Data are overlaid with cells that were not sequenced (gray), for reference. Colors indicate presence or absence of Ighv region premature stop codons in cells of the indicated phenotype (pos, positive, neg, negative). (C) BCR and CXCR4 levels on individual indexed sequenced cells. Results are normalized for rare population enrichments performed during FACS sorting. Numbers of cells sequenced are indicated above bars and the proportion of total DZ stop codons that are in the BCR low subset is marked below. (B) Summary of results from multiple mice and experiments. (A) Frequency (mean ± S.E.M., n = 5) of premature stop codons in BCR high and BCR low CXCR4 high CD83 low DZ populations is shown. Single IgD low CD95 + GL7 + (mice 1-3), or IgD low CD95 + (mice 4 or 5), CD45.1/2 + SW HEL GC B cells were index sorted on day 8 and their Ighv region genes were PCR amplified, Sanger sequenced, and analyzed for the presence of premature stop codons. See also Figure S1.ĭZ Cells Acquiring Detrimental Mutations in Their Immunoglobulin V Region Genes Accumulate in BCR low Gates SW HELxFucci2 B cells were co-transferred with OT-II CD4 + T cells into WT hosts that were subsequently immunized with HEL-OVA/adjuvant. Numbers in (C–E) and (H) represent mean ± SEM from 7 (C and D), 10 (E) and 11 (H) mice from 2–4 experiments. (H) Surface BCR levels on Aicda +/+ and Aicda −/− IgD low CD95 + GL7 + GC cells from mixed bone marrow chimeric mice 8 days after SRBC immunisation. Numbers of cells in each group per experiment are indicated. Plots for additional mice are shown in Figure S1. The plots for cells that did not grow, or that grew did not secrete detected antibody, are also shown and overlaid with the total GC B cell population for reference. (F) The BCR and CXCR4 staining profiles (at the time of sort) of single GC B cells that grew and secreted detectable antibody after culture are shown. (F and G) Single CXCR4 high BCR high and CXCR4 high BCR low DZ SW HEL GC B cells (gated IgD low, CD38 low, GL7 +, CD45.1 +, or CD45.2 +) were index sorted and cultured for 8 days on NB21 feeder cells. (D and E) DZ cells with low BCR levels in MD4 transgenic (D) and SW HEL gene targeted GC B cell populations (E). (C) Surface and intracellular BCR (IgMa) levels were co-stained in fixed/permeabilized MD4 GC B cells revealing a subset of cells that is negative for both. Splenic IgD low CD95 + GL7 + CD45.1 + GC B cells were analyzed on day 7.
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(C–G) HEL-specific SW HEL or MD4 B cells were adoptively co-transferred with OT-II CD4 + T cells and mice were immunized with HEL-OVA/adjuvant adjuvant. (B) DZ and LZ cells are gated as shown and BCR levels overlaid with IgD + CD95 low follicular B cells. The gate indicates a BCR low DZ population. (A) BCR levels on DZ populations (CXCR4 high) were analyzed and compared to B220 neg non-B cells. Reduced BCR Levels on DZ GC B Cells (A and B) Mice were immunized with SRBCs and splenic IgD low CD95 + GL7 + GC B cells were analyzed on day 7. Thus, BCR replacement and pre-screening in DZs prevents the accumulation of clones with non-functional receptors and facilitates selection in the LZ.Ĭopyright © 2018 The Author(s). Ectopic expression of the anti-apoptotic gene Bcl2 was not sufficient for cells with damaging mutations to reach the LZ, suggesting that BCR-dependent cues may actively facilitate the transition. Instead, apoptosis was triggered preferentially in late G1, a stage wherein cells with functional BCRs re-entered cell cycle or reduced surface expression of the chemokine receptor CXCR4 to enable LZ migration. We found that most GC B cells degrade pre-SHM receptors before leaving the DZ, and that B cells acquiring crippling mutations during SHM rarely reached the LZ. Accurate selection requires that cells fully replace surface B cell receptors (BCRs) following SHM, but whether this happens before LZ entry is not clear. Adaptive immunity involves the development of bespoke antibodies in germinal centers (GCs) through immunoglobulin somatic hypermutation (SHM) in GC dark zones (DZs) and clonal selection in light zones (LZs).