RESUMO
Patients chronically infected with hepatitis C virus (HCV) frequently develop mixed cryoglobulinemia (MC), a monoclonal expansion of immunoglobulin M (IgM)+ autoreactive B cells, and also have an increased B-cell lymphoma risk. Whether HCV infection also impacts the B-cell compartment and the B-cell receptor repertoire in patients not affected by MC or lymphomas is poorly understood. Flow cytometric analysis of peripheral blood B cells of 30 MC-negative HCV-infected patients and 15 healthy controls revealed that frequencies of class-switched memory B cells were increased in the patients, whereas frequencies of transitional and naive B cells were decreased. For 22 HCV+ patients and 7 healthy controls, we performed high-throughput sequencing of immunoglobulin heavy chain VDJ rearrangements of naive, mature CD5+, IgM+ memory, and class-switched memory B cells. An increased usage of several IGHV genes, including IGHV1-69 and IGHV4-59, which are closely linked to MC and HCV-associated lymphomas, was specifically seen among IgM+ memory B cells of the patients. Moreover, many, and partly very large, expanded clones were seen predominantly among IgM+ memory B cells of all HCV-infected patients analyzed. Thus, chronic HCV infection massively disturbs the B-cell compartment even in patients without clinically detectable B-cell lymphoproliferation and generates many large B-cell clones, especially among non-class-switched memory B cells. Because B-cell clones in MC and lymphomas derive from this B-cell subset, this establishes IgM+ memory B cells as a general target of lymphoproliferation in HCV+ patients, affecting apparently all patients.
Assuntos
Linfócitos B/fisiologia , Evolução Clonal , Genes de Cadeia Pesada de Imunoglobulina , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Éxons VDJ/genética , Adulto , Estudos de Casos e Controles , Proliferação de Células/genética , Evolução Clonal/genética , Evolução Clonal/imunologia , Células Clonais/metabolismo , Células Clonais/patologia , Feminino , Hepatite C Crônica/sangue , Humanos , Contagem de Linfócitos , MasculinoRESUMO
Chronic infection with hepatitis C virus (HCV) often affects the B-cell compartment, leading to the occurrence of autoimmunity and B-cell lymphoproliferation, in particular mixed cryoglobulinemia and B-cell lymphomas. HCV presumably causes these lymphoproliferations by chronic antigenic stimulation and/or direct mutagenic effects on B cells. It has been speculated that the interaction of HCV with B cells and the expansion of antigen-triggered B cells happens in germinal center-like structures in the livers of HCV carriers. We studied rearranged immunoglobulin V(H) genes from seven B-cell follicles microdissected from the livers of three unselected chronic HCV patients. The follicles consisted of polyclonal naive and memory B-cell populations with only rare indication of minor clonal expansions and no evidence for active somatic hypermutation. Frequent detection of V(H) rearrangements using the VH1-69 gene segment nevertheless indicated that at least a fraction of the B cells is HCV-specific and/or autoreactive. Thus, the typical intrahepatic B-cell follicles in chronic HCV carriers do not function as ectopic germinal centers for clonal expansion and affinity maturation of B cells. Hence, autoreactive and HCV-specific B-cell clones might either develop in secondary lymphoid organs or in intrahepatic follicles only under particular, yet undefined, circumstances.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Fígado/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Sequência de Bases , Feminino , Centro Germinativo/patologia , Hepacivirus/genética , Hepatite C Crônica/genética , Hepatite C Crônica/patologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Hipermutação Somática de Imunoglobulina/genéticaRESUMO
The ability to accurately characterize DNA variant proportions using PCR amplification is key to many genetic studies, including studying tumor heterogeneity, 16S microbiome, viral and immune receptor sequencing. We develop a novel generalizable ultrasensitive amplicon barcoding approach that significantly reduces the inflation/deflation of DNA variant proportions due to PCR amplification biases and sequencing errors. This method was applied to immune receptor sequencing, where it significantly improves the quality and estimation of diversity of the resulting library.
Assuntos
Código de Barras de DNA Taxonômico/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase/métodos , Viés , DNA/genética , Primers do DNA/genética , Biblioteca Gênica , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de DNA/métodosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.