The formation of mutated IgM memory B cells in rat splenic marginal zones is an antigen dependent process

Abstract

Previous studies in rodents have indicated that only a minor fraction of the immunoglobulin heavy chain variable region (IGHV-Cμ) transcripts carry somatic mutations and are considered memory B cells. This is in marked contrast to humans where nearly all marginal zone B (MZ-B) cells are mutated. Here we show in rats that the proportion of mutated IgM+ MZ-B cells varies significantly between the various IGHV genes analyzed, ranging from 27% mutated IGHV5 transcripts to 65% mutated IGHV4 transcripts. The observed data on mutated sequences in clonally-related B cells with a MZ-B cell or follicular B (FO-B) cell phenotype indicates that mutated IgM+ MZ-B and FO-B cells have a common origin. To further investigate the origin of mutated IgM+ MZ-B cells we determined whether mutations occurred in rearranged IGHV-Cμ transcripts using IGHV4 and IGHV5 genes from neonatal rat MZ-B cells and FO-B cells. We were not able to detect mutations in any of the IGHV4 and IGHV5 genes expressed by MZ-B cells or FO-B cells obtained from neonatal rat spleens. Germinal centres (GCs) are absent from neonatal rat spleen in the first few weeks of their life, and no mutations were found in any of the neonatal sequences, not even in the IGHV4 gene family which accumulates the highest number of mutated sequences (66%) in the adult rat. Therefore, these data do not support the notion that MZ-B cells in rats mutate their IGHV genes as part of their developmental program, but are consistent with the notion that mutated rat MZ-B cells require GCs for their generation. Our findings support that the splenic MZ of rats harbors a significant number of memory type IgM+ MZ-B cells with mutated IGHV genes and propose that these memory MZ-B cells are probably generated as a result of an antigen driven immune response in GCs, which still remains to be proven.

Fig 1. Four colour cytometry of FO-B cells and MZ-B cells.

Single cell suspensions of spleen from rats were stained with FITC conjugated anti-rat IgM, biotinylated anti-rat IgD, APC conjugated anti-rat CD90/Thy1.1 and PE-conjugated anti-rat TCRαβ, TCRγδ, CD161a/NKRa. Biotinylated monoclonal antibodies were revealed with streptavidin conjugated to the tandem fluorochrome PE-Cy5.5. Viable lymphocytes were gated by forward scatter and side scatter profiles. Acquisition gates were set to exclude PE positive cells (T cells and NK cells) and CD90 positive (immature) B cells. Mature FO-B cells, defined as CD90^-IgD^highIgM^low and MZ-B defined as CD90^-IgM^highIgD^lowwere sorted. Post sort reanalysis showed that the purity of FO-B cells and MZ-B cells was >95%.

Fig 2. Percentage of mutated IgM⁺ FO-B cells and MZ-B cells within different IGHV gene families.

Analysis of the proportion of mutated sequences (>2 mutations compared to the closest germline gene) shows that MZ-B cells express more mutated sequences than FO-B cells, when all sequences from the three IGHV gene families are combined (i.e. “total”) (Fisher’s exact test: P < 0.001). This difference between MZ-B cells and FO-B cells was largely due to a significant difference in the percentage of mutated sequences within the IGHV3 family (Fisher’s exact test: P = 0.016). There are relatively more mutated sequences found in the IGHV4 gene family compared to IGHV3 and IGHV5 both for the MZ-B cells (Fisher’s exact test: P < 0.001) and FO-B cells (Fisher’s exact test: P = 0.023).

Fig 3. Mutation frequency of IGHV-Cμ transcripts of different IGHV families within MZ-B cells and FO-B cells.

Analysis of the distribution of the number of mutations of all (“total”) IGH-Cμ transcripts shows that MZ-B cells have significantly more mutations per transcript than FO-B cells (Mann-Whitney P = 0.046). Within the MZ-B cell subset, IGHV4 sequences contained more mutations than in IGHV3 or IGHV5 sequences (Kruskal-Wallis, P = 0.011).

Fig 4. Three-colour cytometry was used to analyze FO-B cells and MZ-B cells.

A single neonatal rat splenic cell suspension was stained with FITC conjugated anti-rat IgM (HIS40; eBioscience, San Diego, CA, USA), biotinylated anti-rat IgD (MaRD3; AbD Serotec, Oxford, UK), and APC anti-rat CD90/Thy1.1 (HIS51; eBioscience). Biotinylated mAb were revealed with streptavidin conjugated to the tandem fluorochrome PE-Cy5.5 (Ebioscience). Lymphocytes were sequentially gated by forward scatter and side scatter. Acquisition gates were set to exclude the unwanted immature B cells (CD90⁺-APC). Gate settings were set appropriately for FO-B cells (CD90^negIgD^highIgM^low) and MZ-B (CD90^negIgM^highIgD^low) cells.