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-IgDhighIgMlow and MZ-B defined as CD90-IgMhighIgDlowwere sorted. Post sort reanalysis showed that the purity of FO-B cells and MZ-B cells was >95%.
Table 1.
Sequence analysis of IGHV3,4 and 5-Cμ transcripts expressed by FO-B cells and MZ-B cells from adult BN rat spleen.
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 (CD90negIgDhighIgMlow) and MZ-B (CD90negIgMhighIgDlow) cells.
Table 2.
Neonatal IGHV Cμ mRNA transcripts from FO-B cells and MZ-B cells.