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T–B-cell entanglement and ICOSL-driven feed-forward regulation of germinal centre reaction

Abstract

The germinal centre (GC) reaction supports affinity-based B-cell competition and generates high-affinity bone-marrow plasma cells (BMPCs)1,2. How follicular T-helper (TFH) cells regulate GC selection is not clear3,4. Using competitive mixed chimaera, we show here that, beyond the role in promoting TFH development5,6,7, ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) is important for individual B cells to competitively participate in the GC reaction and to develop into BMPCs. Using intravital imaging aided by a calcium reporter, we further show that ICOSL promotes an ‘entangled’ mode of TFH–B-cell interactions, characterized by brief but extensive surface engagement, productive T-cell calcium spikes, and B-cell acquisition of CD40 signals. Reiterated entanglement promotes outer-zone co-localization of outcompeting GC B cells together with TFH cells, affording the former increased access to T-cell help. ICOSL on GC B cells is upregulated by CD40 signals. Such an intercellular positive feedback between contact-dependent help and ICOSL-controlled entanglement promotes positive selection and BMPC development, as evidenced by observations that higher-affinity B-cell receptor variants are enriched in the ICOSLhigh fraction, that numerically disadvantaged ICOSL-deficient GC B cells or BMPCs exhibit strong affinity compensation in competitive chimaera, and that when GC competition proceeds without ICOSL, selection of high-affinity variants in otherwise normal GC reactions is impaired. By demonstrating entanglement as the basic form of GC TFH–B-cell interactions, identifying ICOSL as a molecular linkage between T–B interactional dynamics and positive selection for high-affinity BMPC formation, our study reveals a pathway by which TFH cells control the quality of long-lived humoral immunity.

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Figure 1: GC T–B entanglement controlled by ICOSL and ICOS.
Figure 2: Coordinated outer-zone localization of outcompeting Icosl+/+ B cells and associated TFH cells.
Figure 3: Dynamic ICOSL regulation and ICOSL-driven feed-forward GC selection.

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Acknowledgements

We thank L. and M. McHeyzer-Williams for providing the VH gene analysis protocol, T. Nagai for YC-Nano reporter constructs9, and H. Wekerle for the Twitch-1CD reporter construct10. H.Q. is indebted to Y. Hong for support. H.Q. was a Tsinghua–Bayer Investigator and a Tsinghua–Janssen Investigator. This work was funded in part by the Ministry of Science and Technology ‘973’ program (2014CB542501), National Natural Science Foundation of China (81330070 and 81361120397), Tsinghua University Initiative Scientific Research Program (20131089224), Institut Mérieux, and the Tsinghua–Peking Center for Life Sciences.

Author information

Authors and Affiliations

Authors

Contributions

D. Liu and H.X. conducted a majority of the experiments; C.S. developed the intravital calcium imaging method and conducted much of the imaging work together with H.X.; Z.W. developed the assay for ICOSL-deficient GCs and conducted related mutational analyses; X.M. performed RNA-seq analysis; W.M. and D. Luo helped with imaging and molecular cloning. H.Q. conceptualized the study, designed the experiments with input from H.X., D. Liu, C.S. and Z.W., and wrote the paper. All authors contributed collectively to interpreting data.

Corresponding author

Correspondence to Hai Qi.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 ICOSL-mediated control of GC and BMPC competencies of antigen-specific B cells and the lack of effects of ICOSL–ICOS interactions on cognate T–B contact duration in vivo.

a, Schematic diagram of the protocol of making 50:50 bone marrow (BM) chimaera using CD45.1 wild-type (WT) BM cells mixed with CD45.2 ICOSL wild type or ICOSL-knockout (KO) bone marrow cells. b, A schematic diagram depicting the role for follicular non-presenting (pMHCneg), bystander B-cell ensemble in promoting ICOS-driven, PI3K-dependent motility of T cells and their recruitment into the follicle from the T–B border. c, A schematic diagram depicting the reduced but non-abrogated effect on follicular recruitment of T cells by the non-presenting bystander B-cell ensemble in the Icosl+/+:Icosl−/− 50:50 mixed bone marrow chimaera. d, Percentages of total GC B cells in CD19+ cells in chimaeras of indicated types at indicated time points after immunization with NP-KLH. These are the same chimaeric mice analysed below. e, Equation for calculating CD45.2 competitive competencies. fh, Pseudo-colour scatter plots show gating strategies and the scatter plots show CD45.2 competency values in individual chimaeric mice for the CD19+GL7highFashigh GC compartment at day 7, 14 and 21 post-immunization (f), the B220lowCD138high BMPC compartment at day 21 (g), and the NP-binding, isotype-switched splenic memory compartment at day 21 (h) following immunization with 100 μg NP-KLH (examples in g and h from the same mouse assayed for both BMPC and memory B cells). Each symbol represents one mouse. Lines denote the means. Data are pooled from 3 (day-7 GC, day-21 memory), 4 (day-14 GC), or 5 (day-21 BMPC) experiments. The percentages in parentheses were ratios of mean values between knockout:wild-type and wild-type:wild-type. i, Percentages of plasma cells developed from Icosl+/+ or Icosl−/− MD4 B cells at day 5 after HEL-OVA immunization in recipient mice that were also given OT-II cells. j, Development of NP-specific splenic plasma cells in knockout:wild-type and wild-type:wild-type mixed chimaera 6 days after NP-KLH immunization. The competitive competency is calculated according to e. k, T–B interactions in the presence or absence of ICOSL on the B-cell partner. Left, the T–B border distribution pattern of CFP OT-II T cells and Icosl+/+ MD4 GFP B cells and Icosl−/− MD4 dsRed B cells 36–72 h post-immunization in the draining lymph node taken after intravital imaging analyses; follicle identified by IgD staining, each CFP+ T cell highlighted with a circle; scale bar, 50 μm. Right, contact duration between OT-II T cells and the two types of MD4 B cells as measured by intravital imaging (See also Supplementary Video 1a). l, Interactions between CFP OT-II T cells and GFP Icosl+/+ or dsRed Icosl−/− MD4 B cells visualized 96–120 h after HEL-OVA immunization. Duration of T–B contacts involving 114 Icosl+/+ and 102 Icosl−/− events pooled from 2 experiments. See Supplementary Video 1b and corresponding contact area analyses in Fig. 1.

Extended Data Figure 2 Comparable TFH entanglement with GFP and dsRed MD4 B cells.

To rule out potential effects of GFP and dsRed transgenes on T–B contact, CFP OT-II T cells and co-transferred GFP Icosl+/+ and dsRed Icosl+/+ MD4 B cells were visualized 96–120 h after HEL-OVA immunization precisely as done in Fig. 1a–f. a, Duration of contacts involving 106 GFP and 102 dsRed Icosl+/+ MD4 B cells pooled from 2 experiments. b, Time-lapse images showing ‘entangled’ behaviours of OT-II T cells (arrowheads) contacting the two types of B cells; time is shown as min:s. See corresponding Supplementary Video 3. Scale bar, 20 μm. c, Scatter plots of the SEI for 106 GFP and 102 dsRed MD4 B cells. d, Perimeters of analysed B cells of the two types. e, f, SEI histograms and fitted Gaussian curves (R2 for goodness of fit: GFP, 0.79; dsRed, 0.79) (e) and the plot of SEI against contact duration (f); the dotted line indicates 38%.

Extended Data Figure 3 FRET-based calcium imaging of TFH cells in vivo.

a, Domain composition of the YC-Nano-50CD calcium reporter, containing the calcium-binding module (CaM–linker–M13) from YC-Nano-50 from ref. 9, and the FRET pair from the codon-diversified CFP and cpCitrine described in ref. 10. b, A diagram of the calcium-sensing FRET process. The normalized 2-photon excitation efficiency of CFP and YFP at 830 nm is 80% and 5%, respectively. c, Formulas for quantitative parameters. df, Demonstration of analysing calcium fluxes of TFH cells in response to antigen in vivo. MD4 GCs were induced and imaged as described in Methods. During the imaging session, 100 μg OVA323–339 peptide was intravenously injected to acutely trigger the OT-II T cells. d, Image sequences of four time points, two before and two after the OVA injection. For each time point, middle: fluorescence overlay; scale bar, 50 μm; left, two representative T cells tracked by iso-surfaces built on YFP fluorescence; right, pseudo-colour FRET ratio image (R image, as defined in c) calculated pixel by pixel by the RatioPlus plug-in of ImageJ. Note cell no. 1 is within the GC proper in the lower left corner, while cell no. 2 is in the area immediately surrounding the GC. e, ΔRt/R0 calculated according to c for the two T cells tracked in d. Dotted lines indicate the time of OVA injection. R0 here is the average R of all trackable time points before OVA injection. Total fluorescence intensity signals from voxels encapsulated within the iso-surface (cell) were used to calculate R0 and Rt . Fluctuations of ΔRt/R0 seen in cell no. 1 before OVA injection likely reflect triggering by endogenous B cells in the GC. f, Mean ΔRt/R0 averaged over the first 5 min after OVA injection for 19 OT-II T cells imaged in 2 independent experiments. The dotted line indicates an average 50% increase in R over R0 . The mean ΔRt/R0 is an internally referenced parameter and can be used to compare cells imaged in different sessions.

Extended Data Figure 4 Calcium imaging of TFH cells in the core LZ densely populated with MD4 B cells.

Intravital imaging of YC-Nano-50CD-transduced OT-II T cells was done as in Fig. 2h–l except with imaging fields covering the core light zone and a change of reference state definition. a, Formulas for quantitative parameters. Of note, R0 ′, as an ensemble average of all T cells outside GCs without ever contacting MD4 B cells in the imaging session, is theoretically equivalent to the average of R0 s of all T cells quantitated in Fig. 2. b, A ΔRt/R0 ′ trace to illustrate the sensitivity of maximum ΔRt/R0 ′ quantitation and the insensitivity of mean ΔRt/R0 ′ quantitation due to averaging of infrequent high-amplitude calcium spikes. c, d, Maximum (c) or mean (d) ΔRt/R0 ′ of 55 OT-II T cells visualized in Icosl+/+ GCs and 67 in Icosl−/− GCs. Data were pooled from three experiments. e, f, For comparison, maximum (e) or mean (f) ΔRt/R0 of all 88 OT-II T cells contacting Icosl+/+ MD4 GC B cells and 88 contacting Icosl−/− cells that were analysed in Fig. 2h–l are shown. Note comparable sensitivities of c and e in detecting differences between T cells under the two conditions. The apparent failure of d to detect differences between the two conditions is consistent with prevailing short GC T–B contacts and infrequent high-amplitude calcium spikes averaged out by relatively idle periods.

Extended Data Figure 5 ICOS co-stimulates calcium signalling and CD40L mobilization.

a, b, Calcium fluxes detected by Indo-1 in T-cell blasts that were incubated on ice with biotinylated anti-CD3 or anti-ICOS or isotype control antibodies separately (a) or incubated with the indicated mixture of anti-CD3 anti-ICOS or isotype control antibodies on ice (b) before streptavidin stimulation at 37 °C. Data represent three independent experiments. c, Surface staining of CD40L following stimulation for 3 (left) or 5 (right) minutes with 0.25 μg ml−1 anti-CD3 plus 5 μg ml−1 anti-ICOS or isotype or 1 μM ionomycin according to the mobilization assay protocol detailed in the Methods. One of two experiments with similar results is shown. d, Quantitation of four independent ICOS co-stimulated CD40L mobilization assays (left, each matched symbol denoting one experiment) and the histogram overlay from one representative experiment (right). Background-subtracted CD40L MFI on un-stimulated cells is defined as unit 1. The P values were calculated with paired t-tests.

Extended Data Figure 6 Gene set enrichment analysis of Icosl+/+ and Icosl−/− MD4 GC B cells.

B6 mice were transferred with GFP Icosl+/+ and dsRed Icosl−/− MD4 B cells together with OT-II T cells and immunized with HEL-OVA. MD4 GC B cells of the two genotypes were sorted from the draining lymph nodes 120 h post-immunization and subjected to transcriptome analysis by RNA-seq. a, The gating strategy for sorting. bd, GSEA analysis was carried out using GSEA software from Broad Institute17,18. RNA-seq data of the two types of GC B cells were ranked by the log2_Ratio_of_Classes metric. b, Enrichment profiles for those genes that are upregulated (>3-fold) by 2-h (left) or 4-h (middle) anti-CD40 or anti-IgM (right) stimulation as reported in ref. 18. Compared to Icosl−/− GC B cells, Icosl+/+ cells in vivo are strongly imprinted with the transcription signature that is significantly upregulated by prolonged CD40 signalling. c, d, Genes in the core enrichment for the CD40 (4 h) group, presented as a cluster-analysed heatmap of expression levels (log2-transformed RPKM) of the two types of cells (c) or as subclasses manually categorized according to Gene Ontology (http://www.geneontology.org) (d). Nine genes that were either unclassifiable or with unknown functions were omitted.

Extended Data Figure 7 Calculation of the edge localization index (ELI).

To analyse imaging data presented in Fig. 3, the GC volume is detected and marked by an iso-surface using dsRed fluorescence of the competitor GC cell population. Individual T or B cells within the GC are tracked, as represented by the green dot and line trace here. At any time point t, the shortest distance of the cell to the GC iso-surface is calculated by a custom Matlab script as dt . The maximal value of dt for a given cell represents its longest distance away from the GC edge. The maximal radius of the GC on a xy plane is defined as D. The maximum of dt for a given cell divided by D is the edge localization index of the cell. The closer ELI is to 0, the closer the cell is located to the GC edge.

Extended Data Figure 8 A schematic representation of entanglement and ICOSL-driven feed-forward GC selection.

ad, Normally TFH cells display a low level of CD40L on the cell surface, which can in principle be used by any GC B cells that constitutively express the CD40 receptor, regardless of ICOSL expression (a); GC B cells carrying BCR of the same specificity but different affinities present different amounts of peptide–MHC (major histocompatibility complex) complexes (red dot: peptide epitope) (b, c), and utilize the intercellular feed-forward loop (d) differently. More intense peptide–MHC antigen presentation coupled with ICOSL costimulation leads to enhanced calcium response and better entanglement by the T cell (i); the calcium response triggers rapid CD40L externalization from the intracellular store (ii), and far more CD40L becomes available and used by the presenting B cell (iii); CD40L signals and probably additional help signals from the T cells promote ICOSL surface expression (iv), which prepare the B cells for more efficient entanglement with the T cells. Note that one B cell does not need to repeatedly entangle with the same T cells, and there must be a way by which ICOSL is down-modulated even on outcompeting GC B cells. ICOSL downregulation by BCR signalling as suggested in Fig. 3 could be a potential mechanism (not depicted).

Extended Data Table 1 VH mutations in NP-binding GCs at day 9.5 in mixed chimaera
Extended Data Table 2 VH mutations in NP-binding BMPCs at day 21 in mixed chimaera

Supplementary information

Supplementary Information

This file contains Supplementary Notes 1-5. (PDF 142 kb)

Comparable duration of contacts between T cells and Icosl+/+ or Icosl-/- B cells at the T-B border or in the GC

(A) GFP-expressing Icosl+/+ (green), dsRed-expressing Icosl-/- (red) MD4 B cells and CFP-expressing OT-II T cells (white) were visualized 36 to 72 hours after HEL-OVA immunization. Also see Extended Data Fig. 1e. Scale bar, 100 μm. (B) GFP-expressing Icosl+/+ (green), dsRed-expressing Icosl-/- (red) MD4 B cells and CFP-expressing OT-II (white) T cells were visualized in a GC cluster 96 hours after HEL-OVA immunization. Scale bar, 50 μm. Both (A) and (B) are a projection of 60 μm in z direction. (MOV 11489 kb)

Different modes of contact between TFH cells and Icosl+/+ or Icosl-/- B cells in GCs

(A-C) GFP-expressing Icosl+/+ (green), dsRed-expressing Icosl-/- (red) MD4 B cells and CFP-expressing OT-II (white) T cells in two GC clusters were visualized approximately 100 hours after immunization. Arrowheads highlight the “passing” mode of T cell contact with an Icosl-/- B cell and the “entangled” behavior of a T cell with an Icosl+/+ B cell in the small cluster played back with a faster (A) and slower speed (B). Also see corresponding time-lapse images in Fig. 2b. (C) At representative time points, “entangled” T cell contacts in the larger GC cluster are highlighted with arrowheads. Scale bar, 40 μm. A projection of 30 μm in z direction. (D) Another example for different modes of contact between TFH cells and Icosl+/+ or Icosl-/- B cells, with arrowheads highlighting the “passing” mode of T cell contact with an Icosl-/- B cell and “entangled” contacts of T cells with Icosl+/+ B cells. Scale bar, 20 μm. A projection of 20 μm in z direction. (MOV 34883 kb)

Comparable modes of contact between TFH cells and GFP- or dsRed-expressing Icosl+/+ B cells

GFP-expressing (green) and dsRed-expressing (red) WT MD4 B cells and CFP-expressing OT-II (white) T cells were visualized approximately 100 hours after immunization. Arrowheads highlight “entangled” contacts of T cells B cells. Played back at 10 frames per second. Scale bar, 20 μm. A projection of 30 μm in z direction. (MOV 13496 kb)

Acute calcium signaling of OT-II TFH cells induced by exogenous antigen: detection by the YC-Nano-50CD reporter in vivo

YC-Nano-50CD-expressing OT-II and dsRed-expressing MD4 B cells were imaged 120 hours post HEL-OVA immunization, and 100 μg OVA323-339 peptide was intravenously injected at the marked time point during the imaging session. The video is played back twice (version A and B); in each, the left panel is fluorescent overlay and the right panel is pseudo-color FRET ratio image. In (B), the two cells that were quantitatively analyzed in Extended Data Fig. 3 as examples are highlighted along their migration paths. Played back at 5 frames per second. Scale bar, 50 μm. A projection of 60 μm in z direction. (MOV 13662 kb)

Pronounced calcium fluxes in the T cells are associated with entangled contacts but reduced when B cells lack in ICOSL

(A) YC-Nano-50CD-expressing OT-II cells come in passing (#1) or entangled contact (#2) with Icosl+/+ dsRed-expressing MD4 B cells (red). See corresponding time-lapse images in Fig. 2h. A projection of 20 μm in z direction. (B) Another example of calcium signaling of YC-Nano-50CD-expressing OT-II TFH cells entangled with Icosl+/+ MD4 B cells. See corresponding time-lapse images in Fig. 2i. A projection of 60 μm in z direction. (C) An example of calcium signaling of YC-Nano-50CD-expressing OT-II TFH cells entangled with Icosl-/- MD4 B cells. A projection of 30 μm in z direction. See corresponding time-lapse images in Fig. 2i. All played back at 5 frames per second. Scale bar, 20 μm. (MOV 15507 kb)

Calcium fluxes of YC-Nano-50CD-expressing OT-II TFH cells in the core LZ of Icosl+/+ or Icosl-/- GC

The approximate (A) Icosl+/+ or (B) Icosl-/- GC volume encapsulated by a grey iso-surface is overlaid onto the pseudo-color FRET ratio movie. See quantitative analyses in Extended Data Fig. 4. Played back at 10 frames per second. Scale bar, (A) 50 μm, (B) 70 μm. Both are a projection of 30 μm in z direction. (MOV 29560 kb)

Even distribution of Icosl+/+ tester MD4 B cells and OT-II TFH cells in a GC predominantly composed of Icosl-/- competing MD4 B cells 96 hours post immunization

Two examples of GFP-expressing Icosl+/+ (green), dsRed-expressing Icosl-/- (red) MD4 B cells and CFP-expressing OT-II T cells (blue) evenly intermixed in a GC cluster. Scale bar, 100 μm. Projection of 60 μm in z direction. (MOV 10347 kb)

Outer-zone localization of Icosl+/+ tester MD4 B cells and OT-II TFH cells in a GC predominantly composed of Icosl-/- competing cells 120 hours post immunization

GFP-expressing Icosl+/+ (green), dsRed-expressing Icosl-/- (red) MD4 B cells and CFP-expressing OT-II T cells (blue) were visualized in a GC cluster. Scale bar, 100 μm. A projection of 60 μm in z direction. (MOV 11489 kb)

Even distribution of Icosl+/+ tester MD4 GC B cells and OT-II TFH cells in a GC predominantly composed of Icosl+/+ competing cells 120 hours post immunization

GFP-expressing Icosl+/+ (green), dsRed-expressing Icosl+/+ (red) MD4 B cells and CFP-expressing OT-II T cells (blue) were visualized in a GC cluster. Scale bar, 100 μm. A projection of 60 μm in z direction. (MOV 8639 kb)

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Liu, D., Xu, H., Shih, C. et al. T–B-cell entanglement and ICOSL-driven feed-forward regulation of germinal centre reaction. Nature 517, 214–218 (2015). https://doi.org/10.1038/nature13803

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