RESUMO
Long-lived "memory-like" NK cells have been identified in individuals infected by human cytomegalovirus (HCMV), but little is known about how the memory-like NK cell pool is formed. Here, we have shown that HCMV-infected individuals have several distinct subsets of memory-like NK cells that are often deficient for multiple transcription factors and signaling proteins, including tyrosine kinase SYK, for which the reduced expression was stable over time and correlated with epigenetic modification of the gene promoter. Deficient expression of these proteins was largely confined to the recently discovered FcRγ-deficient NK cells that display enhanced antibody-dependent functional activity. Importantly, FcRγ-deficient NK cells exhibited robust preferential expansion in response to virus-infected cells (both HCMV and influenza) in an antibody-dependent manner. These findings suggest that the memory-like NK cell pool is shaped and maintained by a mechanism that involves both epigenetic modification of gene expression and antibody-dependent expansion.
Assuntos
Anticorpos/imunologia , Infecções por Citomegalovirus/genética , Epigênese Genética/imunologia , Memória Imunológica , Células Matadoras Naturais/imunologia , Proliferação de Células , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Metilação de DNA , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Matadoras Naturais/classificação , Células Matadoras Naturais/patologia , Células Matadoras Naturais/virologia , Análise em Microsséries , Subfamília C de Receptores Semelhantes a Lectina de Células NK/deficiência , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Receptores de IgG/deficiência , Receptores de IgG/genética , Receptores de IgG/imunologia , Transdução de Sinais , Quinase SykRESUMO
The â¼28-kb 3' regulatory region (3'RR), which is located at the most distal 3' region of the Ig H chain locus, has multiple regulatory functions that control IgH expression, class-switch recombination (CSR), and somatic hypermutation. In this article, we report that deletion of the entire 3'RR in a mouse B cell line that is capable of robust cytokine-dependent CSR to IgA results in reduced, but not abolished, CSR. These data suggest that 3'RR is not absolutely required for CSR and, thus, is not essential for targeting activation-induced cytidine deaminase to S regions, as was suggested. Moreover, replacing 3'RR with a DNA fragment including only its four DNase I hypersensitive sites (lacking the large spacer regions) restores CSR to a level equivalent to or even higher than in wild-type cells, suggesting that the four hypersensitive sites contain most of the CSR-promoting functions of 3'RR. Stimulated cells express abundant germline transcripts, with the presence or absence of 3'RR, providing evidence that 3'RR has a role in promoting CSR that is unique from enhancing S region transcription.
Assuntos
Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região de Troca de Imunoglobulinas/genética , Animais , Células Cultivadas , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Região de Troca de Imunoglobulinas/imunologia , CamundongosRESUMO
NK cells respond to tumor and virus-infected cells directly through several activation receptors, including natural cytotoxicity receptors, or indirectly through the activating Fc receptor CD16 for antibody-coated cells. Triggering of NK-cell effector functions through these receptors depends on physically associated transmembrane signaling adaptors, such as FcRγ (also known as FcεRIγ) and CD3ζ, both of which have been traditionally believed to be expressed by all mature NK cells. However, we have identified a distinct subset of human NK cells that are deficient for FcRγ expression but express normal levels of CD3ζ. FcRγ-deficient NK cells were readily detectable in about one-third of the healthy individuals examined. The deficiency was confined to the CD56(dim) population and was due to low FcRγ mRNA. FcRγ-deficient NK cells displayed dramatically reduced expression of the natural cytotoxicity receptors NKp46 and NKp30 but still expressed substantial levels of CD16. Compared to FcRγ-expressing NK cells, FcRγ-deficient NK cells showed poor direct reactivity toward tumor targets as measured by cytokine production and degranulation. Unexpectedly, however, FcRγ-deficient NK cells exhibited significantly more robust responsiveness upon stimulation through CD16, particularly for cytokine production, compared to FcRγ-expressing NK cells. Thus, our study reveals FcRγ-deficient NK cells as a novel subset of human NK cells that have remarkably potent responses toward antibody-coated targets. These findings also illustrate a differential contribution of FcRγ and CD3ζ for the expression and functional activity of their associated receptors.
Assuntos
Anticorpos/imunologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Neoplasias/imunologia , Receptores de IgG/metabolismo , Antígenos de Neoplasias/imunologia , Complexo CD3/metabolismo , Células Cultivadas , Citocinas/metabolismo , Humanos , Imunofenotipagem , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Receptor 3 Desencadeador da Citotoxicidade Natural/genética , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Receptor Cross-Talk , Receptores de IgG/deficiência , Receptores de IgG/imunologia , Transdução de SinaisRESUMO
Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) class switch recombination (CSR), somatic hypermutation (SHM), and gene conversion by converting DNA cytosines to uracils at specific genomic regions. In this study, we examined AID footprints across the entire length of an engineered switch region in cells ablated for uracil repair. We found that AID deamination occurs predominantly at WRC hot spots (where W is A or T and R is A or G) and that the deamination frequency remains constant across the entire switch region. Importantly, we analyzed monoallelic AID deamination footprints on both DNA strands occurring within a single cell cycle. We found that AID generates few and mostly isolated uracils in the switch region, although processive AID deaminations are evident in some molecules. The frequency of molecules containing deamination on both DNA strands at the acceptor switch region correlates with the class switch efficiency, raising the possibility that the minimal requirement for DNA double-strand break (DSB) formation is as low as even one AID deamination event on both DNA strands.
Assuntos
Linfócitos B/citologia , Citosina/metabolismo , Switching de Imunoglobulina/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Animais , Citidina Desaminase/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Desaminação/imunologia , Recombinação Genética/genéticaRESUMO
A randomized double-blind placebo-controlled immunity study involving 99 healthy volunteers was performed to investigate the effect of poly- γ -glutamate ( γ -PGA) on human natural killer (NK) cell activity in peripheral blood. The volunteers were randomly assigned to one of three groups and orally treated with solutions (25 mL) containing 0 mg (placebo), 250 mg (low dosage), or 500 mg (high dosage) of γ -PGA. Each volunteer took one dose every 12 hours for 8 weeks. Blood samples were drawn before the initial treatment and at the 4th and the 8th weeks of treatment. NK cell activity was assessed by measuring its degranulation, cytokine production, and cytotoxicity against the K562 cell line. Our results revealed that the cytotoxic activities of NK cells from the high-dosage γ -PGA group were significantly higher (P < 0.05 for all comparisons) compared to the low dosage and placebo groups at weeks 4 and 8 after the initial treatment. This increase in the NK cell activity among peripheral blood mononuclear cells (PBMCs) of healthy individuals was also confirmed in vitro (as assessed by the degranulation and cytokine production). These results suggest that the oral administration of γ -PGA induces a cell-mediated immunity by increasing the NK cell activity in humans.