Abstract
The germinal centre is a dynamic microenvironment in which B cells that express high-affinity antibody variants produced by somatic hypermutation are selected for clonal expansion by limiting the numbers of T follicular helper cells1,2. Although much is known about the mechanisms that control the selection of B cells in the germinal centre, far less is understood about the clonal behaviour of the T follicular helper cells that help to regulate this process. Here we report on the dynamic behaviour of T follicular helper cell clones during the germinal centre reaction. We find that, similar to germinal centre B cells, T follicular helper cells undergo antigen-dependent selection throughout the germinal centre reaction that results in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal centre leads to increased division of T follicular helper cells. Competition between T follicular helper cell clones is mediated by the affinity of T cell receptors for peptide–major-histocompatibility-complex ligands. T cells that preferentially expand in the germinal centre show increased expression of genes downstream of the T cell receptor, such as those required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to marked remodelling of the functional T follicular helper cell repertoire during the germinal centre reaction.
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Data availability
The data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus are accessible through GEO series accession number GSE147182.
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Acknowledgements
We thank A. Miyawaki and T. Kurosaki for the Fucci mice, E. E. Kara and T. Hagglof for the generation and maintenance of Sell-CreERT2 ROSA-tdT reporter mice, H. Zebroski for the generation of the APLs and the NP-APL conjugates in house, T. Eisenreich for help with mouse colony management, and technical help, A. Escolano for advice, B. Zhang and C. Zhao at The Rockefeller University Genomics Resource Center for assistance with 10x genomics and high-throughput sequencing, K. Gordon and K. Chhosphel for assistance with cell sorting, and all members of the Nussenzweig laboratory for discussions. We thank G. Victora for discussions involving the photoactivation experiments. This work was supported by National Institutes of Health (NIH) grant 5R37 AI037526 and NIH Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVID) 1UM1AI144462-01 to M.C.N. J.M. is an EMBO fellow. M.C.N. is an HHMI investigator.
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J.M., S.F. and M.C.N. conceived, designed and analysed the experiments. J.M., S.F. and J.K. carried out all experiments. A.G. and M.C. produced anti-DEC205-conjugates. C.R.N. contributed to paGFP experiments and discussions. H.H. bred and helped generate the Sell-CreERT2 ROSA-tdT mice. V.R. and T.Y.O. performed the bioinformatic analysis. B.T.C., W.Z. and P.D.B.O. helped perform the characterization on APL MHC class II occupancy. J.M. and M.C.N. wrote the manuscript with input from all co-authors.
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Extended data figures and tables
Extended Data Fig. 1 TFH cell gating strategy.
a, Flow cytometry plots detailing the TFH cell gating strategy. Rightmost histogram compares BCL6 expression in naive (grey) and non TFH (blue) versus TFH (red) cell populations on day 10 after immunization.
Extended Data Fig. 2 Production of Sell-CreERT2 ROSA-tdT indicator mice.
a, Targeting strategy and the configuration of the SellIRES-CreERT2 allele. The mice were produced at Rockefeller University and crossed to ROSA tdTomatoloxP/loxP to generate Sell-CreERT2 ROSA-tdT indicator mice. b, Schematic representation of the experimental strategy used in c. c, Flow cytometry plots profiling tdTomato expression in naive and TFH cell splenic compartments in tamoxifen treated mice culled 4 dpi. Rightmost plots compare the percentage of tdTomato labelling in naive T cells (red) and TFH cells (blue) residing in spleens (closed circle) or lymph nodes (open circle) of mice following the regime outlined in b. Data are from 4–5 mice per group and each dot represents one mouse. ****P < 0.0001, one-way ANOVA test. The experiment was performed twice. d, Schematic representation of the experimental setup. e, Flow cytometry plot showing EdU incorporation in labelled (red) or unlabelled (grey) TFH cell populations on 14 dpi. Rightmost plot shows the frequency of EdU positive TFH cells at 14 dpi, in population generated in the first 7 days (grey) or 8 and 14 from day 7 onwards (red) or the cumulative (blue). EdU was administered 3 h before mice were culled. Numbers inside the gates denote the relative representation of EdU+ cells from within tdTomato+ or tdTomato− populations. Each dot represents one mouse. Data are from 14 mice and the experiment was performed twice.
Extended Data Fig. 3 OT-II have disparate abilities to recognize the truncated APLs.
a, Description and sequence alignment of the nested APLs. b, Graph shows OT-II T cell responses to decreasing concentrations of APLs in vitro as measured by CD69 upregulation after 18 h of exposure. Bar graph shows the area under the adjacent response curves (AUC). *P = 0.0130, ***P = 0.0001, ****P < 0.0001, one-way ANOVA test. Each dot represents a distinct experimental well. This experiment was repeated four times. c, Schematic representation of experimental setup used in d–f. d, Bar graph shows percentage of OT-II among all CD4 T cells in adoptive transfer recipients 72 h after immunization with the indicated NP-APLs. e, Bar graph shows percentage of OT-II T cells that become TFH cells 7 days after NP-APL immunization. *P = 0.0186, **P = 0.0039, ***P < 0.001, ****P < 0.0001, one-way ANOVA test. Each dot represents one mouse, with 6 or 7 mice per group and repeated twice. f, Flow cytometry plots showing dilution of CellTrace Violet by OT-II T cells responding to the NP-APL immunization in vivo after 72 h. Each plot is an individual mouse. g, Schematic representation of experimental setup used in h. h, Left, flow cytometry plots profiling Fucci expression in non-boosted (5 mice) control or 18 h after anti-DEC–OVA (7 mice) injection. Right, comparison of the percentage of Fucci+ OT-II TFH cells in the respective conditions. Each dot represents a mouse and this experiment was performed twice. **P = 0.0048, unpaired Student’s t-test.
Extended Data Fig. 4 GC-resident TFH cells proliferate.
a, Schematic representation of experimental set up used in b–e. b, GCs in popliteal lymph nodes, as defined by the fluorescently labelled FDC network (red), were photoactivated (green) 18 h after anti-DEC–OVA injection, stained with a cocktail of fluorescent antibodies to allow analysis by downstream flow cytometry. c, Representative flow cytometry plots showing the gating of photoactivated OT-II TFH cells. d, A single GC from a popliteal lymph node as defined by fluorescently labelled FDC networks (red) before and after photoactivation (left to right, respectively). OT-II GC resident TFH cells were photoactivated (green) 18 h after anti-DEC–OVA injection. e, Representative flow cytometry plots comparing DAPI staining in host (grey) or OT-II GC-resident GFP+ cells (green) in unperturbed mice (left) or 18 h after boosting with an anti-DEC–OVA injection (right). Bar graph shows the percentage of photoactivated OT-II TFH cells that entered the cell cycle (DAPI+) after anti-DEC–OVA injection (5 mice) or in uninjected controls (3 mice). Each dot represents pooled lymph nodes from a single mouse. **P = 0.0086, unpaired Student’s t-test. This experiment was repeated twice.
Extended Data Fig. 5 Increased TCR signalling enforces proliferation supported by a switch in metabolic status.
a, b, GSEA and the rank-ordered gene lists found upregulated in anti-DEC-high versus non-boosted (a) or anti-DEC-high versus anti-DEC-low (b) groups. Nominal P values are indicated.
Extended Data Fig. 6 DO11 and OT-II have different affinities for APLs.
a, Schematic depicts the experimental setup. CTV-labelled DO11.10 and OT-II T cells were adoptively transferred into F1 mice and subsequently immunized with the NP-APLs. b, Representative flow cytometry plots compare the relative distribution and CellTrace Violet dilution of DO11.10 and OT-II T cells in adoptive transfer recipients at 3 dpi. Each panel of plots represents an individual mouse from the group. c, Bar graph shows the aggregate relative contribution of DO11.10 and OT-II T cells 3 days after immunization in 3 mice per condition. Dotted line depicts the input ratio of DO11.10 and OT-II at the time of transfer. ***P = 0.0007, ****P < 0.0001, unpaired Student’s t-test. This experiment was performed twice but data for one experiment are plotted. d, Schematic depicting experiment in which adoptive transfer recipients of DO11.10 and OT-II T cells were immunized with NP-329-339. Adjacent bar graph shows the aggregate relative contribution of DO11.10 and OT-II T cells 6 days after immunization with NP-OVA329–339. e, Representative flow cytometry plots showing the distribution of DO11.10 and OT-II T cells in the TFH cell compartment 6 days after immunization. f, Schematic depicting experiment in which adoptive transfer recipients of DO11.10 and OT-II T cells were immunized with NP-329-339 and boosted with the respective anti-DEC205 chimeric antibodies. Bar graph compares changes in the frequency of DO11.10 versus OT-II TFH cells when boosted with different anti-DEC205 chimeric antibodies at 6 dpi. Each dot represents an individual mouse and group size varied from 3–13 mice per condition. ns, not significant (P = 0.9). **P < 0.01, ***P < 0.001, one-way ANOVA test. g, Histogram overlays compare cell size (FSC-A), Ki67 expression and aCASP3 expression in OT-II and DO11.10 TFH cell populations from individual mice 18 h after anti-DEC–OVA323–339 boosting. These experiments were repeated two or three times.
Extended Data Fig. 7 TCR signalling dictates the quality of T cell help delivered to cognate B cells.
a, Flow cytometric plots showing the gating strategy to isolate positively selected light-zone germinal centre B cells 14 h after anti-DEC–APL antibody injection. b, Top, graphical representation of GSEA and the rank-ordered gene lists found upregulated in anti-DEC-high versus anti-DEC-low boosted and recently selected Fucci+ GC light-zone B cells. Bottom, graphical representation of GSEA and the rank-ordered gene lists found upregulated in anti-DEC-high boosted versus non-boosted controls. Nominal P values are indicated.
Extended Data Fig. 8 Conservation of TFH cell clonal families and clonal dominance in the spleens of day-7 immunized mice.
a, Schematic representation of the experimental strategy used in b, c. b, Flow cytometric plots depict the gating strategy used to define TFH cell populations in c. In brief, wild-type mice were immunized with NP-OVA, and 7 days later TFH cells were purified from the two halves of the spleen and then sequenced. c, Pie charts show expanded clonal families in each half of the spleen. Slices are proportional to the number of clones within a family. Colours indicate shared clonotypes (cells that share the same CDR3 sequence for alpha or/and beta chains) between the two spleen halves, ‘a’ and ‘b’, from an individual mouse. Grey tones indicate unique clones not shared between the two halves. The total number of paired TCR chains recovered is indicated by the number in the centre of the pie charts. Clonal distribution between adjacent spleen halves in mouse 1 (M1) (top) (not significant, P = 0.2279) and mouse 2 (M2) (bottom, P = 0.009), Fisher’s exact test. Adjacent bar graphs show the relative conservation of clonotypes between the two halves of the spleen. ‘Shared’ refers to a clone found in both segments of the spleen; ‘non-shared’ refers to anatomically novel clones.
Extended Data Fig. 9 Hemi-splenectomized mice have normal GC reactions.
a, Schematic representation of the experimental strategy used in b, c. In brief, NP-OVA-immunized C57BL/6 mice were biopsied on 7 dpi and culled on 14 dpi to interrogate changes that might occur in the GC compartment after surgical intervention (left). NP-OVA-immunized C57BL/6 mice who did not receive surgical intervention served as controls for biopsied mice (right). b, Adjacent plots show the frequency of TFH cells within total CD4+ T cells (left) or GC B cells within total B cells (right) in individual biopsied mice between time points 7 dpi (open purple) and 14 dpi (closed purple). P = 0.43 (left) and P = 0.14 (right), paired Student’s t-test. Dotted lines trace individual mice over time. c, Adjacent plots compare the frequency of TFH cells within total CD4 T cells (left) or GC B cells within total B cells (right) at 14 dpi in mice that were biopsied (purple) or C57BL/6 mice that were not (black). P = 0.52 (left), P = 0.13 (right), Student’s t-test. d, Schematic representation of the experimental strategy used in e. e, Flow cytometry plots profiling of tdTomato expression in splenic naive and TFH cell compartments in tamoxifen-treated mice but unimmunized mice (left) or similarly in tamoxifen treated mice 7 days after NP-OVA immunization (right). Rightmost bar graph compares the percentage of tdTomato+ populations between labelled populations in the unimmunized and immunized mice (12 days after tamoxifen administration and 7 dpi).
Extended Data Fig. 10 scRNA-seq reveals extensive clonal evolution of TFH cells.
a, Flow cytometry plots depicting gating strategy used to define responding TFH cells. b, Schematic representation of the experimental strategy used in d. c, Colour-coded indexing for the clonal behaviours categorized in mice between days 7 and 21. Expanded (red), conserved (green), contracted (blue) and singles (grey). Clones that are not coloured were novel to each time point. d, Pie charts show clonal populations of TFH cells within each mouse at each time point. Segments within the pie charts report the proportional representation of each clone. Clonotypes contain the same CDR3 sequence for alpha or/and beta chains. Clonal composition is significantly different between time points within the same mouse. ****P = 0.000016, Fisher exact test. Numbers inside the pie charts refer to the total number of TCR-α and TCR-β sequences recovered.
Supplementary information
Supplementary Table 1
A list of mABs produced and their characteristics.
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Merkenschlager, J., Finkin, S., Ramos, V. et al. Dynamic regulation of TFH selection during the germinal centre reaction. Nature 591, 458–463 (2021). https://doi.org/10.1038/s41586-021-03187-x
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DOI: https://doi.org/10.1038/s41586-021-03187-x
