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Anitbody affinity birth through somatic hypermutation

ABSTRACT: B cell somatic hypermutation (SHM) and selection in germinal center (GC)s enhance antibody affinity to antigen. Conventional understanding holds that SHM enhances pre-existing antibody specificities, limiting the scope of SHM-based antibody evolution to those established in the primary repertoire through V(D)J recombination. By tracking pre-defined non-specific B cells, we observed consistent SHM in non-cognate antibody genes after immunization in settings of diverse, polyclonal B and T cells. Removing B cell competition enabled these non-specific yet somatically mutating antibodies to develop entirely new specificities to diverse antigens. Our findings introduce an updated model, where SHM drives antibody diversification without requiring initial antigen specificity. This reveals that B cell competition, rather than a necessity for specific affinity, limits the emergence of new affinities (termed here as affinity birth) by SHM and highlights the mammalian adaptive immune system’s capacity to explore antibody-antigen interactions beyond the epitopes targeted by the V(D)J-dependent primary antibody repertoire.

ORGANISM(S): Mus musculus

PROVIDER: GSE283094 | GEO | 2024/12/04

REPOSITORIES: GEO

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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.

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Anitbody affinity birth through somatic hypermutation

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