Adaptive NK cell response to human cytomegalovirus: Facts and open issues.

Human cytomegalovirus (HCMV) infection exerts broad effects on the immune system. These include the differentiation and persistent expansion of a mature NK cell subset which displays a characteristic phenotypic and functional profile hallmarked by expression of the HLA-E-specific CD94/NKG2C activating receptor. Based on our experience and recent advances in the field, we overview the adaptive features of the NKG2C+ NK cell response, discussing observations and open questions on: (a) the mechanisms and influence of viral and host factors; (b) the existence of other NKG2C- NK cell subsets sharing adaptive features; (c) the development and role of adaptive NKG2C+ NK cells in the response to HCMV in hematopoietic and solid organ transplant patients; (d) their relation with other viral infections, mainly HIV-1; and (e) current perspectives for their use in adoptive immunotherapy of cancer.

Microvascular inflammation in the absence of human leukocyte antigen-donor-specific antibody and C4d: An orphan category in Banff classification with cytotoxic T and natural killer cell infiltration.

Isolated microvascular inflammation (iMVI) without HLA donor-specific antibodies or C4d deposition in peritubular capillaries remains an enigmatic phenotype that cannot be categorized as antibody-mediated rejection (ABMR) in recent Banff classifications. We included 221 kidney transplant recipients with biopsies with ABMR (n = 73), iMVI (n = 32), and normal (n = 116) diagnoses. We compared peripheral blood leukocyte distribution by flow cytometry and inflammatory infiltrates in kidney transplant biopsies among groups. Flow cytometry showed fewer lymphocytes and total, CD4+, and CD8+ peripheral T cells in iMVI compared with ABMR and normal cases. ABMR and iMVI had fewer total natural Killer (NK) cells but more NKG2A+ NK cells. Immunohistochemistry indicated that ABMR and iMVI had greater CD3+ and CD68+ glomerular infiltration than normal biopsies, whereas CD8+ and TIA1+ cells showed only increased iMVI, suggesting they are cytotoxic T cells. Peritubular capillaries displayed more CD3+, CD56+, TIA1+, and CD68+ cells in both ABMR and iMVI. In contrast, iMVI had less plasma cell infiltration in peritubular capillaries and interstitial aggregates than ABMR. iMVI displayed decreased circulating T and NK cells mirrored by T cell and NK cell infiltration in the renal allograft, similar to ABMR. However, the lesser plasma cell infiltration in iMVI may suggest an antibody-independent underlying stimulus.

Long-Term Evolution of the Adaptive NKG2C+ NK Cell Response to Cytomegalovirus Infection in Kidney Transplantation: An Insight on the Diversity of Host-Pathogen Interaction.

Human CMV infection is frequent in kidney transplant recipients (KTR). Pretransplant Ag-specific T cells and adaptive NKG2C+ NK cells associate with reduced incidence of infection in CMV+ KTR. Expansions of adaptive NKG2C+ NK cells were reported in posttransplant CMV-infected KTR. To further explore this issue, NKG2C+ NK, CD8+, and TcRγδ T cells were analyzed pretransplant and at different time points posttransplant for ≥24 mo in a cohort of CMV+ KTR (n = 112), stratified according to CMV viremia detection. In cryopreserved samples from a subgroup (n = 49), adaptive NKG2C+ NK cell markers and T cell subsets were compared after a longer follow-up (median, 56 mo), assessing the frequencies of CMV-specific T cells and viremia at the last time point. Increased proportions of NKG2C+ NK, CD8+, and TcRγδ T cells were detected along posttransplant evolution in viremia(+) KTR. However, the individual magnitude and kinetics of the NKG2C+ NK response was variable and only exceptionally detected among viremia(-) KTR, presumably reflecting subclinical viral replication events. NKG2C+ expansions were independent of KLRC2 zygosity and associated with higher viral loads at diagnosis; no relation with other clinical parameters was perceived. Increased proportions of adaptive NKG2C+ NK cells (CD57+, ILT2+, FcεRIγ-) were observed after resolution of viremia long-term posttransplant, coinciding with increased CD8+ and Vδ2- γδ T cells; at that stage CMV-specific T cells were comparable to viremia(-) cases. These data suggest that adaptive NKG2C+ NK cells participate with T cells to restore CMV replication control, although their relative contribution cannot be discerned.

Single-reaction multi-antigen serological test for comprehensive evaluation of SARS-CoV-2 patients by flow cytometry.

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.

Pretransplant adaptive NKG2C+ NK cells protect against cytomegalovirus infection in kidney transplant recipients.

Cytomegalovirus (CMV) infection constitutes a complication for kidney transplant recipients (KTR) and CMV-specific T cells reduce the risk of viral replication in seropositive patients. CMV promotes the adaptive differentiation and expansion of an NK cell subset, hallmarked by expression of the CD94/NKG2C receptor with additional characteristic features. We previously reported an association of pretransplant NKG2C+ NK cells with a reduced incidence of CMV infection. We have strengthened the analysis in cryopreserved peripheral blood mononuclear cells from an enlarged KTR cohort (n = 145) with homogeneous immunosuppression, excluding cases at low risk of infection (ie, CMV D-R-) or receiving antiviral prophylaxis. Moreover, adaptive NKG2C+ NK cell-associated markers (ie, NKG2A, CD57, Immunoglobulin-like transcript 2 [LIR1 or LILRB1], FcεRI γ chain, and Prolymphocytic Leukemia Zinc Finger transcription factor) as well as T lymphocyte subsets were assessed by multicolor flow cytometry. The relation of NKG2C+ NK cells with T cells specific for CMV antigens was analyzed in pretransplant patients (n = 29) and healthy controls (n = 28). Multivariate Cox regression and Kaplan-Meier analyses supported that NKG2C+ NK cells bearing adaptive markers were specifically associated with a reduced incidence of posttransplant symptomatic CMV infection; no correlation between NKG2C+ NK cells and CMV-specific T cells was observed. These results support that adaptive NKG2C+ NK cells contribute to control CMV infection in KTR.

Adaptive NKG2C+ NK Cell Response and the Risk of Cytomegalovirus Infection in Kidney Transplant Recipients.

CMV infection in kidney transplant recipients (KTRs) has been associated with an increased risk for graft loss and reduced host survival. CMV promotes persistent expansions of NK cells expressing the CD94/NKG2C receptor. The NKG2C (KLRC2) gene is frequently deleted, and copy number influences the adaptive response of NKG2C+ NK cells. The distribution of NKG2C+ NK cells and NKG2C genotypes (NKG2C+/+, NKG2C+/del, NKG2Cdel/del) were studied in cross-sectional (n = 253) and prospective (n = 122) KTR cohorts. Assessment of CMV viremia was restricted to symptomatic cases in the retrospective study, but was regularly monitored in the prospective cohort. Overall, the proportions of NKG2C+ NK cells were significantly higher in KTRs who had suffered posttransplant symptomatic CMV infection in the cross-sectional study. Yet, along the prospective follow-up (3, 6, 12, and 24 mo), posttransplant NKG2C+ NK cell expansions were not observed in every patient with detectable viremia who received preemptive antiviral therapy, suggesting that the adaptive NK cell response may be inversely related with the degree of CMV control. Remarkably, the incidence of posttransplant viremia was reduced among cases with high pretransplant levels of NKG2C+ NK cells. The NKG2C genotype distribution was comparable in KTR and healthy controls, and greater proportions of NKG2C+ cells were detected in NKG2C+/+ than in NKG2C+/del patients. Yet, a trend toward increased NKG2C+/del and reduced NKG2C+/+ frequencies associated with symptomatic infection was appreciated in both cohorts. Altogether, our results indirectly support that adaptive NKG2C+ NK cells are involved in the control of CMV in KTRs.

Relationship of NKG2C Copy Number with the Distribution of Distinct Cytomegalovirus-Induced Adaptive NK Cell Subsets.

CD94/NKG2C and lack of FcεRγ (FcRγ) expression are considered markers of the adaptive NK cell response to human CMV (HCMV) infection. Despite the fact that FcRγ(-) and NKG2C(bright) NK cells share some phenotypic, epigenetic, and functional features, their relationship remains unclear. To address this issue, a systematic analysis of NKG2C(bright) and FcRγ expression was carried out in NK cells from a cohort of healthy young adults (n = 81) considering NKG2C copy number, previously related to the magnitude of NKG2C(+) NK cell expansion. NKG2C(bright) and FcRγ(-) NK cells coincided in a subgroup of HCMV(+) individuals, pointing to a common host-virus interaction pattern. Even though FcRγ loss was often confined to expanded NKG2C(bright) NK cells, both markers appeared occasionally dissociated, consistent with the existence of distinct adaptive NK cell subsets. Remarkably, FcRγ loss was mostly accumulated within the NKG2C(bright) subset in NKG2C(+/+) subjects, whereas NKG2C(-)FcRγ(-) NK cell subpopulations were more frequently detected in NKG2C(+/del) donors and also in NKG2C(del) (/del) individuals, independently of activating killer Ig-like receptor expression. The distribution of other NK receptors (i.e., killer Ig-like receptor, LILRB1, or CD57) supported a sequential differentiation from NKG2C(bright)FcRγ(+) to NKG2C(bright)FcRγ(-) NK cells. Noticeably, NKG2C(bright) NK cells produced more TNF-α in response to Ab-dependent activation, regardless of their FcRγ levels. Moreover, the TNF-α response of NKG2C(-)FcRγ(-) subpopulations was lower than that of concurrent NKG2C(bright)FcRγ(-) NK cells, further supporting that FcRγ levels and enhanced potential for cytokine production are uncoupled. Overall, our data extend the characterization of adaptive NK cell subsets that differentiate in response to HCMV, supporting a relationship between their distribution and NKG2C copy number.

The CD94/NKG2C+ NK-cell subset on the edge of innate and adaptive immunity to human cytomegalovirus infection.

Human cytomegalovirus (HCMV) causes a highly prevalent and lifelong infection, with a multifaceted impact in human health. NK cells play an important role in the immune response to HCMV and the virus has reciprocally developed a variety of immune evasion strategies. We originally reported that HCMV infection promotes, to a variable degree in healthy individuals, a redistribution of the NK-cell receptor (NKR) repertoire which persists under steady-state conditions. Its hallmark is an expansion of a mature NK-cell subset displaying high surface levels of the CD94/NKG2C activating receptor, with additional distinctive phenotypic and functional features. Such adaptation of host NK cells to HCMV infection, confirmed in different clinical settings, is particularly magnified in immunocompromised patients and influenced by NKG2C gene copy number. The mechanism(s) underlying the differentiation and proliferation of NKG2C+ NK cells, the basis for the individual differences in the magnitude of their expansion, and their precise role in anti-viral defence remain open issues. Moreover, the possibility that the impact of HCMV infection on the NK-cell compartment may exert a broader influence on immunity deserves further attention.

Antibody-mediated response of NKG2Cbright NK cells against human cytomegalovirus.

Human CMV (HCMV) infection promotes a variable and persistent expansion of functionally mature NKG2C(bright) NK cells. We analyzed NKG2C(bright) NK cell responses triggered by Abs from HCMV(+) sera against HCMV-infected MRC5 fibroblasts. Specific Abs promoted the degranulation (i.e., CD107a expression) and the production of cytokines (TNF-α and IFN-γ) by a significant fraction of NK cells, exceeding the low natural cytotoxicity against HCMV-infected targets. NK cell-mediated Ab-dependent cell-mediated cytotoxicity was limited by viral Ag availability and HLA class I expression on infected cells early postinfection and increased at late stages, overcoming viral immunoevasion strategies. Moreover, the presence of specific IgG triggered the activation of NK cells against Ab-opsonized cell-free HCMV virions. As compared with NKG2A(+) NK cells, a significant proportion of NKG2C(bright) NK cells was FcεR γ-chain defective and highly responsive to Ab-driven activation, being particularly efficient in the production of antiviral cytokines, mainly TNF-α. Remarkably, the expansion of NKG2C(bright) NK cells in HCMV(+) subjects was related to the overall magnitude of TNF-α and IFN-γ cytokine secretion upon Ab-dependent and -independent activation. We show the power and sensitivity of the anti-HCMV response resulting from the cooperation between specific Abs and the NKG2C(bright) NK-cell subset. Furthermore, we disclose the proinflammatory potential of NKG2C(bright) NK cells, a variable that could influence the individual responses to other pathogens and tumors.

NK Cell and Ig Interplay in Defense against Herpes Simplex Virus Type 1: Epistatic Interaction of CD16A and IgG1 Allotypes of Variable Affinities Modulates Antibody-Dependent Cellular Cytotoxicity and Susceptibility to Clinical Reactivation.

HSV-1 latently infects most humans, causing a variable clinical picture that depends, in part, on host genetic factors. Both IgG and its cellular FcRs, CD16A and CD32A-C (encoded by FCGR3A and FCGR2A-C, respectively, on chromosome 1), display polymorphisms that could affect their defensive function. Of potential relevance are a FCGR3A dimorphism resulting in CD16A-valine/phenylalanine-158 allotypes with different IgG affinity, variations conditioning NK cell expression of CD32B or CD32C, and IgG1 H chain (IGHG1) and kappa-chain (IGKC) polymorphisms determining allotypes designated G1m and Km. In this study, we assessed the contribution of Ig genetic variations and their interaction with FcR polymorphism to HSV-1 susceptibility, as well as their impact on NK cell-mediated Ab-dependent cellular cytotoxicity (ADCC). Our results show an epistatic interaction between IGHG1 and FCGR3A such that the higher affinity CD16A-158V/V genotype associates with an asymptomatic course of HSV-1 infection only in homozygotes for G1m3. Furthermore, CD16A-158V and G1m3 allotypes enhanced ADCC against opsonized HSV-1-infected fibroblasts. Conversely, Km allotypes and CD32B or CD32C expression on NK cells did not significantly influence HSV-1 susceptibility or ADCC. NK cells degranulating against immune serum-opsonized HSV-1-infected fibroblasts had heterogeneous phenotypes. Yet, enhanced ADCC was observed among NK cells showing a differentiated, memory-like phenotype (NKG2C(bright)NKG2A(-)CD57(+)FcRγ(-)), which expand in response to human CMV. These results extend our knowledge on the importance of immunogenetic polymorphisms and NK cell-Ab interplay in the host response against HSV-1 and point to the relevance of interactions between immune responses elicited during chronic coinfection by multiple herpesviruses.

Rapidity of fibrosis progression in liver transplant recipients with recurrent hepatitis C is influenced by toll-like receptor 3 polymorphism.

Liver transplantation activates the innate immune system through toll-like receptors (TLRs), potentially leading to allograft rejection and graft failure. We evaluated the association of single-nucleotide polymorphisms in TLR genes with the severity of hepatitis C virus recurrence after liver transplantation (LT). This is a two-center study of 176 adult patients who received a first LT from deceased donors for hepatitis C virus (HCV) cirrhosis. Eleven polymorphisms were evaluated by real-time polymerase chain reaction and melting curves analyses: TLR1 (Asp248Ser and Ser602Ile), TLR2 (Arg753Gln), TLR3 (Leu412Phe), TLR4 (Asp299Gly), TLR5 (Arg392Stop), TLR6 (Ser249Pro), TLR7 (Gln11Leu), TLR8 (Met1Val), and TLR9 (-1237T/C and -1486C/T). The CC genotype of TLR3 Leu412Phe in liver recipients was associated with severe recurrence (odds ratio (OR) = 2.01, 95% confidence interval (95% CI) = 1.02-3.93, p = 0.04). We also analyzed this polymorphism in 72 of their donors but no association was found with severity of HCV recurrence (p = 0.89). Multivariate analysis showed donor age older than 40 yr (OR=2.93; 95% CI = 1.49-5.8, p = 0.002) and the TLR3 Leu412Phe CC genotype (OR=2.02, 95%CI=1.01-4.05, p = 0.046) were independently associated with severe HCV recurrence. Our results show that the TLR3 Leu412Phe CC genotype is independently associated with severity of hepatitis C recurrence after LT.

NK cell killer Ig-like receptor repertoire acquisition and maturation are strongly modulated by HLA class I molecules.

The interaction between clonally distributed inhibitory receptors and their activating counterparts on NK cells and HLA class I molecules defines NK cell functions, but the role of HLA class I ligands in the acquisition of their receptors during NK development is still unclear. Although some studies demonstrated that HLA-C affects the expression of killer Ig-like receptors (KIR), other studies showed that NK cells acquire their KIR repertoire in a stochastic manner. Only when infected with human CMV is an expansion of self-specific KIR(+) NKG2C(+) NK cells detected. To gain more insight into this question, we compared the coexpression of different KIR molecules, NKG2A, CD8, and CD57, on NK cells in healthy donors and seven patients with deficient HLA class I expression due to mutations in one of the TAP genes. Our results show a correlation between the presence/absence of HLA class I molecules and the coexpression of their receptors. In an HLA class I low-expression context, an increase in KIR molecules' coexpression is detected on the NKG2A(+) CD8(+) subset. In functional assays, hyporesponsiveness was observed for TAP-deficient NK cells derived from four patients. In contrast, NK cells from patient five were functional, whereas CD107a(+) and IFN-γ(+) CD56(dim) NK cells presented a different pattern of HLA class I receptors compared with healthy donors. Taken together, our results provide strong evidence for the role of HLA class I molecules in NK cell maturation and KIR repertoire acquisition.

Haplo-cord transplantation using CD34+ cells from a third-party donor to speed engraftment in high-risk patients with hematologic disorders.

Among the strategies to optimize engraftment of cord blood (CB) stem cell transplantation (SCT), single CB with the coinfusion of CD34(+) stem cells from an HLA-mismatched auxiliary donor (haplo-cord) provides a valid alternative for adult patients without a suitable donor. A total of 132 high-risk adult patients with hematological malignancies from 3 Spanish institutions underwent myeloablative haplo-cord SCT. The median age was 37 years and median weight was 70 kg; 37% had active disease. The median number of postprocessing CB total nucleated and CD34(+) cells was 2.4 × 10(7)/kg (interquartile range [IQR], 1.8 to 2.9) and 1.4 × 10(5)/kg (IQR, .9 to 2), respectively. Neutrophil engraftment occurred in a median of 11.5 days (IQR, 10.5 to 16.5) and platelet engraftment at 36 days (IQR, 25.5 to 77). Graft failure was 2% overall and only 9% for CB. Cumulative incidence of acute graft-versus-host disease (GHVD) grades II to IV was 21% and cumulative incidence of chronic GVHD was 21%. Median follow-up was 60 months (range, 3.5 to 163). Overall survival was 43.5%, event-free survival was 38.3%, nonrelapse mortality was 35%, and relapse was 20% at 5 years. Myeloablative haplo-cord SCT results in fast engraftment of neutrophils and platelets, low incidences of acute and chronic GVHD, and favorable long-term outcomes using single CB units with relatively low cell content. Moreover, CB cell dose had no impact on CB engraftment and survival in this study. Therefore, haplo-cord SCT expands donor availability while reducing CB cell dose requirements.

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.

NKG2C zygosity influences CD94/NKG2C receptor function and the NK-cell compartment redistribution in response to human cytomegalovirus.

Human cytomegalovirus (HCMV) infection promotes a persistent expansion of a functionally competent NK-cell subset expressing the activating CD94/NKG2C receptor. Factors underlying the wide variability of this effect observed in HCMV-seropositive healthy individuals and exacerbated in immunocompromized patients are uncertain. A deletion of the NKG2C gene has been reported, and an apparent relation of NKG2C genotype with circulating NKG2C(+) NK-cell numbers was observed in HCMV(+) children. We have assessed the influence of NKG2C gene dose on the NK-cell repertoire in a cohort of young healthy adults (N = 130, median age 19 years). Our results revealed a relation of NKG2C copy number with surface receptor levels and with NKG2C(+) NK-cell numbers in HCMV(+) subjects, independently of HLA-E dimorphism. Functional studies showed quantitative differences in signaling (i.e. iCa(2+) influx), degranulation, and IL-15-dependent proliferation, in response to NKG2C engagement, between NK cells from NKG2C(+/+) and hemizygous subjects. These observations provide a mechanistic interpretation on the way the NKG2C genotype influences steady-state NKG2C(+) NK-cell numbers, further supporting an active involvement of the receptor in the HCMV-induced reconfiguration of the NK-cell compartment. The putative implications of NKG2C zygosity over viral control and other clinical variables deserve attention.

Host genetic factors in susceptibility to herpes simplex type 1 virus infection: contribution of polymorphic genes at the interface of innate and adaptive immunity.

HSV-1 establishes life-long latency that can result in clinical relapses or in asymptomatic virus shedding. Although virtually all adults have been exposed to HSV-1, the clinical course varies remarkably. Genetic host variability could be related to this clinical diversity. In this study, we analyzed the contribution of gene families in chromosomes 1, 6, 12, and 19, which encode key regulators of the innate and adaptive immunity, in a cohort of 302 individuals. Class I and class II alleles of the HLA system, the copy-number variation of NK cell receptor genes (KIR and NKG2C), the combinations of killer cell Ig-like receptor and their HLA ligands, and CD16A and CD32A allotypes of variable affinity for IgG subclasses were all studied. Although no major susceptibility locus for HSV-1 was identified, our results show that the risk of suffering clinical HSV-1 infection is modified by MHC class I allotypes (B*18, C*15, and the group of alleles encoding A19), the high-affinity receptor/ligand pair KIR2DL2/HLA-C1, and the CD16A-158V/F dimorphism. Conversely, HLA class II and CD32A polymorphisms and NKG2C deletion did not seem to influence the clinical course of herpetic infection. Collectively, these findings support an important role in host defense against herpetic infection for several polymorphic genes implicated in adaptive immunity and in surveillance of its subversion. They confirm the crucial role of cytotoxic cells (CTL and NK) and the contribution of genetic diversity to the clinical course of HSV-1 infection.

Influence of congenital human cytomegalovirus infection and the NKG2C genotype on NK-cell subset distribution in children.

Human cytomegalovirus (HCMV) has been reported to reshape the NK-cell receptor (NKR) distribution, promoting an expansion of CD94/NKG2C(+) NK and T cells. The role of NK cells in congenital HCMV infection is ill-defined. Here we studied the expression of NKR (i.e., NKG2C, NKG2A, LILRB1, CD161) and the frequency of the NKG2C gene deletion in children with past congenital infection, both symptomatic (n = 15) and asymptomatic (n = 11), including as controls children with postnatal infection (n = 11) and noninfected (n = 20). The expansion of NKG2C(+) NK cells in HCMV-infected individuals appeared particularly marked and was associated with an increased number of LILRB1(+) NK cells in cases with symptomatic congenital infection. Increased numbers of NKG2C(+), NKG2A(+), and CD161(+) T cells were also associated to HCMV infection. The NKG2C deletion frequency was comparable in children with congenital HCMV infection and controls. Remarkably, the homozygous NKG2C(+/+) genotype appeared associated with increased absolute numbers of NKG2C(+) NK cells. Moreover, HCMV-infected NKG2C(+/+) children displayed higher absolute numbers of NKG2A(+) and total NK cells than NKG2C(+/-) individuals. Our study provides novel insights on the impact of HCMV infection on the homeostasis of the NK-cell compartment in children, revealing a modulatory influence of NKG2C copy number.

Multiple viral ligands naturally presented by different class I molecules in transporter antigen processing-deficient vaccinia virus-infected cells.

The transporter associated with antigen processing (TAP) delivers the viral proteolytic products generated by the proteasome in the cytosol to the endoplasmic reticulum lumen that are subsequently recognized by cytotoxic T lymphocytes (CTLs). However, several viral epitopes have been identified in TAP-deficient models. Using mass spectrometry to analyze complex human leukocyte antigen (HLA)-bound peptide pools isolated from large numbers of TAP-deficient vaccinia virus-infected cells, we identified 11 ligands naturally presented by four different HLA-A, HLA-B, and HLA-C class I molecules. Two of these ligands were presented by two different HLA class I alleles, and, as a result, 13 different HLA-peptide complexes were formed simultaneously in the same vaccinia virus-infected cells. In addition to the high-affinity ligands, one low-affinity peptide restricted by each of the HLA-A, HLA-B, and HLA-C class I molecules was identified. Both high- and low-affinity ligands generated long-term memory CTL responses to vaccinia virus in an HLA-A2-transgenic mouse model. The processing and presentation of two vaccinia virus-encoded HLA-A2-restricted antigens took place via proteasomal and nonproteasomal pathways, which were blocked in infected cells with chemical inhibitors specific for different subsets of metalloproteinases. These data have implications for the study of the effectiveness of early empirical vaccination with cowpox virus against smallpox disease.