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Class-switched memory B cells remodel BCRs within secondary germinal centers

Nature Immunology volume 16, pages 296–305 (2015)Cite this article

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Abstract

Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive rediversification of B cell receptors (BCRs), but the underlying mechanisms remain unresolved. Here, the integrated specificity and function of individual memory B cell progeny revealed ongoing evolution of polyclonal antibody specificities through germinal center (GC)-specific transcriptional activity. At the clonal and subclonal levels, single-cell expression of the genes encoding the costimulatory molecule CD83 and the DNA polymerase Polη segregated the secondary GC transcriptional program into four stages that regulated divergent mechanisms of memory BCR evolution. Our studies demonstrate that vaccine boosts reactivate a cyclic program of GC function in class-switched memory B cells to remodel existing antibody specificities and enhance durable immunological protection.

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Figure 1: Class-switched memory B cells form robust secondary GCs.
Figure 2: Diversification of switched BCRs and secondary GC transcription.
Figure 3: Expression of Cd83 and Polh assorts four cyclic stages of GC activity.
Figure 4: ‘Adaptive radiation’ during subclonal BCR evolution in the GC.
Figure 5: De novo secondary GC formation.
Figure 6: Class-switched memory B cells can form secondary GCs after transfer and recall.
Figure 7: Class-switched memory B cells are dominant precursor cells for secondary GCs.

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Acknowledgements

Supported by the Foundation Bettencourt-Schueller (P.J.M.), the Swiss National Science Foundation (S.L.O.), the Novartis Jubliaeumsstiftung (S.L.O.), the Roche Research Foundation (S.L.O.) and the US National Institutes of Health (AI047231, AI040215 and AI071182 to M.G.M.-W.). This is The Scripps Research Institute manuscript number 26086.

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  1. Pierre J Milpied & Shinji L Okitsu

    Present address: Present addresses: Centre d'Immunologie de Marseille-Luminy. Marseille, France (P.J.M.), and EMD Serono Research and Development Institute, Billerica, Massachusetts, USA (S.L.O.).,

  2. Louise J McHeyzer-Williams and Pierre J Milpied: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA

    Louise J McHeyzer-Williams, Pierre J Milpied, Shinji L Okitsu & Michael G McHeyzer-Williams

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Contributions

L.J.M.-W., P.J.M. and M.G.M.-W. designed and performed experiments, analyzed the data and wrote the paper; and S.L.O. designed and performed the experiments in Supplementary Figure 1c.

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Correspondence to Michael G McHeyzer-Williams.

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Integrated supplementary information

Supplementary Figure 1 Antigen-specific secondary GCs from mice primed with different adjuvants and different doses of antigen.

(a) Mice were primed with 400μg NP-KLH prepared in Alum (top row), CFA (middle row) or MPL (bottom row) adjuvants and rested for 70 days before boosting with soluble NP-KLH. NP-specific GC B cells (λ1+ NP+ B220+ CD138– CD38– GL7+ class-switched B cells) were detected in draining lymph nodes for all adjuvant-primed mice 4 days after boost. Numbers indicate mean ± sem percentage across 3 or more mice per group.

(b) Total class-switched NP-specific B cells (top) and NP-specific GC B cells (bottom) per draining lymph node of primed animals before (day 70) or 4 days after soluble NP-KLH boost in Alum, CFA and MPL-primed groups. Bars represent mean ± sem. Fold increases after boost are indicated.

(c) Mice were primed with 4μg (top row) or 40μg (bottom row) NP-KLH prepared in MPL adjuvant and rested for >240 days before boosting with NP-KLH in adjuvant. NP-specific GC B cells (λ1+ NP+ B220+ CD138– CD38– GL7+ class-switched B cells) were detected in draining lymph nodes for all doses of primed mice 5 days after boost. Numbers indicate mean ± sem percentage across 3 mice per group.

(d) Total class-switched NP-specific B cells (left) and NP-specific GC B cells (right) of mice primed with 4μg or 40μg NP-KLH in MPL and boosted with NP-KLH in adjuvant as in c. Bars represent mean ± sem.

Supplementary Figure 2 Phylogenetic tree of rearranged BCR-encoding genes from individual antigen-specific secondary GC cells.

Mice were primed with NP-KLH prepared in MPL adjuvant and rested for 70 days before boosting. Single NP-specific GC B cells (λ1+ NP+ B220+ CD138– CD38– GL7+ class-switched B cells were sorted from draining lymph nodes of mice 8 days after boost with NP-KLH in adjuvant. Nucleotide sequence of VH186.2, DH and JH joints amplified from the mRNA was compared to the germline VH186.2. Displayed is the BCR VH186.2 amino acid sequence for each cell. Only amino acids differing from the germline sequence (top) are presented with the identical amino acid indicated by dashes. The DH and JH regions are identified at the right of each sequence with the total number of VH nucleotide mutations. All boost day 8 germinal center single cells were near-neighbor sequence aligned using Clustal Omega and based on nucleotide sequence in BCR junctional regions assigned a clone and cell ID. A phylogenetic tree generated with Dendroscope 3 is displayed to the left of each cell sequence. n= 133 single cells from 4 mice. Boost day 8 clone G is highlighted in blue.

Supplementary Figure 3 Broad dynamic range for single-cell quantitative RT-PCR with minimal loss of sensitivity.

(a) Representative single cell qPCR analysis of antigen-specific GC B cells day 8 after boost (GR1– CD3–, B cell+, IgD– IgM–, λ1+, NP+, CD138–, B220+, CD38–, GL7+). 10, 3 and 1 cell were sorted per well and assayed across 96 genes. Wells with 0 sorted cells were processed as negative controls. Specific target amplification for cDNA synthesis and PCR pre-amplification was followed by qPCR using nanofluidics technology. Heatmap of relative Ct value showing representative 10,3 and 1 cell for the indicated genes is displayed.

(b) Comparison of gene expression values (30-Ct) measured by single cell qPCR in 10-cell and 1-cell samples of day 8 GC B cells. For every gene in the analysis panel (n=87), the actual gene expression value measured from a 10-cell sample is plotted according to the value expected from single cell expression values for the same gene in the same population. To compute the expected value (30-Ct), we used the following formula: Expected = log2[10 x percentage of positive cells x mean (230-Ct observed in positive single cells)]. The resulting comparison has a strong correlation coefficient (r = 0.9287), and the equation of the corresponding linear regression is Actual = (0.9882 ± 0.04822) x Expected + (0.9440 ± 0.7388). Thus for the vast majority of assays, there is no sensitivity loss when measuring expression in single cell samples.

Supplementary Figure 4 Principal-component analysis for GC B cells emphasizing the distribution of the expression of Cd83 and Polh at the single-cell level.

(a) Single cell gene expression values (30-Ct, 87 genes) of n=372 GC B cells from days 4 and 8 after boost (soluble and adjuvant) were used for principal component analysis (PCA). Dot plots show the position of every cell on the space defined by the first two principal components (PC1 and PC2) (left). Cells positive for Cd83 (middle left) or Polh (middle right) expression are highlighted in yellow. Cells expressing distinct combinations of Cd83 and Polh are highlighted (right, Cd83– Polh– black, Cd83+ Polh– blue, Cd83+ Polh+ white, Cd83– Polh+ yellow).

(b) The same cells were subjected to PCA using only expression of 9 genes in the dataset previously published to be significantly different between DZ and LZ GC B cells (Victora et al, Cell 2010). PC1 from this analysis separates DZ and LZ GC B cells and tightly correlates with PC2 computed with all genes (left, correlation coefficient r = -0.7559, p < 0.0001). LZ and DZ gates were set according to the expression levels of Cd83 and Polh as shown (right).

(c) Based on the gates defined in b, relative proportions of GC B cells in the DZ or the LZ in primary GC (day 70) or secondary GC (days 4 and 8, soluble and adjuvant) were computed. Bars indicate mean ± sem (n=3 experiments).

(d) Based on the gates defined in b, the differences in gene expression between LZ and DZ GC B cells for every gene as described in Materials and Methods was computed. A “volcano” plot was generated to display genes significantly up-regulated in LZ (left side) or DZ (right side) B cells. The dotted line shows a p-value of 0.05. Most significantly up- or down-regulated genes are labeled on the plot.

Supplementary Figure 5 Distribution of the frequency and level of gene expression across the DZ and LZ of the GC.

Single cell gene expression values (30-Ct) of n=372 GC B cells from days 4 and 8 after boost (soluble and adjuvant) were used for principal component analysis (PCA). The cells were subjected to PCA using only expression of 9 genes previously published to be significantly different between DZ and LZ GC B cells (Victora et al, Cell 2010). GC B cells were distributed in 8 consecutive bins according to their PC1 (9 genes) values. Within each bin, the percentage of positive cells and the mean expression level (30-Ct) of positive cells was computed for every gene. We display the resulting graphs for 12 selected genes, with bars corresponding to percentage positive (left Y axis), and blue line corresponding to expression levels (right Y axis). Gene expression variation between DZ and LZ (low PC1 to high PC1) is displayed.

Supplementary Figure 6 Cyclic changes in the expression of Cd83 and Polh in GC transcriptional programming.

(a) GC B cells (from boost day 4 and day 8, n=372) were combined and clustered in a two-dimensional display using t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using Cd83, Polh, Aicda and Mki67 expression, the major sub-groups are displayed and labeled 1 through 4 as described in Figure 3b.

(b) Using the graph-based trajectory algorithm, Wanderlust (Bendall, et al, Cell, 2014), Cd83– Polh– cells were set as the user-defined starting point (Stage1). Wanderlust then computed a trajectory from these starting cells set at 0 through to the end-point at 1. The upper panel shows the trajectory expression patterns for Cd83 (white line) and Polh (grey dashed line). The lower panel displays t-SNE1 and t-SNE2 for the 372 GC cells. The Wanderlust predicted trajectory is displayed by the color code from blue through to dark red. The predicted trajectory is indicated as Cd83– Polh– (Stage 1, blue), Cd83+ Polh– (Stage 2, green), Cd83+ Polh+ (Stage 3, light red), Cd83– Polh+ (Stage 4, dark red).

(c) Proposed schematic for GC B cell stages and transitions in cyclic activity.

Supplementary Figure 7 Class-switched memory B cells form GCs upon adoptive transfer into Rag1–/– hosts.

(a) C57BL/6 mice were immunized 70 days prior with NP-KLH in adjuvant and cells sorted for adoptive transfer of class-switched memory B cells into B6.Rag1–/– hosts. Representative gates used for sorting donor memory B cells (CD8– Gr1– IgM– IgD– CD4– CD19+ GL7–) and memory T cells (CD8– Gr1– IgM– IgD– CD19– CD4+ CD44high) are shown.

(b) Sorted memory B and T cells were transferred, along with congenically labeled spleen cells from B6.MD4 BCR transgenic mice, into Rag1–/– hosts that were subsequently immunized with NP-KLH in MPL adjuvant (n=5) or MPL alone (n=2). Representative plots of memory-derived B cells in host spleens 14 days after transfer are shown. Antigen-specific (λ1+ NP+) total B cell progenies of class-switched memory B cells in recipients boosted with adjuvant only (MPL, top left) or with antigen and adjuvant (NP-KLH in MPL, bottom left). GC B cells (CD19+ B220+ CD38– GL7+) progenies of class-switched memory B cells in NP-KLH boosted recipients for all donor-derived B cells (top right) or antigen-specific (bottom right) B cells. Numbers in gates indicate mean ± sem percentage.

(c) Total numbers of non-specific (top) and NP-specific (bottom) GC B cells in recipients are summarized in the column dot plots. Each dot represents an individual mouse and the bar is set at the mean.

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McHeyzer-Williams, L., Milpied, P., Okitsu, S. et al. Class-switched memory B cells remodel BCRs within secondary germinal centers. Nat Immunol 16, 296–305 (2015). https://doi.org/10.1038/ni.3095

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