Dissection of the NKG2C NK cell response against Puumala Orthohantavirus.

BACKGROUND: Infections with the Puumala orthohantavirus (PUUV) in humans may cause hemorrhagic fever with renal syndrome (HFRS), known as nephropathia epidemica (NE), which is associated with acute renal failure in severe cases. In response to PUUV-infections, a subset of potent antiviral NKG2C+ NK cells expand, whose role in virus defence and pathogenesis of NE is unclear. NKG2C+ NK cell proliferation is mediated by binding of NKG2C/CD94 to HLA-E on infected cells. The proliferation and activation of NKG2C+ NK cells via the NKG2C/HLA-E axis is affected by different NKG2C (NKG2Cwt/del) and HLA-E (HLA-E*0101/0103) alleles, which naturally occur in the human host. Homozygous (NKG2Cdel/del) and heterozygous (NKG2Cwt/del) deletions of the NKG2C receptor results in an impaired NKG2C/CD94 mediated proliferation and activation of NKG2C+ cells. We therefore analyzed the PUUV-mediated NKG2C+ NK cell responses and the impact of different NKG2C and HLA-E alleles in NE patients. METHODOLOGY/PRINCIPAL FINDINGS: NKG2C+ NK cell expansion and effector functions in PUUV-infected cells were investigated using flow cytometry and it was shown that PUUV-infected endothelial cells led to a NKG2C/CD94 mediated NKG2C+ NK cell activation and expansion, dependent on the HLA-G-mediated upregulation of HLA-E. Furthermore, the NKG2Cdel and HLA-E*0101/0103 alleles were determined in 130 NE patients and 130 matched controls, and it was shown that in NE patients the NKG2Cwt/del allele was significantly overrepresented, compared to the NKG2Cwt/wt variant (p = 0.01). In addition, in vitro analysis revealed that NKG2Cwt/del NK cells exhibited on overall a lower proliferation (p = 0.002) and lower IFNγ expression (p = 0.004) than NKG2Cwt/wt NK cells. CONCLUSIONS/SIGNIFICANCE: Our results corroborate the substantial impact of the NKG2C/HLA-E axis on PUUV-specific NK cell responses. A weak NKG2C+ NK cell response, as reflected by NKG2Cwt/del variant, may be associated with a higher risk for a severe hantavirus infections.

NK cell receptor and ligand composition influences the clearance of SARS-CoV-2.

To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry-based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.

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.

The CD94/NKG2A inhibitory receptor educates uterine NK cells to optimize pregnancy outcomes in humans and mice.

The conserved CD94/NKG2A inhibitory receptor is expressed by nearly all human and ∼50% of mouse uterine natural killer (uNK) cells. Binding human HLA-E and mouse Qa-1, NKG2A drives NK cell education, a process of unknown physiological importance influenced by HLA-B alleles. Here, we show that NKG2A genetic ablation in dams mated with wild-type males caused suboptimal maternal vascular responses in pregnancy, accompanied by perturbed placental gene expression, reduced fetal weight, greater rates of smaller fetuses with asymmetric growth, and abnormal brain development. These are features of the human syndrome pre-eclampsia. In a genome-wide association study of 7,219 pre-eclampsia cases, we found a 7% greater relative risk associated with the maternal HLA-B allele that does not favor NKG2A education. These results show that the maternal HLA-B→HLA-E→NKG2A pathway contributes to healthy pregnancy and may have repercussions on offspring health, thus establishing the physiological relevance for NK cell education. VIDEO ABSTRACT.

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.

HLAs, TCRs, and KIRs, a Triumvirate of Human Cell-Mediated Immunity.

In all human cells, human leukocyte antigen (HLA) class I glycoproteins assemble with a peptide and take it to the cell surface for surveillance by lymphocytes. These include natural killer (NK) cells and γδ T cells of innate immunity and αß T cells of adaptive immunity. In healthy cells, the presented peptides derive from human proteins, to which lymphocytes are tolerant. In pathogen-infected cells, HLA class I expression is perturbed. Reduced HLA class I expression is detected by KIR and CD94:NKG2A receptors of NK cells. Almost any change in peptide presentation can be detected by αß CD8+ T cells. In responding to extracellular pathogens, HLA class II glycoproteins, expressed by specialized antigen-presenting cells, present peptides to αß CD4+ T cells. In comparison to the families of major histocompatibility complex (MHC) class I, MHC class II and αß T cell receptors, the antigenic specificity of the γδ T cell receptors is incompletely understood.

Adaptive Features of Natural Killer Cells in Multiple Sclerosis.

Human cytomegalovirus (HCMV) has been recently related with a lower susceptibility to multiple sclerosis (MS). HCMV promotes an adaptive development of NK cells bearing the CD94/NKG2C receptor with a characteristic phenotypic and functional profile. NK cells are proposed to play an immunoregulatory role in MS, and expansion of the NKG2C(+) subset was recently associated with reduced disability progression. To further explore this issue, additional adaptive NK cell markers, i.e., downregulation of FcεRIγ chain (FcRγ) and PLZF transcription factor, as well as antibody-dependent NK cell activation were assessed in controls and MS patients considering HCMV serology and clinical features. In line with previous reports, increased proportions of NKG2C(+), FcRγ(-), and PLZF(-) CD56dim NK cells were found in HCMV(+) cases. However, PLZF(-) NK cells were detected uncoupled from other adaptive markers within the CD56bright subset from HCMV(+) cases and among CD56dim NK cells from HCMV(-) MS patients, suggesting an additional effect of HCMV-independent factors in PLZF downregulation. Interferon-ß therapy was associated with lower proportions of FcRγ(-) CD56dim NK cells in HCMV(+) and increased PLZF(-) CD56bright NK cells in HCMV(-) patients, pointing out to an influence of the cytokine on the expression of adaptive NK cell-associated markers. In addition, proportions of NKG2C(+) and FcRγ(-) NK cells differed in progressive MS patients as compared to controls and other clinical forms. Remarkably, an adaptive NK cell phenotype did not directly correlate with enhanced antibody-triggered degranulation and TNFα production in MS in contrast to controls. Altogether, our results provide novel insights into the putative influence of HCMV and adaptive NK cells in MS.

Development and characterization of a canine-specific anti-CD94 (KLRD-1) monoclonal antibody.

Natural killer (NK) cells are non-T, non-B lymphocytes are part of the innate immune system and function without prior activation. The human NK cell surface determinant, CD94, plays a critical role in regulation of NK cell activity as a heterodimer with NKG2 subclasses. Canine NK cells are not as well defined as the human and murine equivalents, due in part to the paucity of reagents specific to cell surface markers. Canines possess NK/NKT cells that have similar morphological characteristics to those found in humans, yet little is known about their functional characteristics nor of cell surface expression of CD94. Here, we describe the development and function of a monoclonal antibody (mAb) to canine (ca) CD94. Freshly isolated canine CD94+ cells were CD3+/-, CD8+/-, CD4-, CD21-, CD5low, NKp46+, and were cytotoxic against a canine target cell line. Anti-caCD94 mAb proved useful in enriching NK/NKT cells from PBMC for expansion on CTAC feeder cells in the presence of IL-2 and IL-15. The cultured cells were highly cytolytic with co-expression of NKp46 and reduced expression of CD3. Transmission electron microscopy revealed expanded CD94+ lymphocytes were morphologically large granular lymphocytes with large electron dense granules. Anti-caCD94 (mAb) can serve to enrich NK/NKT cells from dog peripheral blood for ex vivo expansion for HCT and is a potentially valuable reagent for studying NK/NKT regulation in the dog.

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.

Targeting Checkpoint Receptors and Molecules for Therapeutic Modulation of Natural Killer Cells.

Among the most promising therapeutic modalities for cancer treatment is the blockade of immune checkpoint pathways, which are frequently co-opted by tumors as a major mechanism of immune escape. CTLA-4 and PD-1 are the representative examples, and their blockade by therapeutic antibodies leads to enhanced anti-tumor immunity with durable clinical responses, but only in a minority of patients. This has highlighted the need to identify and target additional immune checkpoints that can be exploited to further enhance immune responses to refractory cancers. These emerging targets include natural killer (NK) cell-directed checkpoint receptors (KIR and CD94/NKG2A) as well as the NK- and T cell-expressed checkpoints TIM-3, TIGIT, CD96, and LAG-3. Interestingly, the potentiation of anti-tumor immunity by checkpoint blockade relies not only on T cells but also on other components of the innate immune system, including NK cells. NK cells are innate lymphoid cells that efficiently kill tumor cells without MHC specificity, which is complementary to the MHC-restricted tumor lysis mediated by cytotoxic T cells. However, the role of these immune checkpoints in modulating the function of NK cells remains unclear and somewhat controversial. Unraveling the mechanisms by which these immune checkpoints function in NK cells and other immune cells will pave the way to developing new therapeutic strategies to optimize anti-tumor immunity while limiting cancer immune escape. Here, we focus on recent findings regarding the roles of immune checkpoints in regulating NK cell function and their potential application in cancer immunotherapy.

Donor Cell Composition and Reactivity Predict Risk of Acute Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation.

Background. Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (HSCT). We designed a functional assay for assessment of individual risk for acute GVHD. Study Design and Methods. Blood samples were collected from patients and donors before HSCT. Two groups of seven patients each were selected, one in which individuals developed acute GVHD grades II-IV and one in which none showed any clinical signs of GVHD. Peripheral blood mononuclear cells (PBMCs) isolated from donors were incubated in mixed lymphocyte cultures (MLCs) with recipient PBMCs. The cells were characterized by flow cytometry before and after MLC. Results. Samples from donors in the GVHD group contained significantly lower frequencies of naïve γδ T-cells and T-cells expressing NK-cell markers CD56 and CD94. Donor samples in this group also exhibited lower frequencies of naïve CD95+ T-cells compared to controls. After MLC, there were dissimilarities in the CD4/CD8 T-cell ratio and frequency of CD69+ T-cells between the two patient groups, with the non-GVHD group showing higher frequencies of CD8+ and CD69+ T-cells. Conclusion. We conclude that a thorough flow cytometric analysis of donor cells for phenotype and allogeneic reactivity may be of value when assessing pretransplant risk for severe acute GVHD.

The Activating NKG2C Receptor Is Significantly Reduced in NK Cells after Allogeneic Stem Cell Transplantation in Patients with Severe Graft-versus-Host Disease.

Natural killer (NK) cells play a central role in the innate immune system. In allogeneic stem cell transplantation (alloSCT), alloreactive NK cells derived by the graft are discussed to mediate the elimination of leukemic cells and dendritic cells in the patient and thereby to reduce the risk for leukemic relapses and graft-versus-host reactions. The alloreactivity of NK cells is determined by various receptors including the activating CD94/NKG2C and the inhibitory CD94/NKG2A receptors, which both recognize the non-classical human leukocyte antigen E (HLA-E). Here we analyze the contribution of these receptors to NK cell alloreactivity in 26 patients over the course of the first year after alloSCT due to acute myeloid leukemia, myelodysplastic syndrome and T cell Non-Hodgkin-Lymphoma. Our results show that NK cells expressing the activating CD94/NKG2C receptor are significantly reduced in patients after alloSCT with severe acute and chronic graft-versus-host disease (GvHD). Moreover, the ratio of CD94/NKG2C to CD94/NKG2A was reduced in patients with severe acute and chronic GvHD after receiving an HLA-mismatched graft. Collectively, these results provide evidence for the first time that CD94/NKG2C is involved in GvHD prevention.

Development of the adaptive NK cell response to human cytomegalovirus in the context of aging.

Human cytomegalovirus (HCMV) establishes a highly prevalent life-long latent infection. Though generally subclinical, HCMV infection may have severe consequences during fetal development and in immunocompromised individuals. Based on epidemiological studies HCMV(+) serology has been associated with the development of atherosclerosis, immune senescence and an increase mortality rate in elderly people. Such long-term detrimental effects of the viral infection presumably result from an inefficient immune control of the pathogen, depending on the quality and evolution of the individual host-pathogen relationship. Together with antigen-specific T lymphocytes, NK cells play an important role in anti-viral immune defense. HCMV promotes in some individuals the differentiation and persistent steady state expansion of an NK cell subset bearing the CD94/NKG2C activating receptor. The relationship between this adaptive NK cell response to HCMV and aging is overviewed.

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.

A Conserved HIV-1-Derived Peptide Presented by HLA-E Renders Infected T-cells Highly Susceptible to Attack by NKG2A/CD94-Bearing Natural Killer Cells.

Major histocompatibility class I (MHC-I)-specific inhibitory receptors on natural killer (NK) cells (iNKRs) tolerize mature NK cell responses toward normal cells. NK cells generate cytolytic responses to virus-infected or malignant target cells with altered or decreased MHC-I surface expression due to the loss of tolerizing ligands. The NKG2A/CD94 iNKR suppresses NK cell responses through recognition of the non-classical MHC-I, HLA-E. We used HIV-infected primary T-cells as targets in an in vitro cytolytic assay with autologous NK cells from healthy donors. In these experiments, primary NKG2A/CD94(+) NK cells surprisingly generated the most efficient responses toward HIV-infected T-cells, despite high HLA-E expression on the infected targets. Since certain MHC-I-presented peptides can alter recognition by iNKRs, we hypothesized that HIV-1-derived peptides presented by HLA-E on infected cells may block engagement with NKG2A/CD94, thereby engendering susceptibility to NKG2A/CD94(+) NK cells. We demonstrate that HLA-E is capable of presenting a highly conserved peptide from HIV-1 capsid (AISPRTLNA) that is not recognized by NKG2A/CD94. We further confirmed that HLA-C expressed on HIV-infected cells restricts attack by KIR2DL(+) CD56(dim) NK cells, in contrast to the efficient responses by CD56(bright) NK cells, which express predominantly NKG2A/CD94 and lack KIR2DLs. These findings are important since the use of NK cells was recently proposed to treat latently HIV-1-infected patients in combination with latency reversing agents. Our results provide a mechanistic basis to guide these future clinical studies, suggesting that ex vivo-expanded NKG2A/CD94(+) KIR2DL(-) NK cells may be uniquely beneficial.

Microenvironmental stresses induce HLA-E/Qa-1 surface expression and thereby reduce CD8(+) T-cell recognition of stressed cells.

Hypoxia and glucose deprivation are often observed in the microenvironment surrounding solid tumors in vivo. However, how they interfere with MHC class I antigen processing and CD8(+) T-cell responses remains unclear. In this study, we analyzed the production of antigenic peptides presented by classical MHC class I in mice, and showed that it is quantitatively decreased in the cells exposed to either hypoxia or glucose deprivation. In addition, we unexpectedly found increased surface expression of HLA-E in human and Qa-1 in mouse tumor cells exposed to combined oxygen and glucose deprivation. The induced Qa-1 on the stressed tumor model interacted with an inhibitory NKG2/CD94 receptor on activated CD8(+) T cells and attenuated their specific response to the antigen. Our results thus suggest that microenvironmental stresses modulate not only classical but also nonclassical MHC class I presentation, and confer the stressed cells the capability to escape from the CD8(+) T-cell recognition.

NK Cell Proliferation Induced by IL-15 Transpresentation Is Negatively Regulated by Inhibitory Receptors.

IL-15 bound to the IL-15Rα-chain (IL-15Rα) is presented in trans to cells bearing the IL-2Rß-chain and common γ-chain. As IL-15 transpresentation occurs in the context of cell-to-cell contacts, it has the potential for regulation by and of other receptor-ligand interactions. In this study, human NK cells were tested 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-15Rα. 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 KIR-HLA-C interactions did not reduce the proliferation induced by soluble IL-15. Therefore, transpresentation of IL-15 is subject to downregulation 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. Coengagement of inhibitory receptors, either KIR2DL1 or CD94-NKG2A, did not inhibit phosphorylation of Stat5 but inhibited selectively phosphorylation of Akt and S6 ribosomal protein. IL-15Rα was not excluded from, but was evenly distributed across, inhibitory synapses. These findings demonstrate a novel mechanism to attenuate IL-15-dependent NK cell proliferation and suggest that inhibitory NK cell receptors contribute to NK cell homeostasis.