Neus Romo

NKp46 and DNAM-1 NK-cell receptors drive the response to human cytomegalovirus-infected myeloid dendritic cells overcoming viral immune evasion strategies

Information on natural killer (NK)-cell receptor-ligand interactions involved in the response to human cytomegalovirus (HCMV) is limited and essentially based on the study of infected fibroblasts. Experimental conditions were set up to characterize the NK response to HCMV-infected myeloid dendritic cells (DCs). Monocyte-derived DCs (moDCs) infected by the TB40/E HCMV strain down-regulated the expression of human leukocyte antigen class I molecules and specifically activated autologous NK-cell populations. NKG2D ligands appeared virtually undetectable in infected moDCs, reflecting the efficiency of immune evasion mechanisms, and explained the lack of antagonistic effects of NKG2D-specific monoclonal antibody. By contrast, DNAM-1 and DNAM-1 ligands (DNAM-1L)-specific monoclonal antibodies inhibited the NK response at 48 hours after infection, although the impact of HCMV-dependent down-regulation of DNAM-1L in infected moDCs was perceived at later stages. moDCs constitutively expressed ligands for NKp46 and NKp30 natural cytotoxicity receptors, which were partially reduced on HCMV infection; yet, only NKp46 appeared involved in the NK response. In contrast to previous reports in fibroblasts, human leukocyte antigen-E expression was not preserved in HCMV-infected moDCs, which triggered CD94/NKG2A(+) NK-cell activation. The results provide an insight on key receptor-ligand interactions involved in the NK-cell response against HCMV-infected moDCs, stressing the importance of the dynamics of viral immune evasion mechanisms.

Influence of human cytomegalovirus infection on the NK cell receptor repertoire in children

Human cytomegalovirus (hCMV) infection is usually asymptomatic but may cause disease in immunocompromised hosts. It has been reported that hCMV infection may shape the NK cell receptor (NKR) repertoire in adult individuals, promoting a variable expansion of the CD94/NKG2C+ NK cell subset. We explored the possible relationship between this viral infection and the expression pattern of different NKR including CD94/NKG2C, CD94/NKG2A, immunoglobulin‐like transcript 2 (ILT2, CD85j), KIR2DL1/2DS1, KIR3DL1, and CD161 in peripheral blood lymphocytes from healthy children, seropositive (n=21) and seronegative (n=20) for hCMV. Consistent with previous observations in adults, a positive serology for hCMV was associated with increased numbers of NKG2C+ NK and T cells as well as with ILT2+ T lymphocytes. Moreover, the proportions of CD161+ and NKG2C+CD56−CD3− NK cells also tended to be increased in hCMV+ individuals. Excretion of the virus was associated with higher proportions of NKG2C+ NK cells. Altogether, these data reveal that hCMV may have a profound influence on the NKR repertoire in early childhood.

Natural killer cell-mediated response to human cytomegalovirus-infected macrophages is modulated by their functional polarization

MΦ comprise a heterogeneous population of cells, which contribute to host defense and maintenance of immune homeostasis. MΦ may be infected by human cytomegalovirus (HCMV), which has evolved different strategies to subvert the immune response. In the present study, we comparatively analyzed the natural killer (NK) cell response against HCMV (TB40E)‐infected proinflammatory (M1) and antinflammatory (M2) MΦ, derived from autologous monocytes, cultured in the presence of GM‐CSF and M‐CSF, respectively. M1 MΦ were more resistant to infection and secreted IL‐6, TNF‐α, IFN‐α, and IL‐12; by contrast, in HCMV‐infected M2 MΦ, proinflammatory cytokines, IL‐10, and IFN‐α production were limited and IL‐12 was undetectable. NK cell degranulation was triggered by interaction with HCMV‐infected M1 and M2 MΦ at 48 h postinfection. The response was partially inhibited by specific anti‐NKp46, anti‐DNAM‐1, and anti‐2B4 mAb, thus supporting a dominant role of these activating receptors. By contrast, only HCMV‐infected M1 MΦ efficiently promoted NK cell‐mediated IFN‐γ secretion, an effect partially related to IL‐12 production. These observations reveal differences in the NK cell response triggered by distinct, HCMV‐infected, monocyte‐derived cell types, which may be relevant in the immunopathology of this viral infection.

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. iCa2+ 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.

IL-12-Dependent Inducible Expression of the CD94/NKG2A Inhibitory Receptor Regulates CD94/NKG2C+ NK Cell Function

The inhibitory CD94/NKG2A and activating CD94/NKG2C killer lectin-like receptors specific for HLA-E have been reported to be selectively expressed by discrete NK and T cell subsets. In the present study, minor proportions of NK and T cells coexpressing both CD94/NKG2A and CD94/NKG2C were found in fresh peripheral blood from adult blood donors. Moreover, CD94/NKG2A surface expression was transiently detected upon in vitro stimulation of CD94/NKG2C+ NK cells in the presence of irradiated allogeneic PBMC or rIL-12. A similar effect was observed upon coculture of NKG2C+ NK clones with human CMV-infected autologous dendritic cell cultures, and it was prevented by an anti-IL-12 mAb. NKG2A inhibited the cytolytic activity of NKG2C+ NK clones upon engagement either by a specific mAb or upon interaction with a transfectant of the HLA class I-deficient 721.221 cell line expressing HLA-E. These data indicate that beyond its constitutive expression by an NK cell subset, NKG2A may be also transiently displayed by CD94/NKG2C+ NK cells under the influence of IL-12, providing a potential negative regulatory feedback mechanism.

Association of Atherosclerosis With Expression of the LILRB1 Receptor By Human NK and T-Cells Supports the Infectious Burden Hypothesis

Objective—The contribution of human cytomegalovirus (HCMV) to vascular disease may depend on features of the immune response not reflected by the detection of specific antibodies. Persistent HCMV infection in healthy blood donors has been associated with changes in the distribution of NK cell receptors (NKR). The putative relationship among HCMV infection, NKR distribution, subclinical atherosclerosis, and coronary heart disease was assessed. Methods and Results—NKR expression was compared in acute myocardial infarction (AMI) patients (n=70) and a population-based control sample (n=209). The relationship between NKR expression and carotid intima-media thickness (CIMT) in controls (n=149) was also studied. HCMV infection was associated with higher proportions of NKG2C+ and LILRB1+ NK and T-cells. In contrast, only LILRB1+ NK and CD56+ T-cells were found to be increased in AMI patients, independent of age, sex, conventional vascular risk factors, and HCMV seropositivity. Remarkably, LILRB1 expression in NK and T-cells significantly correlated with CIMT in controls. Conclusion—The association of overt and subclinical atherosclerotic disease with LILRB1+ NK and T-cells likely reflects a relationship between the immune challenge by infections and cardiovascular disease risk, without attributing a dominant role for HCMV. Our findings may lead to the identification of novel biomarkers of vascular disease.

Functional impact of A91V mutation of the PRF1 perforin gene

Perforin (PRF1) gene mutations have been associated with Familial Hemophagocytic Lymphohistiocytosis type 2 (FHL2). Substitution p.A91V (c.272C>T) in exon 2 was first described as a neutral polymorphism. Nonetheless, recent clinical evidence and functional assays, suggest a potential pathogenic role for p.A91V, especially in compound heterozygous individuals. Moreover, p.A91V homozygosity has been linked to various pathological states including FHL and lymphocytic leukaemias. In the present report we evaluated the impact of this mutation in a compound heterozygous A91V/G149S 31 year-old asymptomatic female. Functional assays revealed low perforin expression levels, as well as an impaired NK cell-mediated cytotoxicity, partially reconstituted after incubation with IL-2. These results support that p.A91V mutation, associated to another mutated PRF1 allele, may potentially predispose seemingly healthy carriers to suffer a milder FHL2 clinical phenotype, including later onset of the disease. Thus, clinical monitoring of p.A91V carrier individuals bearing another mutation in PRF1 is warranted.

Natural killer cell receptor expression reflects the role of human cytomegalovirus in the pathogenesis of a subset of CD4 T-cell large granular lymphocytosis

A high frequency of CD4(+) T-cell large granular lymphocyte (T-LGL) lymphocytosis occurs in human leukocyte antigen (HLA) -DRB1*0701 individuals displaying monoclonal expansions of Vβ13.1+ CD4(+) T-cell clones, which specifically respond to human cytomegalovirus (HCMV) antigens. We previously reported the expression of natural killer (NK)-cell associated receptors (NKR) by HCMV-specific cytolytic CD4(+) T cells from healthy donors. In the present study a high expression of different NKR (i.e., NKG2D, killer Ig-like receptors (KIR), CD94, ILT2) was observed in CD4(+) T cells from both Vβ13.1- and Vβ13.1+ CD4(+) T-LGL cases. Remarkably, elevated numbers of CD94/NKG2C+ NK cells, previously shown to expand in association to HCMV infection, were preferentially found in Vβ13.1+ T-LGL, further supporting its role in the pathogenesis of a subset of CD4(+) T-LGL.