DL2/3 and the C1 epitope of HLA-C. KhoeSan hunter-gatherers of Southern Africa represent the earliest population divergence known and are the most genetically diverse indigenous people, qualities reflected in their KIR and HLA genes. Of the ten KhoeSan KIR2DL1 alleles, KIR2DL1*022 and KIR2DL1*026 likely originated in the KhoeSan, and later were transmitted at low frequency to the neighboring Zulus through gene flow. These alleles arose by point mutation from other KhoeSan KIR2DL1 alleles that are more widespread globally. Mutation of KIR2DL1*001 gave rise to KIR2DL1*022, causing loss of C2 recognition and gain of C1 recognition. This makes KIR2DL1*022 a more avid and specific C1 receptor than any KIR2DL2/3 allotype. Mutation of KIR2DL1*012 gave rise to KIR2DL1*026, causing premature termination of translation at the end of the transmembrane domain. This makes KIR2DL1*026 a membrane-associated receptor that lacks both a cytoplasmic tail and signaling function. At higher frequencies than their parental allotypes, the combined effect of the KhoeSan-specific KIR2DL1*022 and KIR2DL1*026 is to reduce the frequency of strong inhibitory C2 receptors and increase the frequency of strong inhibitory C1 receptors. Because interaction of KIR2DL1 with C2 is associated with risk of pregnancy disorder, these functional changes are potentially advantageous. Whereas all other KhoeSan KIR2DL1 alleles are present on a wide diversity of centromeric KIR haplotypes, KIR2DL1*026 is present on a single KIR haplotype and KIR2DL1*022 is present on two very similar haplotypes. The high linkage disequilibrium across their haplotypes is consistent with a recent emergence for these KIR2DL1 alleles that have distinctive functions.

KIR2DL3 and KIR2DL1 show similar impact on licensing of human NK cells.

Killer cell immunoglobulin-like receptor/HLA class I (KIR/HLA-I) combinations are associated with disease risk, implicating functional roles for NK cells (NKCs) or KIR(+) T cells. KIR/HLA-I interactions can act through inhibition of NKC activation by target cells and NKC licensing for greater intrinsic responsiveness. We compared licensing conferred by the weaker, HLA-C group 1/KIR2DL3, and the stronger, HLA-C group 2/KIR2DL1, inhibitory combinations. The "rheostat model" predicts weaker licensing by HLA-C1/KIR2DL3 interactions than HLA-C2/KIR2DL1. We analyzed degranulation in NKC subsets expressing single and multiple receptors for HLA-I. NKG2A had the strongest licensing impact, while KIR2DL3, KIR2DL1, and KIR3DL1 were weaker, and not significantly different to each other. Presence of one or two matched HLA-C allotypes did not alter licensing of KIR2DL3(+) and KIR2DL1(+) NKC. Coexpression of activating KIR2DS1 disarmed KIR2DL3(+) and KIR2DL1(+) NKC to a similar extent. KIR3DL1 and NKG2A combined for more enhanced licensing of double-positive NKC than the combination of KIR2DL3 and KIR2DL1. Thus, KIR2DL3 and KIR2DL1 have similar capacity to license NKC, suggesting that inhibitory signal strength and amount of available HLA-C ligands do not correlate with NKC licensing. Altogether, our results show that the basis for disease associations of HLA-C and KIR2DL likely encompasses factors other than licensing.

Missing or altered self: human NK cell receptors that recognize HLA-C.

Natural killer (NK) cells are fast-acting and versatile lymphocytes that are critical effectors of innate immunity, adaptive immunity, and placental development. Controlling NK cell function are the interactions between killer-cell immunoglobulin-like receptors (KIRs) and their HLA-A, HLA-B and HLA-C ligands. Due to the extensive polymorphism of both KIR and HLA class I, these interactions are highly diversified and specific combinations correlate with protection or susceptibility to a range of infectious, autoimmune, and reproductive disorders. Evolutionary, genetic, and functional studies are consistent with the interactions between KIR and HLA-C being the dominant control mechanism of human NK cells. In addition to their recognition of the C1 and C2 epitopes, increasing evidence points to KIR having a previously unrecognized selectivity for the peptide presented by HLA-C. This selectivity appears to be a conserved feature of activating KIR and may partly explain the slow progress made in identifying their HLA class I ligands. The peptide selectivity of KIR allows NK cells to respond, not only to changes in the surface expression of HLA-C, but also to the more subtle changes in the HLA-C peptidome, such as occur during viral infection and malignant transformation. Here, we review recent advances in understanding of human-specific KIR evolution and how the inhibitory and activating HLA-C receptors allow NK cells to respond to healthy cells, diseased cells, and the semi-allogeneic cells of the fetus.

A single amino acid change in inhibitory killer cell Ig-like receptor results in constitutive receptor self-association and phosphorylation.

Signaling by immunoreceptors is often 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 ITIMs results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work showed 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. In this article, we show that substitution of His-36 in the first Ig domain of KIR2DL1 with alanine (KIR2DL1-H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, as well as recruitment of tyrosine phosphatase SHP-1. Furthermore, substitution of His-36 with a similar bulky amino acid, phenylalanine, maintained the receptor in its unphosphorylated state, suggesting that steric hindrance by the His-36 side chain prevents constitutive KIR2DL1 self-association and ITIM phosphorylation. 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 that 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 did expression of wild-type KIR2DL1. These results 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.