ABSTRACT
NK cells monitor altered molecular patterns in tumors and infected cells through an ample array of receptors. Two families of evolutionarily distant receptors have converged to enable human NK cells to sense levels of HLA class I ligands, frequently abnormal in altered cells. Whilst different forms of polymorphism are a hallmark of killer-cell immunoglobulin-like receptors and their classic HLA-A, B, and C ligands, genetic diversity of killer-cell lectin-like receptors for the non-classical HLA-E (CD94/NKG2 heterodimers) is less conspicuous and has attracted less attention. A common pattern of diversification in both receptor families is evolution of pairs of inhibitory and activating homologs for a common ligand, the genes encoding activating receptors being more frequently affected by copy number variation (CNV). This is exemplified by the gene encoding the activating NKG2C subunit (KLRC2 or NKG2C), which marks an NK-cell subpopulation that differentiates or expands in response to cytomegalovirus. We have studied NKG2C diversity in 240 South European individuals, using polymerase chain reaction and sequencing methods to assess both gene CNV and single-nucleotide polymorphisms (SNPs) affecting its promoter, coding and 3'-untranslated (3'UT) regions. Sequence analysis revealed eight common SNPs-one in the promoter, two in the coding sequence, and five in the 3'UT region. These SNPs associate strongly with each other, forming three conserved extended haplotypes (frequencies: 0.456, 0.221, and 0.117). Homo- and heterozygous combination of these, together with complete gene deletion (0.175) and additional haplotypes with frequencies lower than 0.015, generate a diversity of NKG2C genotypes of potential immunological importance.
Subject(s)
DNA Copy Number Variations , Histocompatibility Antigens Class I , Humans , 3' Untranslated Regions , Alleles , Genotype , Haplotypes , Histocompatibility Antigens Class I/genetics , HLA-A Antigens/genetics , Immunoglobulins/genetics , Lectins/genetics , Ligands , NK Cell Lectin-Like Receptor Subfamily C/geneticsABSTRACT
CD247 (or CD3-ζ chain) is an essential adaptor and signal-transducing molecule of the T-cell antigen receptor (TCR) complex, and it also couples to NK-cell activating receptors such as NKp46, NKp30 and CD16A (FcγRIII). Noncoding sequence polymorphisms and variations in CD247 expression, a tightly regulated process, have been related with an altered immune response in multiple health conditions. A single nucleotide polymorphism (T > A) at nucleotide 844 of the CD247 3'-untranslated region, rs1052231, has been related with lower CD247 gene expression and it has been investigated as a potential biomarker of autoimmune disease. We present here a simple, accurate, reliable, time-efficient, and cost-effective method for CD247-rs1052231 genotyping. Using this method, based on polymerase chain reaction with confronting two-pair primers (PCR-CTPP), we have also characterized the CD247-rs1052231 genotypes in a panel of worldwide available cell lines, which should facilitate study of the role of this polymorphism in immunity and human health.
Subject(s)
Multimorbidity , Polymorphism, Single Nucleotide , Alleles , CD3 Complex/genetics , Cross-Sectional Studies , Genotype , Humans , Untranslated RegionsABSTRACT
Fcγ receptors (FcγR), cell-surface glycoproteins that bind antigen-IgG complexes, control both humoral and cellular immune responses. The FCGR locus on chromosome 1q23.3 comprises five homologous genes encoding low-affinity FcγRII and FcγRIII, and displays functionally relevant polymorphism that impacts on human health. Recurrent events of non-allelic homologous recombination across the FCGR locus result in copy-number variation of ~82.5 kbp-long fragments known as copy-number regions (CNR). Here, we characterize a recently described deletion that we name CNR5, which results in loss of FCGR3A, FCGR3B, and FCGR2C, and generation of a recombinant FCGR3B/A gene. We show that the CNR5 recombination spot lies at the beginning of the third FCGR3 intron. Although the FCGR3B/A-encoded hybrid protein CD16B/A reaches the plasma membrane in transfected cells, its possible natural expression, predictably restricted to neutrophils, could not be demonstrated in resting or interferon γ-stimulated cells. As the CNR5-deletion was originally described in an Ecuadorian family from Llano Grande (an indigenous community in North-Eastern Quito), we characterized the FCGR genetic variation in two populations from the highlands of Ecuador. Our results reveal that CNR5-deletion is relatively frequent in Llano Grande (5 carriers out of 36 donors). Furthermore, we found a high frequency of two strong-phagocytosis variants: the FCGR3B-NA1 haplotype and the CNR1 duplication, which translates into an increased FCGR3B and FCGR2C copy-number. CNR1 duplication was particularly increased in Llano Grande, 77.8% of the studied sample carrying at least one such duplication. In contrast, an extended haplotype CD16A-176V - CD32C-ORF+2B.2 - CD32B-2B.4 including strong activating and inhibitory FcγR variants was absent in Llano Grande and found at a low frequency (8.6%) in Ecuador highlands. This particular distribution of FCGR polymorphism, possibly a result of selective pressures, further confirms the importance of a comprehensive, joint analysis of all genetic variations in the locus and warrants additional studies on their putative clinical impact. In conclusion, our study confirms important ethnic variation at the FCGR locus; it shows a distinctive FCGR polymorphism distribution in Ecuador highlands; provides a molecular characterization of a novel CNR5-deletion associated with CD16A and CD16B deficiency; and confirms its presence in that population.
Subject(s)
DNA Copy Number Variations , Genetics, Population , Polymorphism, Single Nucleotide , Receptors, IgG/genetics , Alleles , Cell Line , Ecuador , GPI-Linked Proteins/genetics , Gene Expression , Genetic Loci , Genetic Variation , Genotype , Granulocytes/metabolism , HumansABSTRACT
The novel NKG2C*03 allele encodes a hybrid of the NKG2C*01 and NKG2C*02 primary structures.
Subject(s)
Killer Cells, Natural , NK Cell Lectin-Like Receptor Subfamily C , Alleles , Genomics , Humans , NK Cell Lectin-Like Receptor Subfamily C/geneticsABSTRACT
Following the publication of the original article [1], the authors have requested to amend the Abstract and Discussion section as follows.
ABSTRACT
Inhibitory Killer-cell Immunoglobulin-like Receptors (KIR) specific for HLA class I molecules enable human natural killer cells to monitor altered antigen presentation in pathogen-infected and tumor cells. KIR genes display extensive copy-number variation and allelic polymorphism. They organize in a series of variable arrangements, designated KIR haplotypes, which derive from duplications of ancestral genes and sequence diversification through point mutation and unequal crossing-over events. Genomic studies have established the organization of multiple KIR haplotypes-many of them are fixed in most human populations, whereas variants of those have less certain distributions. Whilst KIR-gene diversity of many populations and ethnicities has been explored superficially (frequencies of individual genes and presence/absence profiles), less abundant are in-depth analyses of how such diversity emerges from KIR-haplotype structures. We characterize here the genetic diversity of KIR in a sample of 414 Spanish individuals. Using a parsimonious approach, we manage to explain all 38 observed KIR-gene profiles by homo- or heterozygous combinations of six fixed centromeric and telomeric motifs; of six variant gene arrangements characterized previously by us and others; and of two novel haplotypes never detected before in Caucasoids. Associated to the latter haplotypes, we also identified the novel transcribed KIR2DL5B*0020202 allele, and a chimeric KIR2DS2/KIR2DL3 gene (designated KIR2DL3*033) that challenges current criteria for classification and nomenclature of KIR genes and haplotypes.
Subject(s)
Genotype , Killer Cells, Natural/metabolism , Receptors, KIR/genetics , Alleles , DNA Copy Number Variations , Europe , Flow Cytometry , Gene Frequency , Genetics, Population , Haplotypes , Humans , Polymorphism, Genetic , Receptors, KIR/metabolism , Recombination, Genetic , Spain , TranscriptomeABSTRACT
BACKGROUND: C3 hypocomplementemia and the presence of C3 nephritic factor (C3NeF), an autoantibody causing complement system over-activation, are common features among most patients affected by Barraquer-Simons syndrome (BSS), an acquired form of partial lipodystrophy. Moreover, BSS is frequently associated with autoimmune diseases. However, the relationship between complement system dysregulation and BSS remains to be fully elucidated. The aim of this study was to provide a comprehensive immunological analysis of the complement system status, autoantibody signatures and HLA profile in BSS. Thirteen subjects with BSS were recruited for the study. The circulating levels of complement components, C3, C4, Factor B (FB) and Properdin (P), as well as an extended autoantibody profile including autoantibodies targeting complement components and regulators were assessed in serum. Additionally, HLA genotyping was carried out using DNA extracted from peripheral blood mononuclear cells. RESULTS: C3, C4 and FB levels were significantly reduced in patients with BSS as compared with healthy subjects. C3NeF was the most frequently found autoantibody (69.2% of cases), followed by anti-C3 (38.5%), and anti-P and anti-FB (30.8% each). Clinical data showed high prevalence of autoimmune diseases (38.5%), the majority of patients (61.5%) being positive for at least one of the autoantibodies tested. The HLA allele DRB1*11 was present in 54% of BSS patients, and the majority of them (31%) were positive for *11:03 (vs 1.3% in the general population). CONCLUSIONS: Our results confirmed the association between BSS, autoimmunity and C3 hypocomplementemia. Moreover, the finding of autoantibodies targeting complement system proteins points to complement dysregulation as a central pathological event in the development of BSS.
Subject(s)
Lipodystrophy/immunology , Lipodystrophy/metabolism , Adolescent , Adult , Aged , Autoimmunity/physiology , Child , Complement C3/metabolism , Complement C3 Nephritic Factor/metabolism , Complement C4/metabolism , Complement Factor B/metabolism , Female , Humans , Male , Middle Aged , Properdin/metabolism , Young AdultABSTRACT
Natural killer (NK) cells can orchestrate effective antitumor immunity. The presence of tumor-infiltrating NK cells in diagnostic biopsies predicts pathologic complete response (pCR) to HER2-specific therapeutic antibodies in patients with primary breast cancer. Here, we analyzed whether diversity in circulating NK cells might influence tumor infiltration and HER2-specific therapeutic antibody efficacy. We found that numbers of circulating CD57+ NK cells inversely correlated with pCR to HER2-specific antibody treatment in patients with primary breast cancer independently of age, traditional clinicopathologic factors, and CD16A 158F/V genotype. This association was uncoupled from the expression of other NK-cell receptors, the presence of adaptive NK cells, or changes in major T-cell subsets, reminiscent of cytomegalovirus-induced immunomodulation. NK-cell activation against trastuzumab-coated HER2+ breast cancer cells was comparable in patients with high and low proportions of CD57+ NK cells. However, circulating CD57+ NK cells displayed decreased CXCR3 expression and CD16A-induced IL2-dependent proliferation in vitro Presence of CD57+ NK cells was reduced in breast tumor-associated infiltrates as compared with paired peripheral blood samples, suggesting deficient homing, proliferation, and/or survival of NK cells in the tumor niche. Indeed, numbers of circulating CD57+ were inversely related to tumor-infiltrating NK-cell numbers. Our data reveal that NK-cell differentiation influences their antitumor potential and that CD57+ NK cells may be a biomarker useful for tailoring HER2 antibody-based therapeutic strategies in breast cancer.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Breast Neoplasms/blood , Breast Neoplasms/metabolism , CD57 Antigens/metabolism , Drug Resistance, Neoplasm , Killer Cells, Natural/metabolism , Lymphocyte Count , Adult , Aged , Biopsy , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , CD57 Antigens/genetics , Female , Genotype , Humans , Immunomodulation , Immunophenotyping , Killer Cells, Natural/immunology , Lymphocyte Activation/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Middle Aged , Neoplasm Staging , Receptors, IgG/geneticsABSTRACT
Nucleosomes cover eukaryotic genomes like beads on a string and play a central role in regulating genome function. Isolated strings of nucleosomes have the potential to compact and form higher order chromatin structures, such as the well-characterized 30-nm fiber. However, despite tremendous advances in observing chromatin fibers in situ it has not been possible to confirm that regularly ordered fibers represent a prevalent structural level in the folding of chromosomes. Instead, it appears that folding at a larger scale than the nucleosome involves a variety of random structures with fractal characteristics. Nevertheless, recent progress provides evidence for the existence of structural motifs in chromatin fibers, potentially localized to strategic sites in the genome. Here we review the current understanding of chromatin fiber folding and the emerging roles that oligonucleosomal motifs play in the regulation of genome function.
Subject(s)
DNA/metabolism , Genome/physiology , Nucleosomes/metabolism , Animals , DNA/chemistry , Models, Molecular , Nucleosomes/chemistryABSTRACT
Natural killer (NK) cells play a dual role in the defense against viral pathogens by directly lysing infected cells as well as by regulating anti-viral T cell immunity. Infection by human cytomegalovirus (HCMV) promotes a persistent expansion of NKG2C+ adaptive NK cells which have been shown to display enhanced antibody-dependent responses against infected targets and associated to viral control in transplanted patients. Based on gene expression data showing increased transcription of CIITA and several genes related to the MHC class II pathway in adaptive NK cells, we explored their putative capacity for antigen presentation to CD4+ T cells. Phenotypic analysis confirmed a preferential steady-state expression of HLA-DR by circulating NKG2C+ adaptive NK cells in healthy individuals. Expression of HLA-DR in NKG2C+ adaptive NK cells was variable and unrelated to the expression of activation (i.e., CD69 and CD25) or differentiation (i.e., FcRγ chain, CD57) markers, remaining stable over time at the individual level. Incubation of purified NK cells with HCMV complexed with serum specific antibodies induced an up-regulation of surface HLA-DR concomitant to CD16 loss whereas no changes in CD80/CD86 co-stimulatory ligands were detected. In addition, surface CX3CR1 decreased upon antigen-loading while HLA-DR+ NK cells maintained a CCR7-, CXCR3low homing profile. Remarkably, HCMV-loaded purified NK cells activated autologous CD4+ T cells in an HLA-DR dependent manner. The fraction of T lymphocytes activated by antigen-loaded NK cells was smaller than that stimulated by monocyte-derived dendritic cells, corresponding to CD28-negative effector-memory CD4+ T cells with cytotoxic potential. Antigen presentation by NK cells activated a polyfunctional CD4+ T cell response characterized by degranulation (CD107a) and the secretion of Th1 cytokines (IFNγ and TNFα). Overall, our data discloses the capacity of NKG2C+ adaptive NK cells to process and present HCMV antigens to memory CD4+ cytotoxic T cells, directly regulating their response to the viral infection.
Subject(s)
Antigen Presentation , Antigens, Viral/immunology , CD4-Positive T-Lymphocytes/immunology , Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , HLA-DR Antigens/immunology , Immunologic Memory , Killer Cells, Natural/immunology , NK Cell Lectin-Like Receptor Subfamily C/immunology , Adult , CD4-Positive T-Lymphocytes/pathology , Cytomegalovirus Infections/pathology , Female , Humans , Killer Cells, Natural/pathology , MaleABSTRACT
Classical Hodgkin lymphoma (cHL) is frequently related to Epstein-Barr virus (EBV) infection. Its malignant capacity is attributed to disruption of an EBV-host balance influenced by environmental and genetic drivers. EBV structures activate Type I interferon (IFN) pathway of the innate immunity, therefore, genetic polymorphisms could influence this response. We explored the impact of four single nucleotide polymorphisms (SNPs) on EBV-associated cHL susceptibility. Toll-like receptors 9 (TLR9_rs5743836), and 3 (TLR3_rs3775291), Interleukin-28B (IL28B_rs12979860), and DEAD-box polypeptide 58 (DDX58_rs10813831) were genotyped in 73 EBV-positive and 106 EBV-negative cHL patients and 396 controls. Only DDX58_rs10813831 T-allele was decreased among EBV-positive cHL compared to controls. A stratified analysis in EBV-positive cHL showed that the reduced rate was associated with younger age and nodular sclerosis. In conclusion, DDX58_rs10813831 T-allele may be associated with a reduced risk of nodular sclerosis EBV-related cHL, which suggests a role for RIG-I (retinoic acid-inducible gene I), encoded by DDX58, in these cases.
Subject(s)
Alleles , DEAD Box Protein 58/genetics , Epstein-Barr Virus Infections/complications , Herpesvirus 4, Human , Hodgkin Disease/etiology , Hodgkin Disease/pathology , Polymorphism, Single Nucleotide , Adolescent , Adult , Aged , Case-Control Studies , Cell Transformation, Viral , DEAD Box Protein 58/metabolism , Female , Gene Frequency , Genetic Predisposition to Disease , Genotype , Hodgkin Disease/metabolism , Humans , Male , Middle Aged , Odds Ratio , Receptors, Immunologic , Sclerosis , Young AdultABSTRACT
Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.
ABSTRACT
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.
Subject(s)
Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , Gene Dosage , Killer Cells, Natural/immunology , Lymphocyte Subsets/immunology , NK Cell Lectin-Like Receptor Subfamily C/metabolism , Adaptive Immunity , Adult , Cell Proliferation , Cells, Cultured , Cohort Studies , Female , Humans , Killer Cells, Natural/virology , Lymphocyte Activation , Lymphocyte Subsets/virology , Male , NK Cell Lectin-Like Receptor Subfamily C/genetics , Receptors, IgG/genetics , Receptors, IgG/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
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.
Subject(s)
Herpes Simplex/immunology , Herpesvirus 1, Human/immunology , Immunoglobulin G/immunology , Killer Cells, Natural/immunology , Animals , Antibody-Dependent Cell Cytotoxicity/genetics , Antibody-Dependent Cell Cytotoxicity/immunology , Cell Degranulation/genetics , Cell Degranulation/immunology , Cell Line , Disease Susceptibility , Epistasis, Genetic , Gene Expression , Genetic Variation , Genotype , Herpes Simplex/genetics , Humans , Immunoglobulin G/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin kappa-Chains/genetics , Immunoglobulin kappa-Chains/immunology , Immunophenotyping , Phenotype , Polymorphism, Genetic , Receptors, IgG/genetics , Receptors, IgG/metabolism , Virus Activation/immunologyABSTRACT
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.
Subject(s)
Antibodies, Viral/immunology , Antibody Formation/immunology , Cytomegalovirus/immunology , Killer Cells, Natural/immunology , NK Cell Lectin-Like Receptor Subfamily C/immunology , Antibody-Dependent Cell Cytotoxicity/immunology , Cell Line , Cells, Cultured , Fibroblasts/immunology , Fibroblasts/metabolism , Fibroblasts/virology , Flow Cytometry , Humans , Immunoglobulin G/immunology , Interferon-gamma/immunology , Interferon-gamma/metabolism , Killer Cells, Natural/metabolism , NK Cell Lectin-Like Receptor Subfamily C/metabolism , Receptors, IgE/immunology , Receptors, IgE/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism , Virion/immunologyABSTRACT
Cryofibrinogenemia is a rare clinical finding with a yet unknown physiopathogenic mechanism. We describe the case of a woman with cold-induced extensive necrotic lesions that responded poorly to initial corticosteroid and anticoagulant therapies. Serum cryoglobulin determinations were persistently negative. After several years of evolution, she developed severe cold-related skin lesions that required left-leg amputation. Further analysis disclosed the presence of cryofibrinogen and an apparently insignificant serum monoclonal immunoglobulin Gκ peak. We additionally demonstrate that the cold precipitation of fibrinogen was directly related to the monoclonal paraprotein presence. The lesions responded dramatically to a B cell-targeted therapy with intravenous cyclophosphamide and dexamethasone.
Subject(s)
Antibodies, Monoclonal/immunology , Cryoglobulinemia/drug therapy , Cryoglobulinemia/immunology , Cyclophosphamide/therapeutic use , Dexamethasone/therapeutic use , Paraproteins/immunology , Antibodies, Monoclonal/blood , Cryoglobulins/immunology , Drug Therapy, Combination , Female , Fibrinogens, Abnormal/immunology , Humans , Middle Aged , Treatment OutcomeABSTRACT
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.
Subject(s)
Cytomegalovirus Infections , Cytomegalovirus/immunology , Gene Dosage , Killer Cells, Natural/immunology , NK Cell Lectin-Like Receptor Subfamily C , NK Cell Lectin-Like Receptor Subfamily D , Adolescent , Adult , Calcium Signaling/genetics , Calcium Signaling/immunology , Cell Degranulation/genetics , Cell Degranulation/immunology , Child , Child, Preschool , Cytomegalovirus/genetics , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/immunology , Female , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Interleukin-15/genetics , Interleukin-15/immunology , Male , NK Cell Lectin-Like Receptor Subfamily C/genetics , NK Cell Lectin-Like Receptor Subfamily C/immunology , NK Cell Lectin-Like Receptor Subfamily D/genetics , NK Cell Lectin-Like Receptor Subfamily D/immunology , HLA-E AntigensABSTRACT
Cell culture systems are widely used for molecular, genetic and biochemical studies. Primary cell cultures of animal tissues offer the advantage that specific cell types can be studied in vitro outside of their normal environment. We provide a detailed protocol for generating primary neural cell cultures derived from larval brains of Drosophila melanogaster. The developing larval brain contains stem cells such as neural precursors and intermediate neural progenitors, as well as fully differentiated and functional neurons and glia cells. We describe how to analyze these cell types in vitro by immunofluorescent staining and scanning confocal microscopy. Cell type-specific fluorescent reporter lines and genetically encoded calcium sensors allow the monitoring of developmental, cellular processes and neuronal activity in living cells in vitro. The protocol provides a basis for functional studies of wild-type or genetically manipulated primary neural cells in culture, both in fixed and living samples. The entire procedure takes â¼3 weeks.
Subject(s)
Cell Culture Techniques , Drosophila melanogaster/cytology , Embryo, Nonmammalian/cytology , Neurons/cytology , Animals , Drosophila melanogaster/embryology , Larva/cytology , Microscopy, FluorescenceABSTRACT
A recently developed anti-KIR2DL5 (CD158f) antibody has demonstrated KIR2DL5 expression on the surface of NK and T lymphocytes, making it the last functional KIR identified in the human genome. KIR2DL5 belongs to an ancestral lineage of KIR with Ig-like domains of the D0-D2 type, of which KIR2DL4, an HLA-G receptor, is the only other human member. Despite KIR2DL4 and KIR2DL5 being encoded by genes with similar domain usage, several KIR2DL5 functions resemble more closely those of KIR recognizing classical HLA class I molecules - surface-expressed KIR2DL5 inhibits NK cells through the SHP-2 phosphatase and displays a clonal distribution on NK and T lymphocytes. No activating homolog of KIR2DL5 has been described in any species. The genetics of KIR2DL5 is complicated by duplication of its gene in an ancestor of modern humans living â¼1.7 million years ago. Both KIR2DL5 paralogs have undergone allelic diversification; the centromeric gene is most often represented by alleles whose expression is silenced epigenetically through DNA methylation, thus providing a natural system to investigate the regulation of KIR transcription. The role of KIR2DL5 in immunity is not completely understood, in spite of different attempts to define its ligand. Here we revisit the most relevant characteristics of KIR2DL5, an NK-cell receptor possessing a unique combination of genetic, structural, and functional features.