Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9, 3106
eCollection

Expression of the Plasma Cell Transcriptional Regulator Blimp-1 by Dark Zone Germinal Center B Cells During Periods of Proliferation

Affiliations

Expression of the Plasma Cell Transcriptional Regulator Blimp-1 by Dark Zone Germinal Center B Cells During Periods of Proliferation

Daniel Radtke et al. Front Immunol.

Abstract

Long-lived plasma cells (PCs) develop in germinal centers (GCs) by the differentiation of affinity matured B cells. Antibody affinity maturation involves iterative rounds of somatic hypermutation in dark zones (DZs) and selection in light zones (LZs), however the details of where, when and how PC commitment occurs are not well-understood. Fate bifurcation at the time of selection is one possibility, with the very highest affinity GC B cells differentiating as an alternative to DZ re-entry. However, how this model fits with a need to also retain these clones in the response is not clear. Here, we show that subsets of bona fide DZ cells express the plasma cell master regulator Blimp-1 at low levels during periods of proliferation. Ex vivo culture experiments demonstrate that these cells are not yet committed to plasma cell differentiation but that they may be sensitized to go down that route. Contrary to models in which T cells directly select GC B cells to begin expressing Blimp-1, we found that expression of this transcriptional regulator occurred even when follicular helper T cells were ablated. We speculate that Blimp-1 may be induced during proliferation in the DZ, and that as such single selected cells might give rise to both GC and post-GC progeny.

Keywords: B cell; antibody affinity maturation; differentiation; germinal center; humoral immunity.

Figures

Figure 1
Figure 1
A Blimp-1-GFPdim population is found in the germinal center dark zone. Prdm1wt/gfp mice were analyzed by FACs at day 10 after SRBC immunization. (A) Splenic GC B cells were gated as B220+IgDlowCD95+GL7+. (B) Pre-plasma cell differentiation stages were defined based upon Blimp-1-GFP and CD138 levels. The dashed line marks the detection threshold for GFP+ cells. (C) Quantification of populations defined in (B) using the same color code. (D) Frequencies of LZ (CXCR4low CD83high) and DZ (CXCR4high CD83low) cells among Blimp-1-GFPneg and Blimp-1-GFPpos GC B cells. A further gate is shown around cells falling outside of DZ/LZ gates (CXCR4low/neg and CD83low/neg). (E) Summary of results as in (D), from multiple mice and experiments. (F) DZ/LZ frequencies were determined for the different differentiation stages defined in (B). (G) Summary of results as in (F), from multiple mice and experiments. (H) CXCR4 vs. Blimp-1-GFP on total GC B cells. (I) Confocal image of a splenic GC in which Blimp-1-GFP+ cells are identified and marked by arrows, with colors indicating their position. CD21/35 demarcates the LZ. (J) Confocal image in which Blimp-1-GFP+ cells with high intra-IgG levels are highlighted by orange rectangles. (K) Mutation frequency per 100bp of Ighv sequence was determined for the indicated populations I-V. Results for follicular B cells (IgDneg CD95neg GL7neg) and plasma cells (B220int Blimp-1-GFPbright CD138+) are also shown. Plots in (A,B,D,F) and (H) are representative of at least 7 mice from 3 experiments. Numbers shown are percent in gates, ± S.D. For confocal images, varying numbers of GFP+ cells were detected per GC. GC localized Blimp-1-GFP+ cells were detected in 11 separate mice from 2 experiments (I) or 7 mice from 3 experiments (J). Data in H is from two independent experiments but population V was only included in one.
Figure 2
Figure 2
RNA-seq analysis reveals Blimp-1 is expressed by GC B cells while they still retain dark zone and light zone signatures. One hundred cell populations were sorted according to the gating scheme presented in Figures 1A–D but with slightly increased stringency on the LZ/DZ gates to ensure purity. Their transcriptomes were determined by RNA-seq. Sample names have been shortened, e.g., DZ CD138neg Blimp-1-GFPdim cells are referred to as DZdim in the figure. (A) A multidimensional scaling plot showing differences between samples based on top 1,000 genes with the largest standard deviations between samples. (B) log2 RPKM values for various important B cell lineage, GC regulating or plasma cell associated genes. (C) Single cell RT-PCR was performed on index-sorted GC B cells to confirm co-expression of the indicated genes (colored according to legend). Cells not co-expressing at least two of the indicated genes (Aicda, Pax5, Prdm1) are depicted in black. CXCR4 and GFP protein intensities are also indicated on the x and y axis. (D) Gene set enrichment analysis (GSEA) of Blimp-1 activated or Blimp-1 repressed gene sets comparing DZdim and DZneg cells. The nominal p-values (p) and FDR q-values (FDR) are given. (E) Relative expression levels of Blimp-1 directly activated genes. (F) Changes in ASC-related gene expression sorted by log2 fold difference relative to Blimp-1-GFPneg DZ cells. Frequencies represent percent of ASC-genes that are increased in the indicated subset. (G) Differences in DZ-associated genes. Genes discussed in text are shown in red. (H) The top 10 most up-regulated and 10 down-regulated genes between LZ vs. any LZ-like Blimp-1-GFP+ population and DZ vs. any DZ-like Blimp-1-GFP+ population. Additional genes of interest were added to the list (depicted in red). RNAseq was performed using 5 biological replicates (mice) per group except for follicular B cells where 3 were used. Means are shown in (E,G,H).
Figure 3
Figure 3
Blimp-1 is expressed at low levels during periods of proliferation. (A) DNA content based cell cycle analysis was performed on the indicated splenic GC subsets from Prdm1wt/gfp mice on day 10 following SRBC immunization (left). Frequency means (±S.D.) of cells in S/G2/M are indicated on histograms. (B) Summary of results as in (A), from multiple mice and experiments. (C) Confocal micrographs showing EdU incorporation by Blimp-1-GFP+ GC B cells (left). Mice received single i.p. injections of EdU 5 h before analysis. Blimp-1-GFP+EdU+ cells in the GC area are highlighted by orange rectangles and are shown in magnification with or without the EdU channel on. A Prdm1wt/wt non-EdU treated control sample is also shown (right). For (B) a Kruskal-Wallis test with Dunn's test was performed (n = 7; pooled from 3 independent experiments). Horizontal lines indicate means *P < 0.05; **P < 0.01; ***P < 0.001. (C) Is representative of GCs from 4 mice.
Figure 4
Figure 4
GC B cells expressing low Blimp-1 levels retain potential for extensive clonal expansion. (A) Single splenic GC B cells (B220+ IgDlow CD38neg GL7+) from Prdm1wt/gfp mice were index sorted on day 10 after SRBC immunization and cultured ex vivo using the “Nojima culture” method in 96 well plates. Follicular B cells (B220+ IgD+ CD38+ GL7neg) and plasma cells (B220int CD138+ Blimp-1-GFPbright) were also sorted, for comparison. (A) Clonal culture sizes were determined on culture day 9. Solid lines indicate means. Dashed lines indicate the detection limit set according to NB21 feeder cell only wells. (B) Cell numbers recovered after culture were plotted against Blimp-1-GFP level at time of the sort (index FACs data). Vertical dashed lines indicate the detection limit. Blimp-1-GFP detection threshold (horizontal dashed line) was set based on a GFPneg control. GFP intensities gates in (A) were set according to the color code in (B). Pooled data from two independent experiments are shown.
Figure 5
Figure 5
Blimp-1 expression by GC B cells is not acutely dependent upon cues from T cells. (A) Strategy for the temporal ablation of antigen specific T cells during an ongoing response. Lethally irradiated Rag1−/− or TCR β/δ−/− mice were reconstituted with the indicated mixes of bone marrow, then immunized with SRBCs >8 weeks later. Mice received single i.p. injections of diphtheria toxin (DT) 72 or 24 h before analysis on days 11 or 12. (B) CXCR5+ PD-1+ Tfh cells were identified, with and without 24 h DT treatment. Plots are gated on splenic B220neg CD4+ cells and are from individual representative animals. (C) Frequencies of Tfh cells (as a proportion of splenocytes) and, (D) frequencies of GC B cells (as a proportion of total B cells), at the indicated time points post-DT treatment. (E) Representative CD138 vs. Blimp-1-GFP staining patterns on IgDlow CD95+ GL7+ GC B cells, 72 h after T cell ablation. (F) Summary of results as in (E), from multiple mice/experiments. (G,H) RNA-seq analysis was performed on the indicated GC B cell populations from mice with and without DT treatment (72 h). (G) Changes in expression of ASC-related and (H) Blimp-1 activated genes in indicated GC B cell populations were determined and sorted by log2 fold differences in DT treated vs. control mice. Numbers indicate the proportion and direction of ASC or Blimp-1 regulated genes that are changed in treated mice. For (C) no DT n = 38, 24 h DT n = 10 or 72 h DT n = 24 mice. For all populations of (D,F) n = 24 mice from 5 independent experiments. For (C,D,F) a Kruskal-Wallis test with Dunn's test was performed. Horizontal lines indicate means. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 6
Figure 6
“Early” CD138neg Blimp-1-GFPdim cells may be primed to become plasma cells but the differentiation program is not yet hard set. (A) Bcl2-tg+ Prdm1wt/gfp mice were immunized with SRBCs and the indicated splenic GC B cell populations (CD38neg IgDneg GL7+) were sorted on day 10 (500 cells/condition). CD138neg Blimp-1-GFPdim and LZ/DZ gates were set using the scheme outlined in Figures 1B,D, but with slightly increased stringency to ensure purity. Follicular B cells (IgD+CD38+GL7neg) and plasma cells (B220int CD138+ Blimp-1-GFPbright) were also sorted, for comparison. (A) Sorted cells were cultured under “Nojima” conditions for 48 h, before analysis for Blimp-1-GFP and CD138 expression. Numbers indicate the frequencies of cells which were positive or negative for the respective markers. (B) Data from multiple experiments are summarized. Dot colors relate to titles on left. Data is pooled from 4 independent experiments with one animal sorted per experiment. A one-way ANOVA with Dunnett's test was performed **P < 0.01; ***P < 0.001.

Similar articles

See all similar articles

References

    1. Victora GD, Nussenzweig MC. Germinal centers. Annu Rev Immunol. (2012) 30:429–57. 10.1146/annurev-immunol-020711-075032 - DOI - PubMed
    1. Mesin L, Ersching J, Victora GD. Germinal center B cell dynamics. Immunity (2016) 45:471–82. 10.1016/j.immuni.2016.09.001 - DOI - PMC - PubMed
    1. Bannard O, Cyster JG. Germinal centers: programmed for affinity maturation and antibody diversification. Curr Opin Immunol. (2017) 45:21–30. 10.1016/j.coi.2016.12.004 - DOI - PubMed
    1. Allen CDC, Ansel KM, Low C, Lesley R, Tamamura H, Fujii N, et al. . Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat Immunol. (2004) 5:943–52. 10.1038/ni1100 - DOI - PubMed
    1. Victora GD, Schwickert TA, Fooksman DR, Kamphorst AO, Meyer-Hermann M, Dustin ML, et al. . Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell (2010) 143:592–605. 10.1016/j.cell.2010.10.032 - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback