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. 2022 Mar 11;7(69):eabm0775.
doi: 10.1126/sciimmunol.abm0775. Epub 2022 Mar 11.

Competition for refueling rather than cyclic reentry initiation evident in germinal centers

Ziqi Long  1 , Bethan Phillips  1 , Daniel Radtke  1 , Michael Meyer-Hermann  2   3 , Oliver Bannard  1
Affiliations

Competition for refueling rather than cyclic reentry initiation evident in germinal centers

Ziqi Long et al. Sci Immunol. .

Abstract

Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and proliferation in dark zones (DZs) and selection in light zones (LZs). GC B cells exit cell cycle a number of hours before entering LZs; therefore, continued participation in responses requires that they subsequently reenter cell cycle and move back to DZs, a process known as cyclic reentry. Affinity enhancements are thought to arise by B cells having to compete to initiate cyclic reentry each time they enter LZs, with T cell help being a major determinant; however, direct proof is lacking. Using Fucci2 mice, we confirmed an association between B cell receptor affinity and the first step of cyclic reentry, S phase initiation from a resting LZ state. However, neither T cell ablation nor MHCII deletion prevented resting LZ cells from reentering cell cycle, and this late G1-S transition was also not detectably restricted by competition. In contrast, using BATF induction as exemplar, we found that T cells "refueled" LZ cells in an affinity-dependent manner that was limited by both competition and cells' intrinsic antigen-acquiring abilities. Therefore, cyclic reentry initiation and B cell refueling are independently regulated in GCs, which may contribute to permitting cells of different competencies to be sustained alongside each other and allow T cell support to be provided across a dynamic range commensurate with affinity. We speculate that this less binary selection mechanism could help GCs nurture complex antibody maturation pathways and support the clonal diversity required for countering fast-evolving pathogens.

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Conflict of interest statement

Competing Interests

The authors declare no competing interests.

Figures

Figure 1
Figure 1. Identification of light zone cells initiating cyclic re-entry in Fucci2 mice.
Analysis of splenic responses in Fucci2 mice on day 8 post-SRBC immunisation. (A) GC B cell DZ and LZ populations identified as shown. (B) G1 cells and S-G2-M phase cells are identified. mCherry levels accumulate with time post-mitosis, and mVenus accumulates with time in S phase; as such, early S phase cells are mCherryhigh mVenuslow. (C) LZ cells are mostly resting and therefore have higher mCherry levels than DZ cells. Dashed line provided to aid comparison. LZ cells initiating cyclic re-entry (early S phase) are identified by the red box.
Figure 2
Figure 2. Cyclic re-entry initiation is not acutely limited by GC Tfh cell availability.
Analysis of splenic responses in R26-Fucci2 Tcrb/d-/- mice that received an adoptive transfer of CD4-Cre Rosa-DTR T cells and SRBC/LPS immunisation. Analysis is on days 8 and 10, 48hrs post- DT (or vehicle) treatment. (A) GC Tfh cells were gated as shown, (B) and quantitated. (C) The relative absence of PD1+ Tfh cells in GL7+ GCs was determined by IHC (scale bar, 220μm), (D) and enumerated. Each point is a single GC, pooled from 5-6 mice/condition. (E) Frequencies of GC B cells (IgDlow CD95+ GL7+). (F) LZ populations were identified (left, means +/- S.E.M.; no DT 33% +/- 0.9, +DT 35% +/- 1.1) and frequencies of early S phase cells (Green), as well as late G1 (dashed purple), late S-G2-M (dashed orange), determined (right). (G) Results from pooled experiments with means. Results are representative of (A, F), or pooled from (B, E, G), 5 experiments each with 1-3 mice per condition. Analysis, Mann-Whitney U test throughout except (E, no DT vs DT, G) unpaired student’s T test. ****p < 0.0001
Figure 3
Figure 3. Cyclic re-entry initiation does not require MHCII presentation.
(A) Analysis of WT:R26-CreERT2 MHCIIfl/fl Fucci2 mixed BM chimeras on day 8 post-SRBC immunisation, at indicated times after tamoxifen treatment. Representative plots of MHCII deletion by LZ splenic GC B cells (IgDlow CD95+ GL7+). Dashed gates identify WT cells used in (C-D). (B) Antigen acquisition (GFP) and pMHCII presentation (Y-ae mAb staining) by day 8 SWHEL Cd79a-CreERT2 MHCIIfl/fl LZ GC B cells (40hrs post-tamoxifen) following 2 hr ex vivo incubations with HEL-EaGFP or HEL-BSA (control).(C) LZ populations gated in A. Frequencies of late G1 (dashed purple), early S (green) and late S-G2-M (dashed orange) cells determined for WT and MHCII-deleted populations, (D) and summarised. Lines join populations from individual mice. Results show representative FACs plots (A, B, C) or pooled results (D) from 2 experiments each containing 4-5 mice per condition. Analysis, paired two-tailed Student’s t test. *p < 0.05, ****p < 0.0001.
Figure 4
Figure 4. No detectable role for competition in determining cyclic re-entry initiation.
(A) Possible outcomes after competition is relieved in LZs. Models assuming LZ B cells compete for initiating cyclic re-entry (left) predict the fraction of remaining cells (green) entering S phase should increase when competing cells (orange) are removed (middle). Alternatively, if the process is not acutely competitive, cyclic re-entry initiation rates would not change (right). (B) GC B cell analysis after partial DT-mediated GC ablation in AID-DTR:Fucci2 (80:20) mixed BM chimeric mice, on day 8 post-SRBC immunisation. Time points post-DT treatment are indicated, vehicle at 8 hrs. Frequencies of DTR+ (left), Fucci2+ (middle), and total (right) GC B cells. (C) Representative splenic IHC sections (scale bar, 550μm) and (D) quantitation of GC areas, at 12hrs. (E) Fucci2+ LZ cells were gated and (F, G) frequencies of early S cells enumerated. (H, I) PD1+ cells per unit GC area were quantified by immunofluorescence staining at 15hrs post-DT (scale bar, 100μm). Points in D, I represent single GCs. G is pooled from 3 experiments, with 2 mice examined/group/experiment. FACS plots are representative of, and summaries are pooled data from, 2 experiments each containing 5 mice per condition. Analysis, (B, left, center) one-way ANOVA with Tukey’s multiple comparisons, (B right) Kruskal-Wallis with Dunn’s multiple comparisons, (D, I) Mann-Whitney, (G) unpaired two-tailed Student’s t test. **p <0.01, ***p < 0.001, ****p < 0.0001.
Figure 5
Figure 5. Cyclic re-entry initiation is associated with BCR affinity.
Analysis of splenic SWHEL Fucci2 GC B cells responding to HEL3x-SRBC/LPS (or HELWT-SRBCs, staining control) immunization, on day 8 or 9. (A) The frequencies of HEL3x-bright cells within BCR(Igk)+ SWHEL LZ (CXCR4low CD86+ IgDlow GL7+ CD45.1+) populations were determined for each cell cycle stage. (B) Similar analysis was performed for DZ (CXCR4high CD86low) cells. (C) Summary of results pooled from 7 experiments each with 3-5 mice. (D) Similar experiments with SWHEL Cd79a-CreERT2 MHCIIfl/fl B cells, where MHCII could be deleted (42hrs post-tamoxifen). Mice received EdU 30mins before analysis to mark S phase cells. G1 (EdUneg) and S phase (EdU+) populations were gated, for MHCII+ and MHCII-deleted LZ cells, and frequencies of HEL3X-bright cells determined. Results pooled from 3 experiments each with 2-10 mice/group. Lines join populations from single mice. MHCII+ populations include tamoxifen and vehicle mice. Frequencies in (A, B) are from representative mice. Analysis, (C) RM two-way ANOVA with multiple comparisons, (D) paired two-tailed Student’s t test. ****p < 0.0001.
Figure 6
Figure 6. BCR affinity, cell-cell competition and intrinsic antigen acquiring capacity determine T cell-mediated refuelling in LZs.
(A) BATF induction by WT and MHCII-deleted GC (IgDlow CD95+ GL7+) cells in WT: CreERT2+ MHCIIfl/fl on day 8 post-SRBC immunisation, 24hrs after tamoxifen treatment, and (B) enumeration. (C) LZ and DZ gating applied to total GC B cell and BATFhigh populations. (D, E) Frequencies of WT (DTRneg) LZ cells that induced BATF on day 8 post-SRBC immunisation, 15hrs after partial GC ablation to reduce competition (WT:AID-DTR 20:80 BM chimeras). (F) SRBC immunised WT mice received second SRBC injections (or saline) on day 7, and frequencies of BATFhigh LZ cells were assessed on day 8. (G, H) Analysis of splenic SWHEL GC B cells (CD45.1 IgDlow GL7+) responding to HEL3x-SRBC/LPS (or HELWT-SRBCs, staining control) immunization, on day 8. Frequencies of HEL3X-bright cells within BATFlow and BATFhigh LZ populations are shown. (I-J) Frequencies of BATFhigh cells within G1 (Edu- BrdU-) and early S (EdU- BrdU+) LZ populations were determined in SRBC immunised WT mice on day 7. Mice received EdU and BrdU treatments 100 mins and 40 mins before analysis. A, C, D, G, I are representative plots. Pooled results from 2 (B), 3 (E), 3(F), 3(H), 2(J) independent experiments. Lines in (H, J) join points from individual mice. Analysis, (B, E, F) unpaired and, (H, J) paired, two-tailed Student’s t test. **p < 0.01, ***p < 0.001, ****p<0.0001.
Figure 7
Figure 7. Lower affinity cells spend more time resting in LZs before initiating cyclic re-entry.
(A, B) Analysis of splenic SWHEL Fucci2 GC B cells responding to HEL3x-SRBC/LPS (or HELWT-SRBCs, staining control) immunization, on day 8. Very early S phase cells were subdivided into lower (“early”) and higher (“later”) ~50 percentiles based on their mCherry levels (B) Frequencies of HEL3x-bright cells were determined for each of the populations. (C) Summary of pooled results from multiple experiments. (D, E) Day 8 post-SRBC immunisation in WT:Cd83-/- mixed BM chimeric mice. (D) LZ populations were identified and, (E) frequencies of BATFhigh cells were determined. A, B, D are representative FACS plots, lines in C, E join populations from single mice. C, E is pooled from 6 and 2 experiments, each containing 3-6 mice. Analysis, paired two-tailed Student’s t test. ****p < 0.0001.
Figure 8
Figure 8. Alternative models for how T cells support antibody affinity maturation.
GC B cells enter LZs in G1, where they attempt to establish transient interactions with Tfh cells. In one scenario (top), LZ cells reaching a certain threshold of T cell help (dashed line) are triggered to initiate cyclic re-entry. Most cells fail this step and subsequently apoptose, which excludes all but the fittest cells. T cell help strength also determines the subsequent division capacity of cells, however because cyclic re-entry is triggered at a certain help threshold, most cells may undergo this event having received similar doses. Alternatively (bottom), cyclic re-entry initiation is regulated independently of strength of T cell help received (our proposed model). As a result, both lower and higher affinity cells can initiate cyclic re-entry, with cells doing so having been refuelled to differing degrees (which determines division capacity, indicated by arrow weight). Note, our experimental measure of cyclic re-entry initiation was restarting of cell cycle (S phase entry), therefore whether cells can complete cyclic re-entry (i.e., return to DZs) without adequate T cell help remains to be tested.
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