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A Diverse Repertoire of Human Immunoglobulin Variable Genes in a Chicken B Cell Line is Generated by Both Gene Conversion and Somatic Hypermutation.

ABSTRACT: Chicken immune responses to human proteins are often more robust than rodent responses because of the phylogenetic relationship between the different species. For discovery of a diverse panel of unique therapeutic antibody candidates, chickens therefore represent an attractive host for human-derived targets. Recent advances in monoclonal antibody technology, specifically new methods for the molecular cloning of antibody genes directly from primary B cells, has ushered in a new era of generating monoclonal antibodies from non-traditional host animals that were previously inaccessible through hybridoma technology. However, such monoclonals still require post-discovery humanization in order to be developed as therapeutics. To obviate the need for humanization, a modified strain of chickens could be engineered to express a human-sequence immunoglobulin variable region repertoire. Here, human variable genes introduced into the chicken immunoglobulin loci through gene targeting were evaluated for their ability to be recognized and diversified by the native chicken recombination machinery that is present in the B-lineage cell line DT40. After expansion in culture the DT40 population accumulated genetic mutants that were detected via deep sequencing. Bioinformatic analysis revealed that the human targeted constructs are performing as expected in the cell culture system, and provide a measure of confidence that they will be functional in transgenic animals.

SUBMITTER: Leighton PA

PROVIDER: S-EPMC4367436 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A Diverse Repertoire of Human Immunoglobulin Variable Genes in a Chicken B Cell Line is Generated by Both Gene Conversion and Somatic Hypermutation.

Leighton Philip A PA Schusser Benjamin B Yi Henry H Glanville Jacob J Harriman William W

Frontiers in immunology 20150320

Chicken immune responses to human proteins are often more robust than rodent responses because of the phylogenetic relationship between the different species. For discovery of a diverse panel of unique therapeutic antibody candidates, chickens therefore represent an attractive host for human-derived targets. Recent advances in monoclonal antibody technology, specifically new methods for the molecular cloning of antibody genes directly from primary B cells, has ushered in a new era of generating ...[more]

PMID: 25852694

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Project description:Humoral immune perturbations contribute to pathogenic outcomes in persons with HIV-1 infection (PWH). Gut barrier dysfunction in PWH is associated with microbial translocation and alterations in microbial communities (dysbiosis), and IgA, the most abundant immunoglobulin (Ig) isotype in the gut, is involved in gut homeostasis by interacting with the microbiome. We determined the impact of HIV-1 infection on the antibody repertoire in the gastrointestinal tract by comparing Ig gene utilization and somatic hypermutation (SHM) in colon biopsies from PWH (n = 19) versus age and sex-matched controls (n = 13). We correlated these Ig parameters with clinical, immunological, microbiome and virological data. Gene signatures of enhanced B cell activation were accompanied by skewed frequencies of multiple Ig Variable genes in PWH. PWH showed decreased frequencies of SHM in IgA and possibly IgG, with a substantial loss of highly mutated IgA sequences. The decline in IgA SHM in PWH correlated with gut CD4+ T cell loss and inversely correlated with mucosal inflammation and microbial translocation. Diminished gut IgA SHM in PWH was driven by transversion mutations at A or T deoxynucleotides, suggesting a defect not at the AID/APOBEC3 deamination step but at later stages of IgA SHM. These results expand our understanding of humoral immune perturbations in PWH that could have important implications in understanding mucosal immune defects in individuals with chronic HIV-1 infection. IMPORTANCE The gut is a major site of early HIV-1 replication and pathogenesis. Extensive CD4+ T cell depletion in this compartment results in a compromised epithelial barrier that facilitates the translocation of microbes into the underlying lamina propria and systemic circulation, resulting in chronic immune activation. To date, the consequences of microbial translocation on the mucosal humoral immune response (or vice versa) remains poorly integrated into the panoply of mucosal immune defects in PWH. We utilized next-generation sequencing approaches to profile the Ab repertoire and ascertain frequencies of somatic hypermutation in colon biopsies from antiretroviral therapy-naive PWH versus controls. Our findings identify perturbations in the Ab repertoire of PWH that could contribute to development or maintenance of dysbiosis. Moreover, IgA mutations significantly decreased in PWH and this was associated with adverse clinical outcomes. These data may provide insight into the mechanisms underlying impaired Ab-dependent gut homeostasis during chronic HIV-1 infection.

Project description:The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID. Sequencing of the IgL V region and mutationally active GFP transgene in WT, Bcl6-/- Pax5R, and Bcl6-/- Pax5R Bcl6R chicken DT40 cells for evidence of AID dependent mutations. ChIP-seq proiles of RNA Pol II, Spt5, and pSer5 Pol II at a GFP transgene in WT, Bcl6-/- Pax5R, and Bcl6-/- Pax5R Bcl6R chicken DT40 cells.

Dataset Information

A Diverse Repertoire of Human Immunoglobulin Variable Genes in a Chicken B Cell Line is Generated by Both Gene Conversion and Somatic Hypermutation.

Publications

A Diverse Repertoire of Human Immunoglobulin Variable Genes in a Chicken B Cell Line is Generated by Both Gene Conversion and Somatic Hypermutation.

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