Gut-derived antigens trigger immunoglobulin A (IgA) immune responses that are initiated by cognate B cells in Peyer’s patches (PPs). These cells colonize the subepithelial domes (SEDs) of the PPs and subsequently infiltrate pre-existing germinal centers (GCs). Here we defined the pre-GC events and the micro-anatomical site at which affinity-based B cell selection occurred in PPs. Using whole-organ imaging, we showed that the affinity of the B cell antigen receptor (BCR) regulated the infiltration of antigen-specific B cells into GCs but not clonal competition in the SED. Follicular helper-like T cells resided in the SED and promoted its B cell colonization, independently of the magnitude of BCR affinity. Imaging and immunoglobulin sequencing indicated that selective clonal expansion ensued during infiltration into GCs. Thus, in contrast to the events in draining lymph nodes and spleen, in PPs, T cells promoted mainly the population expansion of B cells without clonal selection during pre-GC events. These findings have major implications for the design of oral vaccines.
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All BCR sequencing data generated in this manuscript have been deposited in the European Nucleotide Archive under accession number PRJEB30525. Custom scripts used for data analysis are available upon request. All other data are available in the main text or the supplementary materials.
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Z.S. is supported by the European Research Council (grant No. 677713), Human Frontiers of Science Program (grant No. CDA-00023/2016), Israel Science Foundation (grant No. 1090/18), Azrieli Foundation, Rising Tide Foundation and the Morris Kahn Institute for Human Immunology. Z.S. is a member in the European Molecular Biology Organization Young Investigator Program and is supported by grants from The Benoziyo Endowment Fund for the Advancement of Science, The Sir Charles Clore Research Prize, Comisaroff Family Trust, Irma & Jacques Ber-Lehmsdorf Foundation, Gerald O. Mann Charitable Foundation and David M. Polen Charitable Trust. Imaging was made possible thanks to The de Picciotto-Lesser Cell Observatory in memory of Wolf and Ruth Lesser.
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 Single administration of NP-CT does not increase gut permeability or disrupt the mucosal epithelium.
a, NP-CT (10 μg) dissolved in 3% NaHCO3 or 3% NaHCO3 without NP-CT were given by gavage to wild type mice, indicated as ‘NP-CT’ and ‘Control’, respectively. 24 hours post antigen administration, mice were fed with FITC dextran or left untreated (negative control, NC). FITC fluorescence was measured in blood serum, 2.5 hours after FITC dextran administration. Data were pooled from two independent experiments with three mice in each group (n=6), bar represents mean. P<0.05, one-way ANOVA. b, Diagram representing the experimental protocol in (c-e). c-e, H&E histological section of duodenum (c), jejunum (d), and ileum (e) following NP-CT administration as indicated in (b). Scale bar, 200 μm. Two independent experiments with two mice in each group (n=4) were performed and showed similar results.
Supplementary Figure 2 Analysis of the B cell immune response in MD4 host mice and flow-cytometry gating strategies.
a, Flow cytometry plots and quantification of PP cells derived from wild-type and MD4 mice showing the frequency of GC, SED and TFH cell populations. Data are pooled from two independent experiments with three mice in each experiment for GC and SED staining (n=6); one and two wild-type (n=3), two and three MD4 mice (n=5) for TFH staining analysis. Line represents mean * P<0.05, two-tailed Student’s t test. ns, not significant. b, Representative LSFM images of a PP derived from MD4 mice that were transferred with B1-8hi GFP+ cells and did not receive antigen. Scale bar, 300 μm. Two independent experiments with two mice in each experiment (n=4) showed similar results. c, Representative flow cytometry analysis and gating strategy of adoptively transferred B1-8hi GFP+ and B1-8lo DsRed+ cells in GCs of recipient MD4 mice nine days after NP-CT administration. d, Representative gating and analysis of naïve, GC and SED B cell proliferation by EdU incorporation in wild-type mice (upper panels). Mice were intravenously injected with EdU on day 9 after immunization with NP-CT and analyzed after additional 2.5 hours. Representative EdU analysis gated on these populations is shown with naïve as negative control (lower panels). Similar analysis was held following adoptive transfer of B1-8hi GFP+ and B1-8lo DsRed+ B cells, five days following NP-CT administration as shown in Fig. 2g.
a, TPLSM image showing Blimp-1-YFP cells in a PP under homeostatic conditions. Two experiments with two mice in each experiment (n=4) showing similar results were performed. Scale bar, 100 μm. b, Diagram representing the experimental protocol presented in (c-d). c, TPLSM image showing B1-8hi Blimp-1-YFP+ DsRed+ B cells in the B cell follicle five days following oral antigen administration. Two experiments with two mice in each experiment (n=4) showing similar results were performed. Magnification is shown as marked by the square. Second harmonic generation (SHG) marks collagen and PP boundary. Scale bar, 100 μm. d, PPs of mice imaged in (c) were analyzed by flow cytometry for quantification of Blimp-1-YFP+ cells. Mice that received cell transfer but were not immunized were used as control (left). Percentage shown on the stacked bar represent average Blimp-1-YFP+ frequency. Data are pooled from two independent experiments with two mice in each experiment (n=4), bar represents mean.
a, Images showing GC of AID-GFP:PA-GFP (10:90) chimeric mouse prior to and after photoactivation. Scale bar, 100 μm. b, Flow cytometry plots depict the frequency of total inactive- and active-PA-GFP cells, CD4+ T cell fraction among the active PA-GFP cells, and activated T cells (CD44+CD62L- of active-PA-GFP+CD4+ cells). c, Representative plots showing PD-1hiCXCR5hi TFH cells in the active-PA-GFP CD4+CD44+CD62L- T cell gate. d, Activated and TFH cell frequencies are summarized in the bar graph. e, Ratio of GC (FAS+CD38-) or SED (CD38+CCR6+IgA+, Fig. 3f–j) B cells to TFH cells in each compartment of the PP in the chimeric mice. To represent the full GC population (AID-GFP+ and PA-GFP+ cells) the number of active PA-GFP+ GC cells was normalized to their proportion in the chimeric mouse. Two-tailed Student’s t test. ns, not significant. In b-e, data are pooled from three independent experiments with one mouse in each experiment (n=3), bar represents mean.
a, Diagram representing the experimental protocol presented in (b). b, B1-8hi GFP+ B cell frequency in single isolated PPs one day following cell transfer into wild-type hosts. PPs are numbered from the duodenum to the ileum. Each dot represents mean of two corresponding PPs; Data are pooled from two independent experiments with a total of two mice (n=2). c, Diagram representing the experimental protocol presented in (d). d, Transferred B1-8hi tdTomato+ B cell frequency in the GC (FAS+CD38-) and SED (CD38+CCR6+IgA+) compartments of single isolated PPs, nine days following NP-CT immunization of wild-type or antibiotics (Abx) treated hosts. Antibiotics cocktail was given to the mice in their drinking water for 2 weeks prior to immunization with NP-CT. Data are pooled from two independent experiments with 2–3 mice in each experiment (n=5). Error bars indicate s.e.m. e, Diagram representing the experimental protocol presented in Fig. 5b-f. f, Representative flow cytometry plots showing gating for NP-PE specific B cells staining for Fig. 6b. Unstained sample is used as a negative control. Two independent experiments with three mice in each experiment (n=6) showed similar results.
Supplementary Figure 6 Gating strategies for analysis of transferred B cells and for polyclonal single cell sorting.
a, Representative flow cytometry analysis and gating strategy for GC (upper panel) and SED (lower panel) compartments of recipient wild-type mice, adoptively transferred with B1-8hi GFP+ cells, nine days following antigen administration. b, Representative histogram showing CCR6 expression on GC and SED B cells analyzed as described in (a). c, Gating strategy for cell sorting of GC and SED IgA+ B cells from a single PP harvested from an AicdaCre/+Rosa26Stop-tdTomato/+ mouse.
a,b, Additional supporting data for Fig. 7. All IgA+ B cell clones analyzed from sequences derived from single-sorted cells of the SED and GC compartments, as described in Fig. 7 and in the Methods section. The number of mutations between neighboring nodes is indicated and includes synonymous, non-synonymous, and reverse mutations to the germline sequence. GL, germline. UCA, unique common ancestor, inferred from the sequence analysis. Lineage-trees are shown from two independent experiments with one PP from one mouse in each experiment (n=2), shown as (a) and (b).
Supplementary Figures 1–7, Supplementary Tables 1 and 2
Visualization of all the GC and SED compartments in an intact PP using LSFM. Whole PP imaging of AicdaCre/+Rosa26Stop-tdTomato/+ fate reporter mouse is shown in a volumetric visualization mode. Measure box scaling is 200 μm. Representative video of two independent experiments with two mice in each experiment.
Infiltration of antigen-specific B cells into preformed GCs. DsRed+B1-8hi B cells (red) were transferred into AID-GFP (green) recipient mouse and imaged 9 d following oral delivery of NP-CT. Whole PP is shown in volumetric visualization mode. Measure box scaling is 300 μm. Representative video of two independent experiments with three mice in each experiment.
Antigen-specific B cells are co-localized with CD11c+ cells in the SED. Transferred DsRed+B1-8hi B cells (red) in CD11c-YFP (yellow) recipient mouse 9 d following NP-CT administration. Whole PP is shown in volumetric visualization mode. Measure box scaling is 300 μm. Representative video of two independent experiments with three mice in each experiment.
Antigen-specific B cells are co-localized with CX3CR1+ macrophages in the SED. Transferred DsRed+B1-8hi B cells (red) in Cx3cr1GFP/+ (green) recipient mouse, nine days following NP-CT administration. Whole PP is shown in volumetric visualization mode. Measure box scaling is 300 μm. Representative video of two independent experiments with three mice in each experiment.
B cell bearing high-affinity BCRs infiltrate preformed GCs. Transferred GFP+B1-8hi B cells in the PP of an MD4 recipient mouse, 9 d following NP-CT administration. Whole PP is shown in volumetric visualization mode. Measure box scaling is 300 μm. Representative video of two independent experiments with two mice in each experiment.
B cell bearing low-affinity BCRs colonize the SEDs but fail to progress into the GCs. Transferred DsRed+B1-8lo B cells in the PP of an MD4 recipient mouse, 9 d following NP-CT administration. Whole PP is shown in volumetric visualization mode. Measure box scaling is 300 μm. Representative video of two independent experiments with two mice in each experiment.
Plasmablasts are formed in the subepithelial dome. Transferred DsRed+B1-8hi Blimp-1-YFP B cells in the PP of a wild-type recipient mouse were imaged by TPLSM, 5 d following NP-CT administration. 100μm z-stack is shown with Blimp-1–YFP+ plasmablasts are marked in yellow circles. Scale bar, 100 μm. Representative video of two independent experiments with two mice in each experiment.
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Biram, A., Strömberg, A., Winter, E. et al. BCR affinity differentially regulates colonization of the subepithelial dome and infiltration into germinal centers within Peyer’s patches. Nat Immunol 20, 482–492 (2019). https://doi.org/10.1038/s41590-019-0325-1
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