TLR2/4 are novel activating receptors for SARS-CoV-2 spike protein on NK cells.

Background: In early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s). Methods: SP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs. Results: SPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56brightNK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56dimNK cell functions in COVID-19 recovered, but not in non-infected, individuals. Conclusions: TLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID-19 subjects.

A De Novo CaSR Missense Variant in Combination with Two Inherited Missense Variants in CFTR and SPINK1 Detected in a Patient with Chronic Pancreatitis.

Chronic pancreatitis is often secondary to alcohol abuse, but pancreatitis with no other aetiology is frequently associated with variants in genes encoding proteins related to zymogen granule activation. Our goal was to identify genomic variants in a patient by analyzing an extended panel of genes associated with the intra-pancreatic activation of the trypsin pathway. A 23-year-old woman was addressed at our institution because of chronic pancreatitis of unknown aetiology presenting recurrent episodes since she was the age of four. Next Generation Sequencing was performed to analyze a panel of nine genes associated with pancreatitis (CaSR, CFTR, CPA1, CTRC, CTSB, KRT8, PRSS1, PRSS2, and SPINK1). Three missense variants were found: p.Leu997Phe, maternally inherited, in the CFTR gene; p.Ile73Phe, paternally inherited, in the SPINK1 gene; and p.Phe790Ser, a de novo variant, in the CaSR gene. They were classified, respectively as probably benign, a Variant of Uncertain Significance, and the last one, which has never been described in the literature, as likely being pathogenic following American College of Medical Genetics and Genomics standard guidelines. Extensive intra-pancreatic activation of trypsin pathway gene sequencing detected rare variants that were not found with other gene screening and showed that variants in different genes may interact in contributing to the onset of the pancreatitis phenotype.

The anti-inflammatory cytokine IL-37 improves the NK cell-mediated anti-tumor response.

IL-37 is a member of the IL-1 superfamily exerting anti-inflammatory functions in a number of diseases. Extracellular IL-37 triggers the inhibitory receptor IL-1R8 that is known to regulate different NK cell pathways and functional activities including their anti-tumor effect. However, the effect of IL-37 on human NK cell functions is still to be unveiled. This study aimed to investigate the functional effect of IL-37 in human NK cells activated with IL-15. We found that IL-37 enhanced both NK cell cytotoxic activity against different tumor cell lines and cytokines production. These effects were associated with increased phosphorylation of ERK and NF-Kb. The improved NK cell activity was also strictly related to a time-dependent GSK3ß-mediated degradation of IL-1R8. The enhanced activation profile of IL-37 treated NK cells possibly due to IL-1R8 degradation was confirmed by the results with IL-1R8-silenced NK cells. Lastly, in line with these data, through the analysis of the TNM plot database of a large group of patients, IL-37 mRNA expression was found to be significantly lower in colon and skin cancers than in normal tissues. Colon adenocarcinoma and neuroblastoma patients with higher IL-37 mRNA levels had significantly higher overall survival, suggesting that the presence of IL-37 might be considered an independent positive prognostic factor for this tumor. Our results provide novel information on the mechanisms regulating IL-1R8 function in human NK cells, highlighting the IL-37-IL-1R8 axis as a potential new target to improve the anti-tumor immune response.

Determining Endosomal Toll-Like Receptors Gene Expression in NK Cells After Stimulation with Specific Agonists.

Poor knowledge is currently available about the biology of Toll-like receptors (TLRs) in natural killer (NK) cells. This is particularly due to the old belief that NK cells are unable to specifically eliminate microbes without presensitization. On the contrary, it has been clearly demonstrated that not only they can be activated through the engagement of Toll-like receptors (TLRs) by microbial molecules, but also that this interaction induces NK cells to release cytokines that, in turn, activate other cells of both innate and adaptive immunity. For this reason, immunotherapy based on local infusion of TLRs ligands is currently considered as a novel potential strategy to treat solid tumors. Here, we provide a protocol to efficiently stimulate NK cells via endosomal TLRs agonists and to determine endosomal TLRs gene expression level. This protocol can be used for in vitro investigation into endosomal TLRs function in NK cells under different conditions.

Human toll-like receptor 8 (TLR8) in NK cells: Implication for cancer immunotherapy.

Toll-like receptors (TLR)s are homo- or heterodimeric proteins, whose structure and function were widely described in the antigen presenting cells (APC), such as Dendritic cells (DC). Recently, the expression and the role of TLRs in fighting against pathogens, was described also in NK cells. Their activation and functional properties can be directly and indirectly modulated by agonists for TLRs. In particular CD56bright NK cells subset, that is the most abundant NK cell subset in tissues and tumor microenvironment (TME), was mostly activated in terms of pro-inflammatory cytokine production, proliferation and cytotoxicity, by agonists specific for endosomal TLR8. The interplay between DC and NK, that depends on both cell-to-cell contact and soluble factors such as cytokines, promote both DC maturation and NK cell activation. Based on this concept, a TLR based immunotherapy aimed to activate NK-DC axis, may modulate TME by inducing a pro-inflammatory phenotype, thus improving DC ability to present tumor-associated antigens to T cells, and NK cell cytotoxicity against tumor cells. In this mini-review, we report data of recent literature about TLRs on human NK cells and their application as adjuvant in cancer vaccines or in combined tumor immunotherapy.

Epitope characterization of a monoclonal antibody that selectively recognizes KIR2DL1 allotypes.

Killer immunoglobulin-like receptor (KIR) genes code for a family of inhibitory and activating receptors, finely tuning NK cell function. Numerous studies reported the relevance of KIR allelic polymorphism on KIR expression, ligand affinity, and strength in signal transduction. Although KIR variability, including gene copy number and allelic polymorphism, in combination with HLA class I polymorphism, impacts both KIR expression and NK cell education, only a precise phenotypic analysis can define the size of the different KIRpos NK cell subsets. In this context, reagents recognizing a limited number of KIRs is essential. In this study, we have characterized the specificity of an anti-KIR mAb termed HP-DM1. Testing its binding to HEK-293T cells transfected with plasmids coding for different KIRs, we demonstrated that HP-DM1 mAb exclusively reacts with KIR2DL1. Using site-directed mutagenesis, we identified the four amino acids relevant for HP-DM1 recognition: M44, S67, R68, and T70. HP-DM1 mAb binds to a conformational epitope including M44, the residue crucial for HLA-C K80 recognition by KIR2DL1. Based on the HP-DM1 epitope characterization, we could extend its reactivity to all KIR2DL1 allotypes identified except for KIR2DL1*022 and, most likely, KIR2DL1*020, predicting that it does not recognize any other KIR with the only exception of KIR2DS1*013. Moreover, by identifying the residues relevant for HP-DM1 binding, continuously updating of its reactivity will be facilitated.

IL-1R8 silencing improves the anti-tumor function of freshly isolated human NK cells.

The inhibitory receptor interleukin-1 receptor 8 (IL-1R8) has been recently recognized to be expressed also by human natural killer (NK) cells. This study was aimed to design and optimize IL-1R8 silencing conditions in human NK cells to precisely establish the activity of such receptor in these cells. Electroporation of freshly isolated or IL-2-cultured NK cells with small interfering RNA (siRNA), resulted in a marked, even though variable, IL-1R8-silencing. Although the expression profile revealed downregulation of most genes involved in several intracellular pathways, some genes related to proliferation, expression of some chemokine receptors, antibody-dependent cell cytotoxicity and cytotoxic activity were upregulated in IL-1R8-silenced NK cells. Furthermore, upon IL-15 activation, the majority of genes involved in NK cell function were upregulated in IL-1R8-siRNA-compared with control-siRNA-transfected NK cells. More importantly, in agreement with these findings, the reduction of IL-1R8 gene expression levels resulted in enhanced expression of NK cell activation markers, production of cytokines and chemokines, and cytotoxic activity against several NK cell targets with different susceptibility to NK-mediated lysis. Similar results were obtained following stimulation with IL-18. All together these data, deeply impacting on the main effector functions of human NK cells, can lead to a better understanding of IL-1R8-mediated regulation on these cells and to the design of new strategies for improving NK cell-mediated anti-tumor responses.

Toll-like receptor 8 agonists improve NK-cell function primarily targeting CD56brightCD16- subset.

BACKGROUND: Toll-like receptors (TLRs) are pattern-recognition sensors mainly expressed in innate immune cells that directly recognize conserved pathogen structures (pathogen-associated molecular patterns-PAMPs). Natural killer (NK) cells have been described to express different endosomal TLRs triggered by RNA and DNA sequences derived from both viruses and bacteria. This study was addressed to establish which endosomal TLR could directly mediate NK activation and function after proper stimuli. It was also important to establish the most suitable TLR agonist to be used as adjuvant in tumor vaccines or in combined cancer immunotherapies. METHODS: We assessed endosomal TLR expression in total NK cells by using RT-qPCR and western blotting technique. In some experiments, we purified CD56brightCD16- and CD56dimCD16+ cells subsets by using NK Cell Isolation Kit Activation marker, cytokine production, CD107a expression and cytotoxicity assay were evaluated by flow cytometry. Cytokine release was quantified by ELISA. NK cells obtained from ovarian ascites underwent the same analyses. RESULTS: Although the four endosomal TLRs (TLR3, TLR7/8, and TLR9) were uniformly expressed on CD56brightCD16- and CD56dimCD16+ cell subsets, the TLR7/8 (R848), TLR3 (polyinosinic-polycytidylic acid, Poly I:C) and TLR9 (ODN2395) ligands promoted NK-cell function only in the presence of suboptimal doses of cytokines, including interleukin (IL)-2, IL-12, IL-15, and IL-18, produced in vivo by other environmental cells. We showed that R848 rather than TLR3 and TLR9 agonists primarily activated CD56brightCD16- NK cells by increasing their proliferation, cytokine production and cytotoxic activity. Moreover, we demonstrated that R848, which usually triggers TLR7 and TLR8 on dendritic cells, macrophages and neutrophils cells, activated CD56brightCD16- NK-cell subset only via TLR8. Indeed, specific TLR8 but not TLR7 agonists increased cytokine production and cytotoxic activity of CD56brightCD16- NK cells. Importantly, these activities were also observed in peritoneal NK cells from patients with metastatic ovarian carcinoma, prevalently belonging to the CD56brightCD16- subset. CONCLUSION: These data highlight the potential value of TLR8 in NK cells as a new target for immunotherapy in patients with cancer.

The Latest Approach of Immunotherapy with Endosomal TLR Agonists Improving NK Cell Function: An Overview.

Toll-like receptors (TLRs) are the most well-defined pattern recognition receptors (PRR) of several cell types recognizing pathogens and triggering innate immunity. TLRs are also expressed on tumor cells and tumor microenvironment (TME) cells, including natural killer (NK) cells. Cell surface TLRs primarily recognize extracellular ligands from bacteria and fungi, while endosomal TLRs recognize microbial DNA or RNA. TLR engagement activates intracellular pathways leading to the activation of transcription factors regulating gene expression of several inflammatory molecules. Endosomal TLR agonists may be considered as new immunotherapeutic adjuvants for dendritic cell (DC) vaccines able to improve anti-tumor immunity and cancer patient outcomes. The literature suggests that endosomal TLR agonists modify TME on murine models and human cancer (clinical trials), providing evidence that locally infused endosomal TLR agonists may delay tumor growth and induce tumor regression. Recently, our group demonstrated that CD56bright NK cell subset is selectively responsive to TLR8 engagement. Thus, TLR8 agonists (loaded or not to nanoparticles or other carriers) can be considered a novel strategy able to promote anti-tumor immunity. TLR8 agonists can be used to activate and expand in vitro circulating or intra-tumoral NK cells to be adoptively transferred into patients.

KIR Variation in Iranians Combines High Haplotype and Allotype Diversity With an Abundance of Functional Inhibitory Receptors.

Natural killer (NK) cells are innate lymphocytes that eliminate infected and transformed cells. They discriminate healthy from diseased tissue through killer cell Ig-like receptor (KIR) recognition of HLA class I ligands. Directly impacting NK cell function, KIR polymorphism associates with infection control and multiple autoimmune and pregnancy syndromes. Here we analyze KIR diversity of 241 individuals from five groups of Iranians. These five populations represent Baloch, Kurd, and Lur, together comprising 15% of the ethnically diverse Iranian population. We identified 159 KIR alleles, including 11 not previously characterized. We also identified 170 centromeric and 94 telomeric haplotypes, and 15 different KIR haplotypes carrying either a deletion or duplication encompassing one or more complete KIR genes. As expected, comparing our data with those representing major worldwide populations revealed the greatest similarity between Iranians and Europeans. Despite this similarity we observed higher frequencies of KIR3DL1*001 in Iran than any other population, and the highest frequency of HLA-B*51, a Bw4-containing allotype that acts as a strong educator of KIR3DL1*001+ NK cells. Compared to Europeans, the Iranians we studied also have a reduced frequency of 3DL1*004, which encodes an allotype that is not expressed at the NK cell surface. Concurrent with the resulting high frequency of strong viable interactions between inhibitory KIR and polymorphic HLA class I, the majority of KIR-A haplotypes characterized do not express a functional activating receptor. By contrast, the most frequent KIR-B haplotype in Iran expresses only one functional inhibitory KIR and the maximum number of activating KIR. This first complete, high-resolution, characterization of the KIR locus of Iranians will form a valuable reference for future clinical and population studies.

An Anti-inflammatory microRNA Signature Distinguishes Group 3 Innate Lymphoid Cells From Natural Killer Cells in Human Decidua.

Innate lymphoid cells (ILCs) are a heterogeneous subset of lymphocytes deeply implicated in the innate immune responses to different pathogens, in lymphoid organogenesis and in the maintenance of tissue homeostasis. Group 3 innate lymphoid cells (ILC3) have been detected in human decidua, where they play a role in the early inflammatory phase favoring implantation and tissue remodeling as well as in the subsequent regulatory phase preventing fetal rejection and supporting neoangiogenesis. A balance between inflammation and immune tolerance is required to maintain pregnancy, thus maternal immune system must be controlled by finely tuned mechanisms. microRNAs (miRNAs) are small non-coding RNAs with important regulatory roles in immune cells, but their function in decidual ILC3 (dILC3) and in decidual NK (dNK) cells is still undefined. Here, we examined the miRNome by microarray in these cells during the first trimester of pregnancy and compared with miRNA profiles of peripheral blood NK (pbNK) cells from pregnant women. We show that distinct miRNA profiles could clearly distinguish dILC3 from NK cells. Correlation analyses revealed that dNK and pbNK miRNome profiles are more similar to each other as compared to dILC3. In particular, we identified 302 and 279 mature miRNAs differentially expressed in dILC3 as compared to dNK and pbNK, respectively. The expression of miR-574-3p and the miR-99b/let-7e/miR-125a miRNA cluster resulted the most increased in dILC3. Remarkably, gene ontology analysis and pathway enrichments of miRNA targets revealed an involvement of these miRNAs in the promotion of anti-inflammatory responses. In agreement to this finding, we also found a higher expression of the anti-inflammatory miR-146a-5p in dILC3 with respect to NK cells. Overall, our data identified specific miRNA signatures distinguishing dILC3, dNK, and pbNK cells. Our data suggest the existence of a tight epigenetic control mediated by miRNAs in dILC3, potentially acting as a brake to prevent exaggerated inflammatory responses and to maintain the immune homeostasis in the early phases of pregnancy.

Characterization of Human NK Cell-Derived Exosomes: Role of DNAM1 Receptor In Exosome-Mediated Cytotoxicity Against Tumor.

Despite the pivotal role of natural killer (NK) cells in defenses against tumors, their exploitation in cancer treatment is still limited due to their reduced ability to reaching tumor sites and the inhibitory effects of tumor microenvironment (TME) on their function. In this study, we have characterized the exosomes from IL2- or IL15-cultured human NK cells. Both cytokines induced comparable amounts of exosomes with similar cargo composition. Analysis of molecules contained within or exposed at the exosome surface, allowed the identification of molecules playing important roles in the NK cell function including IFN-γ, Lymphocyte Function-Associated Antigen (LFA-1), DNAX Accessory Molecule-1 (DNAM1) and Programmed Cell Death Protein (PD-1). Importantly, we show that DNAM1 is involved in exosome-mediated cytotoxicity as revealed by experiments using blocking antibodies to DNAM1 or DNAM1 ligands. In addition, antibody-mediated inhibition of exosome cytotoxicity results in a delay in target cell apoptosis. We also provide evidence that NK-exosomes may exert their cytolytic activity after short time interval and even at low concentrations. Regarding their possible use in immunotherapy, NK exosomes, detectable in peripheral blood, can diffuse into tissues and exert their cytolytic effect at tumor sites. This property offers a clue to integrate cancer treatments with NK exosomes.

KIR3DS1-Mediated Recognition of HLA-*B51: Modulation of KIR3DS1 Responsiveness by Self HLA-B Allotypes and Effect on NK Cell Licensing.

Several studies described an association between killer-cell immunoglobulin-like receptor (KIR)/HLA gene combinations and clinical outcomes in various diseases. In particular, an important combined role for KIR3DS1 and HLA-B Bw4-I80 in controlling viral infections and a higher protection against leukemic relapses in donor equipped with activating KIRs in haplo-HSCT has been described. Here, we show that KIR3DS1 mediates positive signals upon recognition of HLA-B*51 (Bw4-I80) surface molecules on target cells and that this activation occurs only in Bw4-I80neg individuals, including those carrying particular KIR/HLA combination settings. In addition, killing of HLA-B*51 transfected target cells mediated by KIR3DS1+/NKG2A+ natural killer (NK) cell clones from Bw4-I80neg donors could be partially inhibited by antibody-mediated masking of KIR3DS1. Interestingly, KIR3DS1-mediated recognition of HLA-B*51 could be better appreciated under experimental conditions in which the function of NKG2D was reduced by mAb-mediated blocking. This experimental approach may mimic the compromised function of NKG2D occurring in certain viral infections. We also show that, in KIR3DS1+/NKG2A+ NK cell clones derived from an HLA-B Bw4-T80 donor carrying 2 KIR3DS1 gene copy numbers, the positive signal generated by the engagement of KIR3DS1 by HLA-B*51 resulted in a more efficient killing of HLA-B*51-transfected target cells. Moreover, in these clones, a direct correlation between KIR3DS1 and NKG2D surface density was detected, while the expression of NKp46 was inversely correlated with that of KIR3DS1. Finally, we analyzed KIR3DS1+/NKG2A+ NK cell clones from a HLA-B Bw4neg donor carrying cytoplasmic KIR3DL1. Although these clones expressed lower levels of surface KIR3DS1, they displayed responses comparable to those of NK cell clones derived from HLA-B Bw4neg donors that expressed surface KIR3DL1. Altogether these data suggest that, in particular KIR/HLA combinations, KIR3DS1 may play a role in the process of human NK cell education.

Sequences of 95 human MHC haplotypes reveal extreme coding variation in genes other than highly polymorphic HLA class I and II.

The most polymorphic part of the human genome, the MHC, encodes over 160 proteins of diverse function. Half of them, including the HLA class I and II genes, are directly involved in immune responses. Consequently, the MHC region strongly associates with numerous diseases and clinical therapies. Notoriously, the MHC region has been intractable to high-throughput analysis at complete sequence resolution, and current reference haplotypes are inadequate for large-scale studies. To address these challenges, we developed a method that specifically captures and sequences the 4.8-Mbp MHC region from genomic DNA. For 95 MHC homozygous cell lines we assembled, de novo, a set of high-fidelity contigs and a sequence scaffold, representing a mean 98% of the target region. Included are six alternative MHC reference sequences of the human genome that we completed and refined. Characterization of the sequence and structural diversity of the MHC region shows the approach accurately determines the sequences of the highly polymorphic HLA class I and HLA class II genes and the complex structural diversity of complement factor C4A/C4B It has also uncovered extensive and unexpected diversity in other MHC genes; an example is MUC22, which encodes a lung mucin and exhibits more coding sequence alleles than any HLA class I or II gene studied here. More than 60% of the coding sequence alleles analyzed were previously uncharacterized. We have created a substantial database of robust reference MHC haplotype sequences that will enable future population scale studies of this complicated and clinically important region of the human genome.

Hematopoietic stem cell transplantation: Improving alloreactive Bw4 donor selection by genotyping codon 86 of KIR3DL1/S1.

KIR3DL1 is a natural killer (NK) cell receptor that recognizes the Bw4 epitope of human leukocyte antigen (HLA) class I molecules. Following hematopoietic stem cell transplantation for patients lacking Bw4, KIR3DL1-expressing NK cells from Bw4-positive donors can be alloreactive and eliminate tumor cells. However, KIR3DL1 alleles having T instead of C at nucleotide 320 (encoding leucine 86 instead of serine 86) are not expressed on the cell surface. Thus, not all individuals testing positive for KIR3DL1 are optimal donors for Bw4-negative recipients. Therefore, we developed a method for genotyping codon 86, which was validated by its perfect correlation with NK cell phenotype for 100 donors of diverse KIR3DL1/S1 genotype. We typed 600 donors and found that ∼12.2% had the KIR3DL1 gene, but did not express cell-surface KIR3DL1. By contrast, high-expressing allotypes were identified when haplotypes from four families with duplicated KIR3DL1/S1 genes were characterized at high resolution. Identifying donors who have KIR3DL1 but lack cell-surface KIR3DL1 would refine donor selection. With this technique, the number of individuals identified who may not be optimal donors for Bw4-negative patients increases by threefold, when compared with standard methods. Taken together, we propose that allele typing of killer cell Ig-like receptor (KIR) polymorphisms should become a standard practice when selecting donors.

TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization.

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection.

Human cytomegalovirus infection promotes rapid maturation of NK cells expressing activating killer Ig-like receptor in patients transplanted with NKG2C-/- umbilical cord blood.

NK cells are the first lymphoid population recovering after allogeneic hematopoietic stem cell transplantation and play a crucial role in early immunity after the graft. Recently, it has been shown that human CMV (HCMV) infection/reactivation can deeply influence NK cell reconstitution after umbilical cord blood transplantation by accelerating the differentiation of mature NKG2A(-) killer Ig-like receptor (KIR)(+) NK cells characterized by the expression of the NKG2C-activating receptor. In view of the hypothesis that NKG2C could be directly involved in NK cell maturation driven by HCMV infection, we analyzed the maturation and function of NK cells developing in three patients with hematological malignancies given umbilical cord blood transplantation from donors carrying a homozygous deletion of the NKG2C gene. We show that HCMV infection can drive rapid NK maturation, characterized by the expansion of CD56(dim)NKG2A(-)KIR(+) cells, even in the absence of NKG2C expression. Interestingly, this expanded mature NK cell subset expressed surface-activating KIR that could trigger NK cell cytotoxicity, degranulation, and IFN-γ release. Given the absence of NKG2C, it is conceivable that activating KIRs may play a role in the HCMV-driven NK cell maturation and that NK cells expressing activating KIRs might contribute, at least in part, to the control of infections after transplantation.