Activating NKG2C Receptor: Functional Characteristics and Current Strategies in Clinical Applications

Vol.:(0123456789) 1 3 Archivum Immunologiae et Therapiae Experimentalis (2023) 71:9 https://doi.org/10.1007/s00005-023-00674-z REVIEW Activating NKG2C Receptor: Functional Characteristics andCurrent Strategies inClinical Applications JagodaSiemaszko1 · AleksandraMarzec‑Przyszlak2,3 · KatarzynaBogunia‑Kubik1 Received: 16 November 2022 / Accepted: 1 February 2023 © The Author(s) 2023 Abstract The interest in NK cells and their cytotoxic activity against tumour, infected or transformed cells continuously increases as they become a new efficient and off-the-shelf agents in immunotherapies. Their actions are balanced by a wide set of activating and inhibitory receptors, recognizing their complementary ligands on target cells. One of the most studied receptors is the activating CD94/NKG2C molecule, which is a member of the C-type lectin-like family. This review is intended to summarise latest research findings on the clinical relevance of NKG2C receptor and to examine its contribution to current and potential therapeutic strategies. It outlines functional characteristics and molecular features of CD94/NKG2C, its interactions with HLA-E molecule and presented antigens, pointing out a key role of this receptor in immunosurveillance, especially in the human cytomegalovirus infection. Additionally, the authors attempt to shed some light on receptor’s unique interaction with its ligand which is shared with another receptor (CD94/NKG2A) with rather opposite properties. Keywords NKG2C· NK cell receptors· NK cells· HLA-E NKG2C: AnActivating Receptor ofNK Cells Natural killer (NK) cells, also known as large granular lymphocytes, constitute a subgroup of recently discovered innate lymphoid cells (ILCs), and together with the ILC1 subpopulation are responsible for type 1 immunity functions, e.g. natural cytotoxicity. Originally considered as components only of the innate immunity, the NK cells have also developed characteristics restrained for adaptive immunity (Pende etal. 2019; Vivier etal. 2011, 2018). The NK cells functions are regulated and properly balanced by a wide set of activating and inhibitory receptors (Quatrini etal. 2021). Among the activating receptors, the CD94/NKG2C remains to belong to the ones of considerable influence on NK functioning. The CD94/NKG2C is a heterodimeric receptor, consisted of two type II proteins and is a member of the C-type lectin-like family. Similar to the other activating NKG2 receptors, the NKG2C has a negatively charged residue (either a lysine or an arginine) in its transmembrane domain. Activation signal is transduced through the DAP-12 adaptor molecule, which contains immunoreceptor tyrosine-based activation motifs. The ligand for CD94/NKG2C is a non-classical HLA-E molecule of major histocompatibility (MHC) class I proteins. Its recognition is crucial for triggering the NK cell cytotoxicity and cytokine production (Glienke etal. 1998; Lanier etal. 1998; Miyashita etal. 2004). NK cell receptors can also be found on some T cells, such as CD8+ T lymphocytes, which express NKG2 receptors of activating (NKG2C, NKG2D) and inhibiting (NKG2A) properties (Patel etal. 2013). The CD94/NKG2C receptor may even operate like an alternative pathway to the T cell receptor in subset of NKG2C+ CD8+ T cells, triggering proliferation and effector functions of those cells (Gumá etal. 2005). The CD94/NKG2C receptor is encoded by the 6 kpb length NKG2C gene, also referred as KLRC2, located within the NK complex on chromosome 12p12.3–13.2 (Fig.1). It consists of six exons and five introns. Protein product * Katarzyna Bogunia-Kubik [email protected] 1 Laboratory ofClinical Immunogenetics andPharmacogenetics, Hirszfeld Institute ofImmunology andExperimental Therapy, Polish Academy ofSciences, Wroclaw, Poland 2 Department ofBiosensors andProcessing ofBiomedical Signals, Faculty ofBiomedical Engineering, Silesian University ofTechnology, Zabrze, Poland 3 Department ofBiomedical Engineering, Faculty ofElectrical Engineering andCommunication, Brno University ofTechnology, Brno, CzechRepublic Archivum Immunologiae et Therapiae Experimentalis (2023) 71:9 1 3 9 Page 2 of 18 has 231 amino acids in length and a molecular mass of 26,159Da. The two homologous proteins of NKG2C are NKG2E and alternatively spliced NKG2H. Both NKG2C and NKG2E genes are highly identical (92.1%) at the genomic level, have duplicated 255-bp length DNA containing a second version of exon III (termed IIIB). The differences in gene sequence were found at the 3’ end of NKG2E, resulting in additional Alu amino acid. The 3’ region of NKG2-A, -E and -F receptor, rich in Alu repeats, is known as coding for the extracellular domain of the NKG2 receptors and may affect ligand specificity (Glienke etal. 1998). It is known that Alu sequences occur with a frequency of approximately one element per 4kb in the human genome, and could cause abrogation of transcriptional activity (Brostjan etal. 2000). It has been assumed that the NKG2 family is rather non-polymorphic although some variabilities in genomic sequences have been described. In our previous report on NKG2D (Siemaszko etal. 2021) we provided a comprehensive summary of the clinically associated genetic variations of its gene. As for the NKG2C gene, two single-nucleotide polymorphisms (SNPs): rs10587111 and rs10588530 have been identified recently in a set of genetic mutations occurred at higher frequency in patients infected with influenza A (H7N9) (Chen etal. 2016). There are three reported alleles of NKG2C. Two of them, named, respectively, *01 and *02 by Shum etal. (2002), were found in individuals of different ethnicities. They differ in two single-nucleotide non-synonymous polymorphisms (c.5G > A, Ser2Asn, and c.305C > T, Ser102Phe). The first one affects the cytoplasmic tail, while the second one is located in the stem connecting the transmembrane region with the ligand-binding domain. Third novel *03 allele, encoding asparagine 2, like NKG2C*02 and serine 102, like NKG2C*01, was recently discovered in two unrelated Caucasoids (Asenjo etal. 2021). One of the most characteristic features of the NKG2C is its genomic mutation resulting in deletion of a ~ 16-kb region encompassing the gene, first observed in a Japanese population (Hikami etal. 2003; Miyashita etal. 2004). The evidence for NKG2C deletion was found due to the polymorphism screening (Hikami etal. 2003) and the breakpoint was detected within the 292‐bp region 1.5–1.8kb telomeric from the 3’ untranslated region of the NKG2A. Fig. 1 Genomic organization of the CD94/NKG2 region on chromosome 12. In the natural killer gene complex, the genes encoding for NKG2A, NKG2C, NKG2E, NKG2F and NKG2D receptors as well as for the CD94 molecule, are located on the short arm of chromosome. The most important single-nucleotide polymorphisms of major receptors are detailed Archivum Immunologiae et Therapiae Experimentalis (2023) 71:9 1 3 Page 3 of 18 9 Deletion haplotype comes with a unique set of nucleotides around the breakpoint with several Alu repeats presented. Although the NKG2C deletion breakpoint is not located in the Alu sequence, the Alu repeats are usually involved in gene recombinations (Miyashita etal. 2004). The NKG2C gene homozygous ~ 16kb deletion is distributed with frequency not exceeding 5% of studied populations (Hikami etal. 2003; Li etal. 2015; López-Botet etal. 2014; Miyashita etal. 2004; Moraru etal. 2012; Muntasell etal. 2013; Thomas etal. 2012; Toson etal. 2022). However, studies on West African populations from Gambia and Guinea-Bissau revealed that NKG2C deletion homozygosity occurred with almost 14% frequency (Goncalves etal. 2016; Goodier etal. 2014). This indicates that the NKG2C gene is not essential for survival and reproduction. Its deletion may be compensated possibly by the NKG2E gene (Hikami etal. 2003; Miyashita etal. 2004). The consequence of NKG2C deletion has been investigated in many studies, reviewed in the next paragraphs. The NKG2C wild-type (wt) allele has a protective effect for human cytomegalovirus (HCMV) viremia and the deletion (del) variant was associated with higher risk of HCMV viremia and disease development (Rangel-Ramírez etal. 2014). The high frequency of viremia and HCMV-disease was also observed after lung transplantation in patients with del/del homozygous genotype (Goodier etal. 2014; Vietzen etal. 2018). It was reported that patients carrying del/del genotype lacked NKG2C expression, while in wt/del heterozygotes the expression was intermediate (Muntasell etal. 2013). Furthermore, as reported by Muntasell etal. (2013), in NKG2C wt/wt HCMV+ individuals the receptor’s expression affects the NKG2Cbright NK cells by increasing their number. The NKG2C genotype also modulates HCMVinduced expansion of NKG2C+ cells. HCMV-seropositive NKG2C wt/wt children and adults express higher numbers of NKG2Cbright cells than hemizygous NKG2C wt/del subjects. Moreover, the quantitative differences in surface expression of NKG2C as well as in the response to receptor engagement were also noticed (López-Botet etal. 2014). Significance ofInteraction Between HLA‑E Molecule andits NKG2A andNKG2C Receptors Being a member of class Ib non-classical human leukocyte antigens (HLA) molecules, HLA-E is expressed constitutively on B and T lymphocytes, NK cells, monocytes, trophoblasts, and also in tumour cells (Coupel etal. 2007). The HLA-E-encoding gene is located in chromosome 6. It is well known that HLA-E is not as polymorphic as the other HLA molecules and it has just two dominant alleles (*01:01 and *01:03) distributed with almost identical frequencies (Iwaszko and Bogunia-Kubik 2011). HLA-E maintains a specific and unique role in modulating immune response: it can interact either with the activating CD94/NKG2C or the inhibitory CD94/NKG2A receptor. Structural studies have revealed that both human inhibitory NKG2A and activating NKG2C receptors possess sequences identical in over 75%, however, inhibitory CD94/NKG2A receptor binds to the HLA-E with approximately six-times higher affinity than CD94/NKG2C (Dissociation constant, KDs ranging from 0.7μM to ∼20μM for NKG2A and KDs ranging from ∼4μM to > 0.1mM for NKG2C) (Kaiser etal. 2005; Valés-Gómez etal. 1999). Variations in the residues forming part of heterodimer interface (165–168 in NKG2C and 167–170 in NKG2A) were found to be responsible for this affinity disparity (Kaiser etal. 2008). Both CD94/NKG2A and CD94/NKG2C bind to the HLA-E α1/α2 domain by recognizing different, but partially overlapping HLA-E epitopes (Wada etal. 2004). A study investigating the effect of HLA-E, expressed by target cells, on a NKG2A+ and NKG2C+/NKG2A– NK cell subsets, revealed that basal levels of HLA-E activate NKG2A+ cells without impact on activation of NKG2C+ NK cells (Kaiser etal. 2005; ValésGómez etal. 1999). Additionally, the relationship between NKG2C copy number and the NK-cell compartment was not dependent on the HLA-E dimorphism (Muntasell etal. 2013). Though NKG2A and NKG2C receptors are not simultaneously expressed on peripheral CD56dim NK cells, however, almost all decidual CD56bright NK cells and peripheral CD56bright NK cells with active NKG2C simultaneously expressed NKG2A and inhibitory receptor prevails (Kusumi etal. 2006; Sáez-Borderías etal. 2009). Summarizing the above, HLA-E upholds the balance of NK cell reactivity, following the principle that if both receptors compete in binding to a specific HLA-E molecule, the inhibitory receptor is the preferable one (Lauterbach etal. 2015). The favoured binding to inhibiting CD94/NKG2A is a crucial feature of monitoring the MHC class I expression on normal cells (Joyce and Sun 2011). Under normal conditions, HLA-E presents conserved peptides derived from HLA-I leader sequences (Fig.2A). HCMV, like other viruses, evolved a strategy to avoid NK cell killing in HLA I class down-regulated conditions. Virus protein gpUL40 delivers a peptide that mimics leader sequences and binds efficiently to HLA-E molecule inducing its expression (Prod'homme etal. 2012; Tomasec etal. 2000; Ulbrecht etal. 2000). Sequencing of viral UL40 DNA from HCMV isolates provided data about heterogeneity in the repertoire of UL40 peptides (Hammer etal. 2018; Heatley etal. 2013). Mutations in UL40 have an impact on NK cell activation, and remain under host immunological pressure. Most frequently occurring strains expressed VMAPRTLIL, VMAPRTLLL and VMAPRTLVL sequences. The HLA-E/ VMAPRTLIL complex is recognized by both NKG2A and NKG2C, however, binding affinity for NKG2C is six-fold Archivum Immunologiae et Therapiae Experimentalis (2023) 71:9 1 3 9 Page 4 of 18 Fig. 2 Impact of the peptide presented by HLA-E on balance between NKG2C and NKG2A contradictory signals. Under normal conditions, with basal levels of HLA-E, the NKG2A/CD94 complex is activated by a broad set of peptides derived from the HLA-I leader sequences. This represents a monitoring mechanism for controlling MHC class I expression on normal cells (A). When HLA-E is loaded with VMAPRTLFL peptide from the HLA-G leader sequence, the binding affinity of such complex is higher for the activating CD94/ NKG2C than for the CD94/NKG2A. Activation signal might cause internalization and degradation of the receptor, preventing excessive NK cell reactivity (B). During HCMV infection, HLA-E presents peptides derived from viral gpUL40 which mimic HLA I leader sequences and induce HLA-E expression. Most frequently occurring strains expressed VMARPRTLIL, VMARPRTLLL and VMARPRTLVL with higher affinity for NKG2A, avoiding therefore NK cell attack (C). VMAPRTLFL peptide from rare HCMV strain is identical to HLA-G peptide and is sufficient for inducing effector functions in NKG2C+ NK cells without co-stimulatory signals (D) Archivum Immunologiae et Therapiae Experimentalis (2023) 71:9 1 3 Page 5 of 18 9 lower than for NKG2A (Heatley etal. 2013). Invitro analysis revealed a gradient in activating properties of UL40 peptides on a subset of NKG2+C NK cells (Hammer etal. 2018). The most frequently occurred peptides have low affinity for NKG2C, while the rare VMAPRTLFL peptide (identical to HLA-G derived signal peptide) is sufficient for inducing effector functions in NKG2C+ NK cells without co-stimulatory signals like interleukin (IL)-12 and IL-18. Viral infections also hamper transporter associated with antigen processing (TAP)-mediated peptide transport. HLA-E can be, therefore, loaded with broad set of unusual peptides with binding motif similar to that of HLA-A*02:01 (Lampen etal. 2013), up to 15 amino acids in length (Hò etal. 2020). More detailed research have demonstrated further impact of the peptide presented by HLA-E on the balance between NKG2C and NKG2A contradictory signals. The CD94/ NKG2A complex has a wide range of interacting peptides resulting in inhibition of NK cells (Lauterbach etal. 2015). In contrast, CD94/NKG2C is regulated by HLA-E loaded with restricted peptide repertoire (Fig.2B). One of best studied is the nonameric peptide (VMAPRTLFL) derived from the signal sequence of HLA-G. Other peptides that have higher binding affinity for NKG2C than for NKG2A, but to a lesser extent are: HLA-B27 leader peptide (Prašnikar etal. 2021), A80 (VMPPRTLLL), B13 (VTAPRTLLL) (Lauterbach etal. 2015), B7 (VMAPRTVLL), B58 (VTAPRTVLL), Cw3 (VMAPRTLIL), Cw4 (VMEPRTLIL), Cw7 (VMAPRALLL) (Valés-Gómez etal. 1999). However, the invitro studies showed that HLA-B2705 and HLA-Cw0702 proteins cannot be considered as efficient (Llano etal. 1998; Navarro etal. 1999). UL40 induces surface expression not only of the HLA-E, but also of the gpUL18, a HCMV-encoded HLA-I homologue (Prod'homme etal. 2012). This protein is capable of forming complexes with β2-microglobulin (Browne etal. 1990) and endogenous peptides (Fahnestock etal. 1995), including UL40-derived sequences. The surface plasmon resonancebinding studies demonstrated that the gpUL18 weakly binds to the CD94/NKG2C but shows no interaction with CD94/ NKG2A receptor invitro (Kaiser etal. 2008). This finding highlighted a potential role of the CD94/NKG2 receptors in direct responses to the virally encoded proteins, like HCMV gpUL18. HLA-E presenting HLA-G nonamer binds to the NKG2 receptors with the highest affinity of any others combination tested (Kaiser etal. 2005, 2008; Lauterbach etal. 2015; Llano etal. 1998; Valés-Gómez etal. 1999). The HLA-Gderived HLA-E ligand appears to interact preferentially with the activating CD94/NKG2C receptor (Heatley etal. 2013; Llano etal. 1998). HLA-G is expressed in immuneprivileged tissues and in virally (e.g. HCMV) infected cells (Onno etal. 2000; Yan etal. 2009), and, as report by Hò etal. (2020), it is recognized by the NKG2A, as well as ILT2 and KIR2DL4. It plays an important role in pregnancy, transplantations and malignancies (Hò etal. 2020), locally inducing immunological tolerance. The HLA-G level increases when exposed to cytokines (such as interferon (IFN)-γ, IL-10 or transforming growth factor-β), hypoxia or heat stress (Jasinski-Bergner etal. 2022). On the other hand, interaction of HLA-G with NKG2C might cause internalization and degradation of the receptor (Lauterbach etal. 2015). This downregulating mechanism might be another one that prevents excessive NK cell reactivity. Peptides with activating potential on NK cells should cause a destabilization CD94/NKG2C heterodimer with simultaneous NKG2C and DAP12 communication adjustment (Prašnikar etal. 2021). Molecular dynamics studies revealed that the influential peptide maintains a unique hydrogen bonding network among receptor-ligand “lock and key”-like complex, appropriate for NK cell activation (Prašnikar etal. 2021). Recently, it has been shown that the peptide derived from the non-structural protein 13 of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) prevents binding of HLA-E to NKG2A inhibitory receptor, leading target cell to be more susceptible to NK cell attack (Hammer etal. 2022). Therefore, high frequencies of NKG2A+ NK cells are found in patients diagnosed with coronavirus disease 2019 (COVID-19), which limits replication of SARS-CoV-2 in lung epithelial cells invitro. Partially similar mechanism occurs when HLA-E presents peptides derived from hsp60. However, such complex interferes with binding with either NKG2A or NKG2C, making itself unrecognizable to both receptors. This indicates that NKG2 receptors are peptide selective (Michaëlsson etal. 2002). NKG2C inViral Infections Human Cytomegalovirus The NKG2C receptor is well known for its role in many viral infections, especially in HCMV infection. An invitro study conducted on peripheral blood lymphocytes cocultured with HCMV-infected fibroblasts resulting in expansion of NKG2C+ NK cells was the very first evidence of expansion of these cells in response to HCMV infection (Gumá etal. 2006a). This effect was confirmed in many subsequent reports (Gumá etal. 2004; Heatley etal. 2013; Hendricks etal. 2014; López-Botet etal. 2014; Lopez-Vergès etal. 2011; Muntasell etal. 2013), highlighting the critical role of NKG2C in HCMV infection, but from the other side uncovered the impact of HCMV on NK cell subsets composition. NKG2C+C NK cells belong to separated NK cell cluster named adaptive NK cells. Transcriptomic profile of adaptive NK cells derived from bone marrow revealed that not every Archivum Immunologiae et Therapiae Experimentalis (2023) 71:9 1 3 9 Page 6 of 18 adaptive NK cell has high NKG2C expression (Yang etal. 2019). When compared to the conventional NK cell subsets, adaptive NK cells displayed a very distinct profile marked by upregulation of NKG2C, CD3E, PATL2 transcription, downregulation of CD7, KLRB1 and FCER1G, low NKp30, CD161, NKG2A surface expression (Rückert etal. 2022). In HCMV+ individuals, adaptive NK cells are characterized by reduction in the expression of FCER1G, ZBTB16, SYK, and EAT-2 compared to other NK subsets (Schlums etal. 2015). Differences in FCER1G, ZBTB16 expression were found between NKG2C+ and NKG2C– NK cells subset of HCMV-seropositive patients (Rückert etal. 2022). Epigenetic characteristics of adaptive NK cells depict unique chromatin remodelling within the NKG2 region, which results in NKG2C upregulation and NKG2A downregulation. Analysis of cis-regulatory elements revealed that adaptive NK cells exhibit increased activity of AP1 motifs, which encode transcription factors involved in defining inflammatory memory in different immune cell types (Larsen etal. 2021). While the expansion of NKG2C+ NK cells in the condition of CMV infection seems to be well proven, little is known about the mechanism behind this phenomenon (Fig.3). There are some examples supporting the idea of NKG2C/CD94 being directly involved in NK cell expansion. In a presence of blocking anti-CD94 monoantibodies, the stimulation of HCMV+ donor peripheral blood mononuclear cells (PBMCs) with virus-infected fibroblasts promoted expansion of NKG2C+ NK cells and the NKG2C deletion also has influence on this effect (López-Botet etal. 2014). Furthermore, the engagement of proinflammatory IL-12 produced by CD14+ monocytes has been regarded as a potential motor agent in NKG2C+ NK cells expansion (Rölle etal. 2014). Despite the fact that some clinical observations indirectly suggest that NKG2C+ cells contribute to control HCMV replication invivo, their invitro response to HCMVinfected cells appears unexpectedly modest and there is no evidence for a triggering role of NKG2C in this system. On the other hand, recent results support that NKG2Cbright NK cells are potent effectors of antibody-dependent cellular cytotoxicity (ADCC), and that HCMV-specific antibodies specifically trigger cytotoxicity and cytokine production against infected cells. Remarkably, little information is available regarding the involvement of ADCC in the immune response to HCMV (López-Botet etal. 2014). As they become more mature, the NK cells express more CD57, known as their maturation marker, which is associated with increased cytokine production and ADCC properties (LopezVergès etal. 2010). The CD57+NKG2C+ NK adaptive cell subset characterises with endurance and resistance to apoptosis. These features correlate with transcriptional changes and epigenetic remodelling, e.g. demethylation of noncoding sequence 1 in the IFN-γ gene locus (Tarantino etal. 2021). The presence of HCMV alters the expression of the CD94/ NKG2 receptors. It has been proved that HCMV-seropositive Fig. 3 Expansion of adaptive CD56dim NKG2C + NK cells driven by HCMV infection. As a subset of adaptive NK cells, the NKG2C + NK cells display distinct expression profiles when compared to conventional NK clusters Archivum Immunologiae et Therapiae Experimentalis (2023) 71:9 1 3 Page 7 of 18 9 adults and children characterise with increased number of NKG2C+ cells (Béziat etal. 2013; Heatley etal. 2013; Monsiváis-Urenda etal. 2010). Acute CMV infection promotes high NKG2C+ NK cells proliferation, often called NKG2C(hi) NK cells, which eventually leads them to acquire CD57. Such a unique NKG2C(hi) CD57+ NK cell subset may be responsible for a specific NK cell memory (Lopez-Vergès etal. 2011). Interestingly, these cells were found to respond specifically to HCMV, but not to Epstein-Barr virus (EBV) infection (Hendricks etal. 2014). NK cells are believed to control the viral infection in the absence of T cells. The NKG2C+ lymphocytosis in a patient suffering from acute HCMV infection coincided with a significant reduction of viremia, when the T cells were absent (Kuijpers etal. 2008). The HCMV seropositivity status increases the numbers of both NKG2C+ NK and T cells (Muntasell etal. 2013). The NKG2C+ NK cells may also regulate the CMV-specific CD8 T cells, which express the HLA-E ligand. In the CMV seropositive individuals, the expansion of CMV-specific CD8 T cells is negatively regulated upon cell activation (Grutza etal. 2020). An invitro study confirmed that, interestingly, in HCMV-infected monocyte-derived dendritic cells cultures, the endogenous IL-12 secretion affected the NKG2A expression in NKG2C+ cells and, therefore, it may be responsible for modulation of the response against HCMVinfected cells. This effect is beneficial for the virus, as the induced expression of CD94/NKG2A strengthens the viral immune evasion mechanisms (Sáez-Borderías etal. 2009). Lack of FcεRγ (FcRγ) expression is another adaptive NK cells marker in response to HCMV. The NKG2Cbright and FcRγ− NK cell population was found in HCMV+ subjects. Moreover, the loss of FcRγ was accumulated within the NKG2Cbright NK cell subset in the NKG2C wt/wt individuals, and the NKG2C−FcRγ− cell population was more frequent in NKG2C wt/del and NKG2C del/del individuals (Muntasell etal. 2016). Controversially, the recent data suggest that individuals who lack expression of NKG2C show an undisturbed immune response to HCMV infection and the NK cell maturation is not altered by the absence of CD94/NKG2C receptor. This suggests that there are some alternative pathways, which provide similar NK cells activity in case of lacking this receptor (Toson etal. 2022). Besides that, many reports reviewed in the next chapters highlight the key role of the HCMV-induced NKG2C+ NK cell expansion observed in other viral infections and inflammatory conditions. Hepatitis B andHepatitis C Virus The expansion of NKG2C+ NK cells in chronic hepatitis patients is associated with an underlying HCMV infection, pointing out the key role of this pathogen in the NK cell activity (Béziat etal. 2012; Malone etal. 2017). Interestingly, there are phenotypic and functional differences in the NK cell repertoire in chronic hepatitis B virus (HBV) infection versus hepatitis C virus (HCV). In case of the HCV infection, the proportion of activated, but more dysfunctional NK cells was higher compared to the HBV-infected individuals. On the other hand, expression of CD94/NKG2C was higher in HBV-infected when compared to HCV-infected and healthy individuals. Additionally, number of circulating NK cells was also lower in HBV and HCV-infected than in control (Oliviero etal. 2009). The NKG2C+ NK cell levels can be considered as a prognostic marker of HBV improved treatment responses. The pegylated interferon (PEG-IFN)-α and entecavir are well-known antiviral drugs for HBV treatment. The clinical study conducted on patients suffering from chronic HBV showed that PEG-IFN-α responders have a significantly higher expression of NKG2C+ NK cells than non-responders, which suggests the reconditioning and activation of innate immune response during IFN treatment (Yan etal. 2015). Human Immunodeficiency Virus The presence of largely controversial reports obscures understanding the role of CD94/NKG2C receptor in HIV susceptibility, infection and progression. The presence of at least one NKG2C delvariant was previously indicated as a risk factor of HIV infection (Thomas etal. 2012). The NKG2C wt/wt and HLA-E*01:01/*01:01 genotypes were found to be involved in faster and more effective recognition of HIVinfected cells and were found less frequently in long-term non-progressors as well as in HIV-infected patients when compared to controls. Recently published results specify that the NKG2C del/del genotype is associated with HIV susceptibility, but only in people living with HIV and not in controls unexposed to HIV or HIV-exposed seronegative subjects (Alsulami etal. 2021; Guzmán-Fulgencio etal. 2013). Unexpectedly, findings from the latest study on HIV-infected Brazilians have questioned any impact of NKG2C genotype on HIV susceptibility (Toson etal. 2022). No association of NKG2C deletion with HIV-1 susceptibility or influence on clinical features was observed in the evaluated cohort. The NKG2C copy number did not correlate with HIV viral load (Alsulami etal. 2021). Therefore, more genetic studies on larger populations are necessary to better understand this phenomenon. Unlike other NK cell receptors, the expression of CD94/ NKG2C and its ligands is upregulated in HIV patients, thus, the NKG2C may be involved in regulating the infection progress. Many studies indicate that changes in NK receptors repertoire in HIV-1 positive patients are derived from an underlying HCMV coinfection. Advanced stages of HIV-1 disease in patients coinfected with HCMV are characterized by downregulation of CD94/NKG2A and Archivum Immunologiae et Therapiae Experimentalis (2023) 71:9 1 3 9 Page 8 of 18 increased expression of CD94/NKG2C (Brunetta etal. 2010; Gumá etal. 2006b; Zhou etal. 2015). This reversed NKG2A/NKG2C ratio is unique for NK cells, but not for CD8+ NKG2C+ T cells (Zeddou etal. 2007). In a Hispanic case study, high level of memory-like NKG2C+ NK cells was associated with the control of HIV-1 viral replication (Climent etal. 2023). However, the high level of NKG2C+ NK cells increases the risk of Kaposi sarcoma in patients with HIV-1 infection (Goodier etal. 2007). In summary, the current knowledge of NKG2C+ NK cells at mucosal genital/anal sites is still limited, and the NK receptor profile of circulating NK cells may differ from the tissue-resident NK which interact with HIV-infected cells (Alsulami etal. 2021). SARS‑CoV‑2 andCOVID‑19 There are studies conducted on COVID-19 patients (Jaiswal etal. 2022a; Vietzen etal. 2021a) indicating that NKG2C deletion is a risk factor for more severe disease outcome. One of those studies showed that both HLA-E*01:01 and NKG2C del variants are more frequently present in hospitalized Austrian COVID-19 patients, and both genotypes are independent risk factors for severe COVID-19 (Vietzen etal. 2021a). The CD94/NKG2C expression may also be related to the severity of infection. Increased number of circulating NKG2C+ NK cells observed in patients suffering from severe COVID-19 was independent of HCMV reactivation and did not correlate with serum levels of anti-CMV IgG. Authors identified upregulated HLA-E in bronchoalveolar lavage (BAL) fluid of COVID-19 patients, and suggested, therefore, a receptor-ligand-driven expansion of adaptive NK cells (Maucourant etal. 2020). Interestingly, even after clearance of SARS-CoV-2, patients with lower recovery of NKG2C+ adaptive NK cells were characterized by HCMV reactivation and increased mortality (Jaiswal etal. 2022a). Interesting results were obtained in a study on Mycobacterium w (Mw), which is used as an immunomodulator in India. The prophylaxis with Mw used in HCMV-seropositive high-risk cohort resulted in six-fold reduction in incidence of symptomatic COVID-19 over a 6-month period. In a response to Mw, the number of NKG2C+ adaptive NK cells increased between 30 and 60days following treatment. Moreover, the lower baseline of NKG2C+ NK cells predisposed to the disease (Jaiswal etal. 2022b). These findings suggest that a higher number of NKG2C+ cells is beneficial and provides a protective effect in SARS-CoV-2 infection, whereas a decreased number of these cells, together with the NKG2C del variant, weakens it. Interestingly, the NK cell subsets differ in mammalian target of rapamycin activity. Study on rapamycin treatment showed that the percentage of mature NKG2A−CD16+ CD57+ cells significantly increased during treatment and that the level of FcRγ was lower in all NK cell subsets (Shemesh etal. 2022). Other Viral Infections The NKG2C deletion did not seem to influence the clinical course of herpetic (herpes simplex type 1 virus) (Moraru etal. 2012), and papillomavirus infection (Vilchez etal. 2013), but predispose to the development of nephropathia epidemica in severe Puumala orthohantavirus cases (Vietzen etal. 2021b). No expansions of circulating NKG2C+ NK cells have been observed in studies of patients with recurrent genital herpes simplex virus type 2 infections (Björkström etal. 2011b), likewise in EBV infection in adults (Hendricks etal. 2014). HCMV+ EBV+ coinfected children had significantly higher proportions of peripheral-blood NKG2C+ NK cells than HCMV+ EBV− children. These results provide further evidence that the expansion of NKG2C+ NK cells is a consequence of CMV-driven immune maturation (Saghafian-Hedengren etal. 2013). NKG2C+ NK cell increased expansion was observed in HCMV coinfection with other acute and chronic viral infections: Hantavirus (Björkström etal. 2011a), Chikungunya (Petitdemange etal. 2011), dengue virus-2 (Petitdemange etal. 2016). But on the contrary, results from the latest study on Puumala orthohantavirus indicate that NKG2C+ NK cell proliferation and effector functions during virus infections can be also induced independent of prior HCMV infection, for example by the cellular stress response (Vietzen etal. 2021b). Pregnancy andRelated Complications Affected byNKG2C Uterine NK cells, accumulated during early pregnancy at the maternal–fetal interface, are engaged in placentation, angiogenesis and regulation of trophoblast invasion (Hanna etal. 2006). It is believed that their dysfunction is a major factor in pathological pregnancies, including pre-eclampsia. The expression of both CD94/NKG2A and CD94/NKG2C receptors is increased on peripheral NK cells from women diagnosed with pre-eclampsia. It might be explained by the scenario in which NK cells try to balance the proper inhibitory/activating receptors’ expression during ongoing systemic inflammation, characterized by high levels of IL-12 and IL-15 (Bachmayer etal. 2009). In accordance, another study revealed expansion of NKG2C+ NK cells in pre-eclampsia women. Furthermore, both CD56– and CD56+ cell subsets were characterized with increased expression of NKG2A receptor. Women suffering from severe pre-eclampsia had an increased percentage of Archivum Immunologiae et Therapiae Experimentalis (2023) 71:9 1 3 Page 9 of 18 9 CD56bright NKG2C+ NK cells (Bueno-Sánchez etal. 2013). The role of NKG2C deletion in pre-eclampsia susceptibility has been excluded in a Brazilian cohort study (Kaminski etal. 2019). Although the HCMV causes the majority of intrauterine viral infections, in the first trimester of pregnancy the congenital infection rate is considered to be low. The first trimester is usually the time when the uterus is infiltrated by decidual NK (dNK) cells, which are propitious for placentation. When exposed to HCMV, these dNK cells, normally responsible for chemokines and cytokines production, become cytotoxic for infected autologous decidual fibroblasts. The dNK cells may be involved in controlling the HCMV intrauterine infection and in limiting the viral spreading into fetal tissues. Over 80% of these dNK cells become NKG2C+ NK cells (Siewiera etal. 2013). Simultaneously, only a minor decrease of NKG2A+ cells was observed. An interesting observation was made in relation to multiple pregnancies. Repeated pregnancies are often related to improved placentation, possibly provided by pregnancy trained decidual NK cells. These cells, involved in vascular remodelling and angiogenesis, are characterized by high expression of NKG2C receptor in HCMV-seropositive women (Gamliel etal. 2018). This observation may lead to question, whether the HCMV serostatus is involved in this NK cells augmentation. Study on endometrium samples from HCMV-seropositive and HCMV-seronegative women showed that endometrial NK cells have an increased pregnancy-induced LILRB1 expression on NKG2C+ cells in HCMV-seropositive women. The HCMV serostatus may be, therefore, involved in the induction of pregnancy-induced memory endometrial NK cells, providing effective placentation (Feyaerts etal. 2019). The NK cell receptors’ distribution is also altered by the postnatal symptomatic HCMV infection in preterm infants, which is characterized by an early increase of total NK cells, as well as NKG2C+ and KIR+ NK cell subsets with a simultaneous decrease of the cells expressing CD94/ NKG2A receptor (Noyola etal. 2015). Furthermore, in children who had symptomatic congenital HCMV infection, the increased number of NKG2C+ NK cells, and lower number of NKG2A+ NK cells was found when compared to asymptomatic or non-infected individuals. What is more interesting, this NKG2C+ cell increase was associated with NKG2C wt genetic variant, and the wt/wt homozygotes had a higher number of circulating NKG2C+ cells than heterozygotes (Noyola etal. 2012). Clinical Outcome ofAllogeneic Transplantation ofHematopoietic Stem Cells andNKG2C Hematopoietic stem cell transplantation (HSCT) is a curative treatment for patients whose bone marrow or immune system is damaged or defective. The commonly used clinical practice in allogeneic setting is donor and recipient HLAtyping and determination of the relationship between various alleles and HSCT outcome. Studies show that the relationship between NK cell receptors and their ligands may play a crucial role in the prediction of HSCT outcome (Bogunia-Kubik and Łacina 2021; Isernhagen etal. 2015; Tsamadou etal. 2017, 2019; Yu etal. 2022). The reactivation of HCMV infection remains to be one of the most common post-transplant complications. It is especially threatening to immunodeficient and immunosuppressed patients (LópezBotet etal. 2014). NK cells are the first lymphoid subset that can be detected in recipients’ peripheral blood after HSC or cord blood transplantations, however, differences between NK cell development patterns may occur. Patients who experienced the reactivation of HCMV infection after cord blood transplantation characterized with a more rapid NK cell maturation, and expression of NKG2C+ NK cells was increased in comparison to patients without reactivation (Della Chiesa etal. 2012). Interestingly, the early reactivation of HCMV infection is related with lower risk of relapse in acute myeloid leukaemia (AML) patients, unleashing the graft-versus-leukaemia properties. This therapeutic effect was observed similarly in patients diagnosed with chronic myeloid leukaemia, under Dasatinib treatment, where the expansion of NK cells is related to HMCV reactivation (López-Botet etal. 2014). Reduced risk of leukaemia relapse may also be associated with expansion of CD56dimCD57+NKG2C+ NK cells. HSCT recipients treated with reduced-intensity conditioning, who developed HCMV reactivation, were characterized with low relapse rate and better disease-free survival when compared to recipients typed as HCMV-seronegative, suggesting the major role of adaptive NK cell in response to infection (Cichocki etal. 2016; Muñoz-Cobo etal. 2014). Development of HCMV replication after allogeneic HSCT was proven to be a strong and independent predictor of a reduced leukemic relapse risk, and increased long-term survival in early and advanced stages of AML (Elmaagacli etal. 2011). Besides the altered expression, the CDD94/NKG2C phenotype is also a major factor in the development of posttransplant complications. The NKG2C del variant in cord blood transplant recipients increases the risk of HCMV reactivation. In recipients without HCMV reactivation, the wild-type variant was dominant (Mehta etal. 2016). NKG2C wt/wt homozygosity was beneficial to the reconstitution and Archivum Immunologiae et Therapiae Experimentalis (2023) 71:9 1 3 9 Page 16 of 18 Jaiswal SR, Bhakuni P, Bhagwati G etal (2020) Alterations in NKG2A and NKG2C subsets of natural killer cells following Epstein-Barr virus reactivation in CTLA4Ig-based haploidentical transplantation is associated with increased chronic graft-versus-host disease. 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