CMV drives the expansion of highly functional memory T cells expressing NK‐cell receptors in renal transplant recipients

Cytomegalovirus (CMV) is a common opportunistic infection encountered in renal transplant recipients (RTRs) and may be reactivated without symptoms at any time post‐transplant. We describe how active and latent CMV affect T‐cell subsets in RTRs who are stable on maintenance therapy. T‐cell responses to CMV were assessed in RTRs (n = 54) >2 years post‐transplant, and healthy controls (n = 38). Seven RTRs had CMV DNA detectable in plasma. CMV antibody and DNA aligned with increased proportions of CD8⁺ T cells and reduced CD4/CD8 ratios. This paralleled an expansion of effector memory T‐cell (T_EM), terminally differentiated T‐cell (T_EMRA) and CD57⁺ T_EMRA cell populations. Expression of NK‐cell receptors, LIR‐1 and KLRG1 on CD4⁺ and CD8⁺ CD57⁺ T_EM and T_EMRA cells correlated with elevated interferon‐γ and cytotoxic responses to anti‐CD3 and increased cytotoxic responses to CMV phosphoprotein (pp) 65 in RTRs who carried CMV DNA. CD8⁺ T cells from all CMV seropositive RTRs responded efficiently to CMV immediate early (IE) ‐1 peptides. The data show that latent and active CMV infection can alter T‐cell subsets in RTRs many years after transplantation, and up‐regulate T‐cell expression of NK‐cell receptors. This may enhance effector responses of CD4⁺ and CD8⁺ T cells against CMV.     

Functional and clinical consequences of changes to natural killer cell phenotypes driven by chronic cytomegalovirus infections

Cytomegalovirus (CMV) infections may affect natural killer (NK) cells and are implicated in age-related disorders—notably poor vascular endothelial function. Changes may be greater in renal transplant recipients (RTR) as they have a high burden of CMV and may influence antibody-dependent cellular cytotoxicity (ADCC) responses to viral antigen. We obtained blood mononuclear cells from RTR stable after transplantation (n = 27) and age- and sex-matched controls (n = 28). Natural killer (NK) cells were assessed for expression of CD107a or TNF-α, after stimulation with autologous antibodies bound to CMV glycoprotein B (measuring ADCC) or anti-CD16 (measuring NK cell activation). Alleles of FCRG3A (encoding CD16; rs396991) were determined by the Taqman assay. The vascular endothelial function was assessed using flow-mediated dilatation (FMD) of the brachial artery. Proportions of NK cells expressing CD16 ex vivo were lower in RTR. Frequencies of NK cells expressing NKG2C or LIR-1 or lacking FcRγ were highest in CMV-seropositive RTR. ADCC was affected by rs396991 genotype and CMV gB antibody levels, but not by RTR status or detection of CMV DNA in plasma. Responses of FcRγ-NK cells to anti-CD16 were lower compared to FcRγ+ NK cells. Increased percentages of LIR-1 ⁺ and FcRγ− NK cells correlated with lower FMD. In summary, CMV evokes substantial and similar ADCC responses in CMV seropositive RTR and controls. The equivalence may reflect higher titers of CMV reactive antibody in RTR, as NK responses stimulated by ligation of CD16 were lower. NK cells that were LIR-1 ⁺ and/or FcRγ− were induced by CMV and correlated inversely with vascular endothelial function.     

Siglec‐7 Defines a Highly Functional Natural Killer Cell Subset and Inhibits Cell‐Mediated Activities

Sialic acid‐binding immunoglobulin‐like lectin‐7 (Siglec‐7) is an inhibitory receptor expressed on natural killer (NK) cells. In this study, we investigated the relationship between Siglec‐7 expression and NK cell functions. Siglec‐7 was highly expressed on NK cells and was preferentially expressed by mature NK cells from peripheral blood of healthy adults. Siglec‐7⁺ NK cells displayed higher levels of activating receptors CD38, CD16, DNAM1, NKp30 and NKp46, but lower levels of inhibitory receptors such as NKG2A and CD158b, compared with Siglec‐7^– NK cells. Functional tests showed that Siglec‐7⁺ NK cells displayed more CD107a degranulation and IFN‐γ production than Siglec‐7^– NK cells. Siglec‐7 inhibited NK cell functions when interacting with specific antibodies. These data suggest that Siglec‐7 defines a highly functional NK cell subset and suppresses NK cell‐mediated functions when cross‐linked with specific antibodies.     

Characterization of Natural Killer Cells in HIV Patients Beginning Therapy with a High Burden of Cytomegalovirus

Background: Active infections with cytomegalovirus (CMV) increase NK cell expression of the inhibitory receptor LIR-1 and the activating receptor NKG2C in transplant recipients. However, the effects of CMV on NK cells are different in HIV patients stable on antiretroviral therapy (ART) and have not been analyzed in young HIV patients beginning ART.

Methodology: We followed a cohort of 78 Indonesian HIV patients beginning ART. CMV antibodies were measured in plasma before ART (baseline), and after 1, 3, 6, and 12 months. CMV DNA was sought in blood granulocytes at baseline by quantitative PCR assay and a deletion in the NKG2C gene was identified by PCR. NK cell profiles were monitored by flow cytometry in 19 patients stratified by the presence of CMV DNA. Healthy controls (n = 17) were assessed once.

Results: All 78 patients were CMV seropositive and 41 had detectable CMV DNA. CMV DNA+ patients had higher proportions of total NK cells and CD16⁺ NK cells at baseline, but similar expression of LIR-1 and NKp30 on NK cells on ART. However, levels of CMV antibody were inversely related to median LIR-1 expression on NK cells. A dramatic elevation in cells expressing NKG2C was restricted to CMV DNA+ patients heterozygous for the NKG2C deletion. Patients with High NKG2C expression had lower levels of CMV antibodies.

Conclusion: A subpopulation of NK cells expressing NKG2C was induced by CMV replication in HIV patients heterozygous for a deletion in this gene. Individuals with an abundant NKG2C⁺ and LIR-1⁺ NK cells displayed lower levels of CMV reactive antibody.     

Adaptive reconfiguration of the human NK‐cell compartment in response to cytomegalovirus: A different perspective of the host‐pathogen interaction

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

Spotlight on NKG2C and the human NK‐cell response to CMV infection

Immune responses to cytomegalovirus (CMV) infection in the mouse and human involve the expansion of specific subsets of natural killer (NK) cells with specific phenotypic characteristics and a heightened ability to produce interferon (IFN)‐gamma. In humans, these NK‐cell responses are largely driven by the activating receptor NKG2C, which recognize human leukocyte antigen (HLA)‐E in complex with leader sequence peptides. In this issue of the European Journal of Immunology, Noyola et al. [Eur. J. Immunol. 2012, 42: 3256‐3266] examine NK‐cell responses in a unique cohort of young children with asymptomatic and symptomatic congenital CMV infection. They also address NK‐cell responses to CMV in relation to NKG2C gene copy number. Children with a symptomatic congenital infection exhibited a marked expansion of NKG2C⁺ NK cells. However, despite having slightly lower frequencies of NKG2C⁺ NK cells, children with a heterozygous deletion of the NKG2C gene seemed to control the virus as efficiently as those with two copies of the NKG2C gene. The present studies shed new light on the role of NKG2C copy number variation on the human NK‐cell response to CMV infection.     

Cytomegalovirus: an unlikely ally in the fight against blood cancers?

Cytomegalovirus (CMV) infection is a potentially fatal complication in patients receiving haematopoietic stem cell transplantation (HSCT), but recent evidence indicates that CMV has strong anti‐leukaemia effects due in part to shifts in the composition of natural killer (NK) cell subsets. NK cells are the primary mediators of the anti‐leukaemia effect of allogeneic HSCT, and infusion of allogeneic NK cells has shown promise as a means of inducing remission and preventing relapse of several different haematological malignancies. The effectiveness of these treatments is limited, however, when tumours express human leucocyte antigen (HLA)‐E, a ligand for the inhibitory receptor NKG2A, which is expressed by the vast majority of post‐transplant reconstituted and ex‐vivo expanded NK cells. It is possible to enhance NK cell cytotoxicity against HLA‐E^pos malignancies by increasing the proportion of NK cells expressing NKG2C (the activating receptor for HLA‐E) and lacking the corresponding inhibitory receptor NKG2A. The proportion of NKG2C^pos/NKG2A^neg NK cells is typically low in healthy adults, but it can be increased by CMV infection or ex‐vivo expansion of NK cells using HLA‐E‐transfected feeder cells and interleukin (IL)‐15. In this review, we will discuss the role of CMV‐driven NKG2C^pos/NKG2A^neg NK cell expansion on anti‐tumour cytotoxicity and disease progression in the context of haematological malignancies, and explore the possibility of harnessing NKG2C^pos/NKG2A^neg NK cells for cancer immunotherapy.     

Intrahepatic and peripheral blood phenotypes of natural killer and T cells: differential surface expression of killer cell immunoglobulin‐like receptors

Deep characterization of the frequencies, phenotypes and functionalities of liver and peripheral blood natural killer (NK), natural killer T (NKT) and T cells from healthy individuals is an essential step to further interpret changes in liver diseases. These data indicate that CCR7, a chemokine essential for cell migration through lymphoid organs, is almost absent in liver NK and T cells. CD56^bright NK cells, which represent half of liver NK cells, showed lower expression of the inhibitory molecule NKG2A and an increased frequency of the activation marker NKp44. By contrast, a decrease of CD16 expression with a potential decreased capacity to perform antibody‐dependent cellular cytotoxicity was the main difference between liver and peripheral blood CD56^dim NK cells. Liver T cells with an effector memory or terminally differentiated phenotype showed an increased frequency of MAIT cells,T‐cell receptor‐γδ (TCR‐γδ) T cells and TCR‐αβ CD8⁺ cells, with few naive T cells. Most liver NK and T cells expressed the homing markers CD161 and CD244. Liver T cells revealed a unique expression pattern of killer cell immunoglobulin‐like receptors (KIR) receptors, with increased degranulation ability and higher secretion of interferon‐γ. Hence, the liver possesses a large amount of memory and terminally differentiated CD8⁺ cells with a unique expression pattern of KIR activating receptors that have a potent functional capacity as well as a reduced amount of CCR7, which are unable to migrate to regional lymph nodes. These results are consistent with previous studies showing that liver T (and also NK) cells likely remain and die in the liver.     

Coordinated expansion of both memory T cells and NK cells in response to CMV infection in humans

NK cells are key players in the fight against persistent viruses. Human cytomegalovirus (HCMV) infection is associated with the presence of a population of CD16⁺ CD56^dim NKG2C⁺ NK cells in both acutely and latently infected individuals. Here, we studied the nature of these terminally differentiated NK cells in different human populations infected with HCMV: healthy donors stratified by age, thymectomized individuals, pregnant women suffering from primary CMV infection, and lung transplant patients. Both CD16⁺ CD56^dim NK‐ and CD8 T‐cell phenotypes as well as functional capacities were determined and stratified according to age and/or CMV event. Similarly to T‐cell responsiveness, we observe an accumulation over time of NKG2C⁺ NK cells, which preferentially expressed CD57. This accumulation is particularly prominent in elderly and amplified further by CMV infection. Latent HCMV infection (without replication) is sufficient for NKG2C⁺ CD57⁺ NK cells to persist in healthy individuals but is not necessarily required in old age. Collectively, the present work supports the emerging concept that CMV shapes both innate and adaptive immunity in humans.     

Robust 8-color flow cytometry panel reveals enhanced effector function of NKG2C+ CD57+ FcεRγ− NK cells in CMV seropositive human blood donors

Increasing evidence suggests that human NK cells may develop memory-like features. Here, we report the establishment of a robust 8-color flow cytometry panel that allows quantification and functional analysis of different memory-like NK cell subsets (NKG2C⁺/CD57⁺, FcεRγ⁻) from relatively small blood samples. We could confirm previous publications reporting an enhanced prevalence of the mentioned memory-like NK cell subsets in CMV seropositive human donors and were able to show a clear congruence between enhanced expression of NKG2C and CD57, the absence of FcεRγ and CMV seropositivity supporting the hypothesis of memory-like NK cell development following viral infections. While we could not detect significant differences in effector functions (i.e. degranulation and production of IFNγ) in regard to age or CMV seropositivity when looking at the overall NK cell population, a significantly enhanced expression of CD107a and IFNγ could be observed in NKG2C⁺/CD57⁺ as well as FcεRγ⁻ NK cell subpopulations in CMV⁺ donors. This enhancement of effector functions was even more pronounced in NKG2C⁺/CD57⁺ NK cells that were also negative for FcεRγ; CMV seropositive donors showed a dramatically increased expression of CD107a as well as IFNγ.With only small-sized volumes of blood needed, our proposed 8-color panel and experimental protocol offers easy handling and a reliable and reproducible option for implementation in accompanying clinical research, e.g. for evaluation of immunosuppressed patients suffering from autoimmune diseases or in allograft recipients.     

Soluble HLA‐I‐mediated secretion of TGF‐β1 by human NK cells and consequent down‐regulation of anti‐tumor cytolytic activity

Soluble HLA class I (sHLA‐I) molecules can regulate survival of NK cells and their anti‐tumor killing activity. Herein, we have analysed whether interaction of sHLA‐I with CD8 and/or different isoforms of killer Ig‐like receptors (KIR) induced secretion of transforming growth factor (TGF)‐β1. CD8⁺KIR^? NK cell clones secreted TGF‐β1 upon the interaction of sHLA‐I with CD8 molecule. sHLA‐Cw4 or sHLA‐Cw3 alleles engaging inhibitory isoforms of KIR, namely KIR2DL1 or KIR2DL2, strongly downregulated TGF‐β1 production elicited through CD8. On the other hand, sHLA‐Cw4 or sHLA‐Cw3 alleles induced secretion of TGF‐β1 by ligation of stimulatory KIR2DS1 or KIR2DS2 isoforms. TGF‐β1 strongly reduced NK cell‐mediated tumor cell lysis and production of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Also, TGF‐β1 inhibited NK cell cytolysis induced by the engagement of stimulatory receptors including NKG2D, DNAM1, 2B4, CD69, NKp30, NKp44 and NKp46. The IL‐2‐dependent surface upregulation of some of these receptors was prevented by TGF‐β1. Furthermore, TGF‐β1 hampered IL‐2‐induced NK cell proliferation but not IL‐2‐mediated rescue from apoptosis of NK cells. Depletion of TGF‐β1 restored all the NK cell‐mediated functional activities analysed. Taken together these findings suggest that sHLA‐I antigens may downregulate the NK cell‐mediated innate response by inducing TGF‐β1 release.     

Differential recruitment and activation of natural killer cell sub‐populations by Mycobacterium bovis‐infected dendritic cells

Through complex interplay with APCs, subsets of NK cells play an important role in shaping adaptive immune responses. Bovine tuberculosis, caused by Mycobacterium bovis, is increasing in incidence and detailed knowledge of host–pathogen interactions in the natural host is essential to facilitate disease control. We investigated the interactions of NK‐cell sub‐populations and M. bovis‐infected DCs to determine early innate mechanisms in the response to infection. A sub‐population of NK cells (NKp46⁺CD2^?) selectively expressing lymphoid homing and inflammatory chemokine receptors were induced to migrate towards M. bovis‐infected DCs. This migration was associated with increased expression of chemokines CCL3, 4, 5, 20 and CXCL8 by M. bovis‐infected DCs. Activation of NKp46⁺CD2^? NK cells and secretion of IFN‐γ was observed, a response reliant on localised IL‐12 release and direct cellular interaction. In a reciprocal manner, NKp46⁺CD2^? cells induced an increase in the intensity of cell surface MHC class II expression on DCs. In contrast, NKp46⁺CD2⁺ NK cells were unable to secrete IFN‐γ and did not reciprocally affect DCs. This study provides novel evidence to demonstrate distinct effector responses between bovine NK‐cell subsets during mycobacterial infection.     

Balance between activating NKG2D,DNAM‐1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast‐like synoviocytes (FLS) are central components of the aggressive, tumour‐like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA‐FLS and NK cells. We used cultured RA‐FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA‐FLS/NK cell cross‐talk. We show that RA‐FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM‐1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA‐FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA‐E expressed on RA‐FLS further enhanced Nishi cell degranulation in co‐culture with RA‐FLS. Using cultured RA‐FLS and the human NK cell line Nishi as an in vitro model system of RA‐FLS/NK cell cross‐talk, our results suggest that cell‐mediated cytotoxicity of RA‐FLS may be one mechanism by which NK cells influence local joint inflammation in RA.     

Increased natural killer cell subsets with inhibitory cytokines and inhibitory surface receptors in patients with recurrent miscarriage and decreased or normal subsets in kidney transplant recipients late post‐transplant

Patients with recurrent miscarriage (RM) show up‐regulated cytotoxic natural killer (NK) cells that are suspected to play a causal role in abortion. In the present study, we investigated counter‐regulating inhibitory mechanisms and compared the results in RM patients with those of healthy controls (HC), patients with end‐stage renal disease (ESRD) and kidney transplant recipients late post‐transplant (TX). NK, NK T and T cell subsets were analysed in the peripheral blood of 31 RM, 14 female ESRD and nine female TX patients as well as 21 female HC using eight‐colour fluorescence flow cytometry. Compared with HC, RM patients showed significantly higher absolute numbers of CD56⁺ NK cells co‐expressing the phenotype interferon (IFN)‐γR⁺, IL‐4⁺, transforming growth factor (TGF)‐β⁺, IL‐4⁺ human leucocyte antigen D‐related (HLA‐DR)⁺, TGF‐β⁺HLA‐DR⁺, IL‐4⁺TGF‐β⁺, IL‐4⁺TGF‐β^–, IFN‐γ⁺ and/or IL‐10^–IFN‐γ⁺ (all P ≤ 0·01), more IL‐17⁺CD56^bright (P = 0·028) NK cells and more CD56^dimCD16⁺ NK cells co‐expressing IFN‐γR, IFN‐γ, IL‐4 and/or TGF‐β (all P ≤ 0·01). When the same cell subsets were analysed in ESRD or TX patients, cytokine‐producing NK cell subsets were not significantly different from those of HC. RM patients showed significantly higher absolute numbers of CD158a⁺, CD158b⁺, CD158a^–CD158e⁺ (all P < 0·05), NKG2D⁺NKG2A⁺, NKG2D ⁺NKG2A^–, NKG2D⁺ and/or NKG2A⁺ (all P ≤ 0·01) CD56⁺ NK cells and higher CD158a⁺, CD158b⁺ (all P < 0·05), NKG2D⁺ and/or NKG2A⁺ (all P < 0·01) CD56^dim+CD16⁺ NK cells than HC. In contrast, ESRD patients had normal and TX recipients had lower CD158a⁺ and NKG2D⁺NKG2A^–CD56⁺ NK cells and lower CD158a⁺CD56^dim+CD16⁺ NK cells (all P < 0·05) than HC. RM patients have abnormally high circulating NK cells expressing inhibitory cytokines and inhibitory surface receptors which might contribute to the pathogenesis of RM.     

Cytomegalovirus vector expressing RAE‐1γ induces enhanced anti‐tumor capacity of murine CD8+ T cells

Designing CD8⁺ T‐cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE‐1γ as CD8⁺ T cell‐based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE‐1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD‐1. CMV vector expressing RAE‐1γ potentiated expansion of KLRG1⁺ CD8⁺ T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long‐term maintenance of epitope‐specific CD8⁺ T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8⁺ T‐cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8⁺ T‐cell sensitive tumors.     

Cis association of leukocyte Ig‐like receptor 1 with MHC class I modulates accessibility to antibodies and HCMV UL18

Leukocyte Ig‐like receptor (LIR) 1 (CD85j/ILT2/LILRB1) is an inhibitory receptor with broad specificity for MHC class I (MHC‐I) and the human CMV MHC‐I homologue UL18. LIR‐1 can inhibit NK cells through the conventional interaction with MHC‐I expressed on a target cell (in trans) but the nature and the effects of LIR‐1 interactions with MHC‐I in cis are not well understood. Here we show that MHC‐I expressed in cis has an impact on the detection of LIR‐1 with various antibodies. We found the cis interaction alters recognition by only one of two antibodies known to block functional trans recognition by LIR‐1 on NK cells. Specifically, we observed an enhancement of recognition with GHI/75 in the presence of various MHC‐I alleles on 721.221 cells. We found that blocking the LIR‐1 contact site with anti‐MHC‐I antibodies decreased detection of LIR‐1 with GHI/75. We also observed a decrease in GHI/75 following acid denaturation of MHC‐I. Finally, disruption of LIR‐1 cis interactions with MHC‐I significantly enhanced UL18‐Fc binding to NK92 cells and enhanced the relative inhibition of NK92 cells by HLA‐G. These results have implications for LIR‐1 function in scenarios such as infection when MHC‐I levels on effector cells may be increased by IFNs.     

NK cells generate memory‐type responses to human cytomegalovirus‐infected fibroblasts

Natural killer (NK) cells are cytotoxic lymphocytes that selectively respond against abnormal cells. Human cytomegalovirus (HCMV) infection causes expansion of NKG2C⁺CD57⁺ NK cells in vivo and NKG2C⁺ NK cells proliferate when cultured with HCMV‐infected cells. This raises the possibility of an NK‐cell subset selectively responding against a specific pathogen and accruing memory. To test this possibility, we compared proliferation, natural cytotoxicity and interferon‐γ (IFN‐γ) production of NK cells from HCMV‐seropositive and HCMV‐seronegative individuals co‐cultured with HCMV‐infected or uninfected MRC‐5 cells. There was no significant difference in proliferation of NK cells from HCMV‐seropositive or seronegative individuals against uninfected MRC‐5 cells, but significantly more NK cells from the HCMV‐seropositive group proliferated in response to HCMV‐infected MRC‐5 cells. Natural cytotoxicity of NK cells against K562 cells increased following co‐culture with HCMV‐infected versus uninfected MRC‐5 only for the HCMV‐seropositive group. After co‐culture with HCMV‐infected MRC‐5 cells, proliferating NK cells from HCMV‐seropositive donors selectively produced IFN‐γ when re‐exposed to HCMV‐infected MRC‐5 cells. Both NKG2C⁺ and NKG2C^? NK cells proliferated in co‐culture with HCMV‐infected MRC‐5 cells, with the fraction of proliferating NKG2C⁺ NK cells directly correlating with the circulating NKG2C⁺ fraction. These data illustrate an at least partly NKG2C‐independent human NK‐cell memory‐type response against HCMV.