Tbcell Entanglement and Icosldriven Feedforward 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 (T_FH) cells regulate GC selection is not clear^3,4. Using competitive mixed chimaera, we show here that, beyond the role in promoting T_FH development^5,6,7, 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 T_FH–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 T_FH 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 ICOSL^high 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 T_FH–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 T_FH cells control the quality of long-lived humoral immunity.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Subscribe to Journal

Get full journal access for 1 year

185,98 €

only 3,65 € per issue

All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Additional access options:

• Log in
• Learn about institutional subscriptions

References

1. Victora, G. D. & Nussenzweig, M. C. Germinal centers. Annu. Rev. Immunol. 30, 429–457 (2012)

   CAS  Article  PubMed  Google Scholar

2. Tarlinton, D. & Good-Jacobson, K. Diversity among memory B cells: origin, consequences, and utility. Science 341, 1205–1211 (2013)

   CAS  Article  ADS  PubMed  Google Scholar

3. Allen, C. D., Okada, T., Tang, H. L. & Cyster, J. G. Imaging of germinal center selection events during affinity maturation. Science 315, 528–531 (2007)

   CAS  Article  ADS  PubMed  Google Scholar

4. Victora, G. D. et al. Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143, 592–605 (2010)

   CAS  PubMed  PubMed Central  Google Scholar

5. Gigoux, M. et al. Inducible costimulator promotes helper T-cell differentiation through phosphoinositide 3-kinase. Proc. Natl Acad. Sci. USA 106, 20371–20376 (2009)

   CAS  Article  ADS  PubMed  PubMed Central  Google Scholar

6. Choi, Y. S. et al. ICOS receptor instructs T follicular helper cell versus effector cell differentiation via induction of the transcriptional repressor Bcl6. Immunity 34, 932–946 (2011)

   CAS  Article  PubMed  PubMed Central  Google Scholar

7. Xu, H. et al. Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility. Nature 496, 523–527 (2013)

   CAS  Article  ADS  PubMed  Google Scholar

8. Qi, H., Cannons, J. L., Klauschen, F., Schwartzberg, P. L. & Germain, R. N. SAP-controlled T–B cell interactions underlie germinal centre formation. Nature 455, 764–769 (2008)

   CAS  Article  ADS  PubMed  PubMed Central  Google Scholar

9. Horikawa, K. et al. Spontaneous network activity visualized by ultrasensitive Ca²⁺ indicators, yellow Cameleon-Nano. Nature Methods 7, 729–732 (2010)

   CAS  Article  PubMed  Google Scholar

10. Mues, M. et al. Real-time in vivo analysis of T cell activation in the central nervous system using a genetically encoded calcium indicator. Nature Med. 19, 778–783 (2013)

    CAS  Article  PubMed  Google Scholar

11. Casamayor-Palleja, M., Khan, M. & MacLennan, I. C. A subset of CD4⁺ memory T cells contains preformed CD40 ligand that is rapidly but transiently expressed on their surface after activation through the T cell receptor complex. J. Exp. Med. 181, 1293–1301 (1995)

    CAS  Article  PubMed  Google Scholar

12. Koguchi, Y., Thauland, T. J., Slifka, M. K. & Parker, D. C. Preformed CD40 ligand exists in secretory lysosomes in effector and memory CD4⁺ T cells and is quickly expressed on the cell surface in an antigen-specific manner. Blood 110, 2520–2527 (2007)

    CAS  Article  PubMed  PubMed Central  Google Scholar

13. Allen, D., Simon, T., Sablitzky, F., Rajewsky, K. & Cumano, A. Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO J. 7, 1995–2001 (1988)

    CAS  Article  PubMed  PubMed Central  Google Scholar

14. Smith, K. G., Light, A., Nossal, G. J. & Tarlinton, D. M. The extent of affinity maturation differs between the memory and antibody-forming cell compartments in the primary immune response. EMBO J. 16, 2996–3006 (1997)

    CAS  Article  PubMed  PubMed Central  Google Scholar

15. Weinstein, J. S. et al. B cells in T follicular helper cell development and function: separable roles in delivery of ICOS ligand and antigen. J. Immunol. 192, 3166–3179 (2014)

    CAS  Article  PubMed  Google Scholar

16. Qi, H., Cannons, J. L., Klauschen, F., Schwartzberg, P. L. & Germain, R. N. SAP-controlled T–B cell interactions underlie germinal centre formation. Nature 455, 764–769 (2008)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar

17. Mootha, V. K. et al. PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nature Genet. 34, 267–273 (2003)

    CAS  Article  ADS  PubMed  Google Scholar

18. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar

19. Zhu, X. et al. Analysis of the major patterns of B cell gene expression changes in response to short-term stimulation with 33 single ligands. J. Immunol. 173, 7141–7149 (2004)

    CAS  Article  PubMed  Google Scholar

20. Jacob J et al. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells. J. of Exp. Med. 178, 1293–1307 (1993)

    Article  Google Scholar

Download references

Acknowledgements

We thank L. and M. McHeyzer-Williams for providing the V_H gene analysis protocol, T. Nagai for YC-Nano reporter constructs⁹, and H. Wekerle for the Twitch-1^CD 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

Author notes

1. Heping Xu & Dan Luo

   Present address: Present addresses: Division of Immunobiology and the Center for Systems Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA (H.X.); College of Life Sciences & Bioengineering, Jiaotong University, 100044 Beijing, China (D. Luo.).,

2. Dan Liu and Heping Xu: These authors contributed equally to this work.

Authors and Affiliations

1. Tsinghua-Peking Center for Life Sciences, Laboratory of Dynamic Immunobiology, School of Medicine, Tsinghua University, Beijing, 100084, China

   Dan Liu, Heping Xu, Changming Shih, Zurong Wan, Weiwei Ma, Dan Luo & Hai Qi

2. School of Life Sciences, Tsinghua University, Beijing, 100084, China

   Xiaopeng Ma

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.

Ethics declarations

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 (pMHC^neg), 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. f–h, Pseudo-colour scatter plots show gating strategies and the scatter plots show CD45.2 competency values in individual chimaeric mice for the CD19⁺GL7^highFas^high GC compartment at day 7, 14 and 21 post-immunization (f), the B220^lowCD138^high 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 T_FH 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 (R ² 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 T_FH cells in vivo.

a, Domain composition of the YC-Nano-50^CD 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. d–f, Demonstration of analysing calcium fluxes of T_FH cells in response to antigen in vivo. MD4 GCs were induced and imaged as described in Methods. During the imaging session, 100 μg OVA_323–339 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, ΔR _t /R ₀ calculated according to c for the two T cells tracked in d. Dotted lines indicate the time of OVA injection. R ₀ 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 R ₀ and R _t . Fluctuations of ΔR _t /R ₀ seen in cell no. 1 before OVA injection likely reflect triggering by endogenous B cells in the GC. f, Mean ΔR _t /R ₀ 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 R ₀ . The mean ΔR _t /R ₀ is an internally referenced parameter and can be used to compare cells imaged in different sessions.

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

Intravital imaging of YC-Nano-50^CD-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, R ₀ ′, 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 R ₀ s of all T cells quantitated in Fig. 2. b, A ΔR _t /R ₀ ′ trace to illustrate the sensitivity of maximum ΔR _t /R ₀ ′ quantitation and the insensitivity of mean ΔR _t /R ₀ ′ quantitation due to averaging of infrequent high-amplitude calcium spikes. c, d, Maximum (c) or mean (d) ΔR _t /R ₀ ′ 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) ΔR _t /R ₀ 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. b–d, GSEA analysis was carried out using GSEA software from Broad Institute^17,18. 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 d _t . The maximal value of d _t 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 d _t 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.

a–d, Normally T_FH 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 V_H mutations in NP-binding GCs at day 9.5 in mixed chimaera

Full size table

Extended Data Table 2 V_H mutations in NP-binding BMPCs at day 21 in mixed chimaera

Full size table

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)

PowerPoint slides

Rights and permissions

About this article

Cite this article

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

Download citation

• Received: 29 October 2013

• Accepted: 27 August 2014

• Published: 15 October 2014

• Issue Date: 08 January 2015

• DOI : https://doi.org/10.1038/nature13803

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

lowgoormes.blogspot.com

Source: https://www.nature.com/articles/nature13803

Share this post