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Nature ImmunologyImmunologyPeer-reviewed journal article

Temporally overlapping mechanisms diversify clonal B cell responses in vivo.

Oliver P. SkinneriD, Saba Asad, Marcela L. MoreiraiD, Hyun Jae Lee, Cameron G. Williams, Zheng RuaniD, Jorene Lim, Ashlyn S. KerriD, Shihan LiiD, Chen ZhuiD, Wang JiniD, Thiago Maass SteineriD, Takahiro Asatsuma, Brooke J. WanrooyiD, Zihan LiuiD, Zhen Wei Marcus TongiD, Megan S. F. SooniD, Jessica A. EngeliD, Kalyan ShobhanaiD, Adam P. UldrichiD, David S. KhouryiD, Zewen Kelvin TuongiD, Hamish W. KingiD, Ashraful HaqueiD

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Abstract

Naive B cells amplify and diversify their responses when activated by cognate antigen, via Myc-dependent clonal expansion, immunoglobulin class switch recombination (CSR), phenotypic variation, and somatic hypermutation (SHM). Whether these mechanisms act combinatorially in vivo to diversify clonal responses to a single pathogen remains unclear. Since diversity in the antigenic targets, functional classes, and production kinetics of parasite-specific antibodies influences immunity to malaria, we test here whether individual B cell clones diversify over time during Plasmodium infection and treatment. During the first week of infection, amid widespread Type I Interferon (IFN)-mediated bystander activation, CSR initiates soon after Myc up-regulation, and overlaps partially with clonal expansion, resulting in isotype variegation amongst clones. During the second week of infection, expanded clones that seed germinal centres (GC) bifurcate into extra-follicular plasmablasts, exhibit isotype variegation, and initiate SHM, revealing substantial intra-clonal diversification. Over the following month, GC clones exhibit SHM at approximately four mutations per week, with IgG mutational diversity and IgM+ cells also preserved in GCs over time. Anti-malarial intervention does not impede SHM, but instead exerts quantitative limits on GC size, plasma cell emergence, circulating IgG levels, and protection against re-infection. Finally, contemporaneous B cell development relocates from bone marrow to spleen during infection and treatment. Thus, multiple temporally overlapping mechanisms combine in vivo to amplify, diversify, and safeguard humoral immune responses. We present this data as a temporal, multi-parameter atlas of B cell differentiation in vivo: https://bcell-dynamics.science.unimelb.edu.au Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/628863v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@c57890org.highwire.dtl.DTLVardef@6e5936org.highwire.dtl.DTLVardef@a2e872org.highwire.dtl.DTLVardef@14abbac_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPartial temporal overlap of CSR with clonal expansion leads to isotype variegation in clones. C_LIO_LIClones seeding GCs bifurcate into plasmablasts and exhibit isotype variegation. C_LIO_LIGC B cells accrue [~]4 mutations/week, a rate unaffected by anti-malarials. C_LIO_LIPlasmodium infection triggers antigen-independent Type I IFN-mediated bystander activation. C_LIO_LIB cell development is preserved in malaria by shifting from bone marrow to spleen. C_LI

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (6 Mar 2026) · CC BY 4.0

Single-cell RNA and BCR sequencing of infected mouse spleens shows class switching overlaps clonal expansion, GC-seeding clones also form plasmablasts, and SHM proceeds at constant rate.

Single-antigen vaccination studies exposed individual B cell diversification mechanisms, but their timing and interplay within one polyclonal in vivo response were unresolved. This work maps them over time during Plasmodium infection and tests whether anti-malarial drugs alter germinal centre (GC) biology.

Key results

  1. ShownSingle naive B cell clones diversify in both fate and isotype: a majority of GC-containing twins also gave a plasmablast, and a substantial fraction of expanded clones showed mixed isotypes.

    Of 721 expanded clones (592 twins), 53 of 92 GC-containing twins (57.6%) also made a plasmablast; 119 of 461 plasmablast twins (25.8%) showed isotype variegation. In one spleen, among 107 clones of 5 or more cells, 100% of the 34 GC-containing clones included plasmablasts; 81.9% were isotype-variegated. · Activated B cell clones seeding GCs bifurcate…

  2. SuggestedClass switch recombination begins soon after Myc upregulation and partially overlaps with clonal expansion, rather than being a separate step.

    At 4 dpi, ~1% of recently activated CD83+CD69+ B cells were IgG2c+, and these had higher Ki67 than unswitched comparators. BGPLVM pseudotime ordering supported overlapping Myc, CSR and cell-cycle programs; flow data n = 9-10 per group, two pooled experiments. · CSR occurring soon after Myc-upregulation…; Figure legends

  3. ShownGC B cells accumulate BCR mutations at a constant weekly rate that anti-malarial treatment does not change, while mutation spread and unswitched IgM+ GC cells persist.

    Median summed heavy+light mutations rose at 3.71 per week (untreated) vs 3.79 per week (drug-treated); counts ranged 0-40 per cell by 42 dpi. Dataset: 72,359 B cells, n = 3 mice per timepoint and arm; GC B cells fell by 36.2% with drug. · GC B cells progressively diversify via a…

  4. SuggestedAnti-malarial treatment reduces the quantitative output of GCs (plasma cells, antibody, protection on re-challenge) without altering SHM rate or GC subset composition.

    At 42 dpi, bone marrow long-lived plasma cells were reduced after drug treatment (n = 12-13 per group, Mann-Whitney); Prg2hi pre-plasma cell frequency, parasite-specific IgG and re-challenge protection also decreased. Statistics for several panels are not given in text. · Anti-malarial treatment exerts quantitative…; Figure legends

  5. ShownProfiling all polyclonal B cells, not only antigen-specific ones, uncovered two concurrent splenic processes: interferon-driven activation of bystander cells and local B cell maturation from precursors.

    SCA-1/Ly6C upregulation on IgD+ cells was abrogated by IFNAR1 blockade and unaffected by B cell Myd88 deficiency. In Py17XNL-infected hosts, transferred splenic CD93+ precursors lost CD93 and gained IgD 21 days later; pre-B cells co-localised with Il7-expressing TRCs, TBRCs and MRCs at 21 dpi. · Plasmablast and GC B cell differentiation…; B cell lymphopoiesis relocates to the spleen…

Methods, data and reproducibility

Paired scRNAseq and BCRseq of FACS-sorted splenic B cells from Plasmodium chabaudi-infected C57BL/6J mice across three experiments (0-42 dpi, with or without anti-malarial drugs), combined with flow cytometry, spatial transcriptomics and Dandelion-based clonal and SHM analysis.

  • Experiment 1 scRNAseq/BCRseq of splenic B cells, 0-14 dpi, post-QC · mouse (C57BL/6J) · n = 40,623 B cells from 20 mice · GSE286215
  • Experiment 2 scRNAseq/BCRseq, IgD-low B cells, 10-42 dpi, with or without anti-malarial treatment · mouse (C57BL/6J) · n = 72,359 B cells from 42 mice · GSE286215
  • Experiment 3 deep scRNAseq/BCRseq of sorted plasmablasts and GC B cells from one 14 dpi spleen · mouse (C57BL/6J) · n = 19,482 post-QC B cells from 1 mouse · GSE286215
  • Stereo-seq spatial transcriptomics of infected spleens at 21 dpi · mouse (C57BL/6J) · n = two independent spleens · GSE286215
  • Public Slide-seqV2 data of infected spleens at 30 dpi with artesunate, used for RCTD deconvolution · mouse · n = n=4 arrays (two arrays per spleen, two mice) · GSE234253
  • Public bone marrow B cell scRNAseq used for scVI integration to identify precursor B cells · GSE168158
  • Public spleen stromal and T cell scRNAseq used as spatial deconvolution references · mouse (stromal data from uninfected mice) · GSE171124; GSE244806; GSE233713
Methods & tools
Cell Ranger 3.1.0 and 9.0.1, Seurat 4 (SCTransform), scVI, Scanpy 1.9.1, GPFates (BGPLVM), Dandelion container v0.3.0, igblastn 1.19.0 with IMGT references, TIgGER 1.0.0, SHaZam, RCTD, ToppGene / WebGestalt, Dash / Plotly
Reagents & environment
Cell Ranger 3.1.0 and 9.0.1; cellranger vdj v6.1.2; R 4.2.0 and 4.3.1; Seurat 4; Scanpy 1.9.1; Dandelion singularity container v0.3.0; igblastn 1.19.0; TIgGER 1.0.0; Python 3.8.8.
Reproducibility gaps
Analysis code is available only on request. Raw and processed data in GSE286215 were provided via a reviewer token at the time of writing.
Limitations (5)
  • No intermediate state between activated B cells and plasmablasts was found, possibly because early timepoints were sparsely sampled. · stated by the authors
  • BCR recovery was lower in GC B cells than in plasmablasts, preventing firm statements about the frequencies with which naive cells choose each fate. · stated by the authors
  • The sorting strategy excluded long-lived plasma cells, so GC plasma cell output was inferred from Prg2-high splenic ASCs as likely pre-plasma cells. · stated by the authors
  • The deep clone-size analysis (clones with 5+ cells) comes from a single spleen of one mouse, limiting generalisation of the high isotype variegation fraction. · noted in this summary
  • Drug effects on GC output combine artesunate and pyrimethamine and lower parasite load; antigen availability was not separated from direct drug effects. · noted in this summary

Read this if you study B cell clonal fate, class switch timing, or germinal centre dynamics during chronic infection using paired single-cell BCR and transcriptome data.

Citations
2
FWCI
2.32
Field percentile
top 11%

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