Class-switch recombination (CSR) and somatic hypermutation (SHM) allow for the production of isotype-switched high-affinity antibodies. Insight into the regulation of these mechanisms is important, as their dysregulation can cause cancer and autoimmunity. Now, a paper in Immunity revises the previously held assumption that both CSR and SHM take place in germinal centre (GC) B cells — a finding with important implications for our understanding of B cell biology.
Carola Vinuesa and colleagues used an immunization model in which mice received adoptively transferred B cells specific for hen egg lysozyme (HEL) as well as mutated HEL protein conjugated to sheep red blood cells (SRBCs) and found that CSR predominantly takes place before either GC or extrafollicular B cell differentiation: the first germline transcripts (GLTs), an indicator of CSR, were observed at the stage of the initial B cell:T cell interactions, a full 2 days before GCs formed. The appearance of GCs, in turn, coincided with a dramatic decline in the expression of GLTs. These results were also confirmed in Cγ1-Cre mice in which GLT expression required for CSR to the IgG1 antibody isotype can be tracked through fluorescent protein expression. Single-cell profiling of GC light zone B cells, previously thought to be the subset in which CSR predominantly takes place, further confirmed that GLTs are not expressed in the vast majority of these cells. Changing immunogen and adjuvant also failed to reveal reactivation or increased rates of CSR at later stages of the GC response.
The authors also investigated the timing of CSR in polyclonal GCs through phylogenetic analysis. By labelling B cells from single GCs through photoactivation, they were able to analyse B cells after they had undergone multiple rounds of division and SHM. Clonal trees of switched and unswitched B cells were established by mRNA sequencing, with the evidence of SHM serving as ‘molecular time stamps’. This analysis confirmed that CSR largely occurs outside of the GCs, before the onset of SHM, and is uncommon after cells have acquired one or more mutations in mature GCs. Multiple mechanisms, including lack of GLT expression and downregulation of the enzyme APE1, likely a target of the transcriptional repressor BCL-6, were proposed to contribute to the repression of CSR in GC B cells.
“CSR largely occurs outside of the GCs, before the onset of SHM”
Interestingly, in silico analysis of CSR and SHM suggested that the determination of the isotype before the GC phase of intense B cell selection and proliferation promotes isotype diversity, whereas ongoing switching in GCs would homogenize the isotype distribution and would be incompatible with the observation of IgM-dominated GCs. The authors also point out that restricting CSR from GCs reduces the risk of GC B cells carrying pathogenic double-stranded breaks becoming long-lived. It also allows production of IgM memory B cells that can switch to protective isotypes upon encounter with antigenically related pathogens.
Roco, J. A. et al. Class-switch recombination occurs infrequently in germinal centers. Immunity https://doi.org/10.1016/j.immuni.2019.07.001 (2019)
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Flemming, A. Class-switch recombination revised. Nat Rev Immunol 19, 596–597 (2019). https://doi.org/10.1038/s41577-019-0214-8