TFH-derived dopamine accelerates productive synapses in germinal centres

Journal name:
Nature
Volume:
547,
Pages:
318–323
Date published:
DOI:
doi:10.1038/nature23013
Received
Accepted
Published online

Abstract

Protective high-affinity antibody responses depend on competitive selection of B cells carrying somatically mutated B-cell receptors by follicular helper T (TFH) cells in germinal centres. The rapid T–B-cell interactions that occur during this process are reminiscent of neural synaptic transmission pathways. Here we show that a proportion of human TFH cells contain dense-core granules marked by chromogranin B, which are normally found in neuronal presynaptic terminals storing catecholamines such as dopamine. TFH cells produce high amounts of dopamine and release it upon cognate interaction with B cells. Dopamine causes rapid translocation of intracellular ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) to the B-cell surface, which enhances accumulation of CD40L and chromogranin B granules at the human TFH cell synapse and increases the synapse area. Mathematical modelling suggests that faster dopamine-induced T–B-cell interactions increase total germinal centre output and accelerate it by days. Delivery of neurotransmitters across the T–B-cell synapse may be advantageous in the face of infection.

At a glance

Figures

  1. Human TFH cells express CgB and contain dense-core vesicles.
    Figure 1: Human TFH cells express CgB and contain dense-core vesicles.

    a, Immunohistochemistry stain of human germinal centres: CgB (brown) (n = 50). Scale bar, 100 μm. b, c, CHGB mRNA by qPCR (b) (normalized to B2M; r.u., relative units) and RNA sequencing (c) in lymphoid cell subsets (c.p.m., counts per million) (n = 3). B mem, memory B (cell); GCB, germinal centre B (cell); PCs, plasma cells; TEFF, effector T (cell); TFR, T follicular regulatory (cell); Treg, T regulatory (cell). d, Immunofluorescence on paraffin-embedded tonsil for CgB (red) and TFH markers (green). Original magnification ×400 (n = 10). e, Flow cytometric plots showing CHGB mRNA in live CD3+ cells and fluorescence intensity within the indicated cell subsets (n = 5). f, CgB stain in IgG4-related disease (n = 5). g, CgB+ cells per square millimetre of tissue. Bars, medians; each dot is the average of ten areas from each patient; NS, not significant; *P ≤ 0.05, **P ≤ 0.01; Mann–Whitney test. h, i, Ultrastructure of dense vesicles (arrows) within germinal centre cells by electron microscopy; ER, endoplasmic reticulum; ex.sp., extracellular space (n = 3). j, k, Immunogold labelling for CgB in germinal centre cells; V, spaces generated during processing; N, nucleus. Scale bar, 2 μm (n = 3). l, Immunofluorescence stain on sorted TFH cells; CgB+ (red) (n = 3).

  2. Human TFH cells produce dopamine.
    Figure 2: Human TFH cells produce dopamine.

    a, Gating strategy for sorting T-cell subsets from human tonsil. DA, dopamine. b, Quantification of catecholamines by GC–MS/MS (n = 2). A, adrenaline; l-DA, l-DOPA; NA, noradrenaline. c, Representative dopamine fluorescence-activated cell sorting (FACS) stain. d, Quantification of dopamine-expressing cells (n = 3). Red bars denote median values. e, Representative immunofluorescence dopamine stains of TFH cells untreated or treated with FSK (n = 5); DAPI, 4′,6-diamidino-2-phenylindole. f, Dopamine expression in untreated or FSK-treated TFH and non-TFH (effector and naive T cells) (n = 5). g, Representative GC/MS/MS peaks showing dopamine content in FSK-treated and untreated TFH cells compared with internal standard ([13C6]dopamine), which controlled for losses during extraction. DA area denotes the area under the peak, measured as the detector response. h, CHGB and TH mRNA expression by qPCR (normalized to RPL13A) in FSK-treated TFH and non-TFH cells (n = 3). f, h, Bars, median values; each dot represents one donor; NS, not significant; **P ≤ 0.01; Mann–Whitney test.

  3. Dopamine is released from TFH cells upon cognate interactions.
    Figure 3: Dopamine is released from TFH cells upon cognate interactions.

    ac, Flow cytometric quantification of dopamine content in FSK-stimulated TFH cells after incubation for 30 min with anti-CD3/CD28 beads (1:1) (a) or autologous or allogeneic germinal centre B cells (1:2) (n = 3) (b) also showing changes in dopamine content in germinal centre B cells (autologous or allogeneic) cultured separately (‘nil’), or together with TFH cells (n = 5); and with or without ICAM-1 (5 μg ml−1) and LFA-1 (10 μg ml−1) block (c) (n = 3); Mann–Whitney test. d, Flow cytometric plots showing plasma cells, identified as CD27hiCD38hi, induced in cultures of germinal centre B cells stimulated for 5 days with anti-CD40 (2 μg ml−1), IL-21 (20 ng ml−1), and different concentrations of freshly prepared dopamine (n = 5). e, Fold changes in plasma cell differentiation from germinal centre B cells stimulated for 2 h with or without dopamine (5 μM) and haloperidol (Haldol, 50 nM), and cultured in the presence of anti-CD40 (2 μg ml−1) and IL-21 (20 ng ml−1) for 5 days; two-tailed Student’s t-test. ac, e, Bars, median values; each dot represents a single experiment conducted in triplicate (n = 5). Two-tailed Student’s t-test; NS, not significant; *P ≤ 0.05, ***P ≤ 0.001.

  4. Dopamine induces ICOSL upregulation on human germinal centre B cells.
    Figure 4: Dopamine induces ICOSL upregulation on human germinal centre B cells.

    a, Gating of germinal centre B cells and fluorescence intensity of specified proteins 30 min after stimulation with dopamine (10 μM) (n = 3). b, Fold changes of surface ICOSL expression with medium control set as unit 1 (n = 8). c, Survival of germinal centre B cells after stimulation with dopamine (n = 8). d, Fold changes of surface ICOSL expression on germinal centre B cells stimulated with dopamine (10 μM), dopamine agonist SKF38393 (10 nM), haloperidol (50 nM), and dopamine antagonist SKF83566 (10 nM) for 30 min, with medium control set as unit 1 (n = 5). e, f, Representative histograms (e) and quantification (f) of surface and intracellular ICOSL on naive, memory, and germinal centre B cells (n = 4); Mann–Whitney test. g, RNA counts per million (c.p.m.) of indicated transcripts in human germinal centre B cells stimulated with or without dopamine (5 μM) for 2 h (n = 3). h, Fold changes of surface ICOSL expression on human germinal centre B cells treated with cycloheximide (CHX, 10 μg ml−1) and stimulated with dopamine (10 μM) for 30 min. i, j, Fold changes of surface ICOSL expression on human germinal centre B cells stimulated with dopamine (10 μM), anti-CD40 (1 μg ml−1), or recombinant CD40L (10 μg ml−1) (i), IL-21 (10, 50, or 100 ng ml−1) or IL-4 (10 ng ml−1) (j) for 30 min (n = 5). b, d, hj, Bars, medians; each dot represents a single experiment conducted in triplicate (n = 10); two-tailed Student’s t-test. NS, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

  5. Effects of ICOS ligation at the immunological synapse.
    Figure 5: Effects of ICOS ligation at the immunological synapse.

    a, Representative images of ICAM-1 ring (white) around CD40L (pseudo-colour scale) in the presence or absence of CD40 and ICOSL at physiological densities on the SLB containing ICAM-1 and UCHT1. Scale bar, 5 μm. b, c, Plots represent CD40L mean fluorescence intensity (MFI) of individual activated human T (b) or TFH (c) cells forming synapses (n = 3). d, Representative images of CgB stain in the presence or absence of ICOSL at the immunological synapse. e, CgB median fluorescence intensity of individual activated TFH and non-TFH cells forming synapses (n = 3). Red bars denote median values. b, c, e, NS, not significant; ***P ≤ 0.001, ****P ≤ 0.0001; non-parametric Mann–Whitney U-test. f, Representative images of CgB+ TFH cells (red) forming synapses with allogeneic B cells (green).

  6. Modelling of dopamine effect on TFH–germinal-centre B-cell synapse and germinal centre output.
    Figure 6: Modelling of dopamine effect on TFH–germinal-centre B-cell synapse and germinal centre output.

    a, Representative ICAM-1 area quantification. b, ICAM-1 area expressed as relative units. ce, Interactions (white) among untreated (green) or FSK-stimulated (blue) TFH cells and allogeneic germinal centre B cells (red) cultured in the same well. For each interaction two frames are shown; numbers indicate time after starting imaging (see corresponding Supplementary Video 1). Plots represent T–B-cell interaction duration (d) and contact area (e). b, d, e, NS, not significant; ***P ≤ 0.001; Mann–Whitney test. f, Impact of the speed of ICOSL upregulation (ΔtICOSL; fast, black and grey lines; slow, coloured lines) in germinal centre B cells onto germinal centre characteristics (mean affinity (left) and produced output (right)) estimated with computer simulations. Simulations were repeated with short (black, red, orange lines) and long (grey, magenta, cyan lines) periods of search for TFH cells. Slow ICOSL upregulation had the tendency to shrink germinal centres (see Ω in Supplementary Table 2, red and magenta lines); therefore, the germinal centre strength was restored by parameter adaptation (see Supplementary Table 2, orange and cyan lines). Lines show mean of 100 simulations, grey shades show the s.d. (details in Methods). g, Graphic model of the proposed positive feedback between human TFH and germinal centre B cells. Upon cognate interactions between TFH and germinal centre B cells (1), dopamine is released from CgB+ granules (2). Dopamine activates DRD1 on germinal centre B cells (3) and induces increase ICOSL surface expression (4), which in turn binds to ICOS on TFH cells, inducing CD40L membrane relocation (5) and CgB+ granule formation (6).

  7. CgB+ cells in human germinal centre.
    Extended Data Fig. 1: CgB+ cells in human germinal centre.

    a, b, Representative immunohistochemistry for CgB (brown) of human lymph node (a) and spleen (b) (n = 10). c, Quantification of CD3+CgB+ cells in human tonsils, lymph nodes (n = 10), and spleens (n = 5). d, Percentage of CgB+ T cells in human reactive and neoplastic conditions. c, d, NS, not significant; *P ≤ 0.05 and **P ≤ 0.01; non-parametric Mann–Whitney U-test. e, Representative double immunohistochemistry for CgB (left) and CD3 (middle) after colour deconvolution. Pseudo-colour image (right) showing signal co-localization. Original magnification ×40. Scale bar, 100 μm (n = 3). f, Representative immunofluorescence images for CD3 (green) and ICOS (red) in human germinal centres.

  8. Mouse CgB expression.
    Extended Data Fig. 2: Mouse CgB expression.

    ai, Immunohistochemistry staining shows no CgB reactivity in mouse germinal centres of immunized wild-type (WT) or Sanroque spleens and Peyer’s patches (n = 3). j, Immunohistochemistry control staining for CgB in mouse pancreas islets. aj, Scale bar, 100 μm; n = 3. k, Relative mouse Chgb mRNA expression in different T-cell subsets with adrenal gland as positive control. T cells were FACS sorted as follows: Tnaive (CD4+CD44loCD25); T effector memory (TEM, CD4+CD44hiCD25PD-1−/lo CXCR5−/lo); TFH (CD4+CD44hiPD-1hiCXCR5hi); Treg (CD4+CD25+CD44int). Gapdh was used as the housekeeping gene (n = 3).

  9. Dopamine β-hydroxylase expression in human and mouse lymphocytes.
    Extended Data Fig. 3: Dopamine β-hydroxylase expression in human and mouse lymphocytes.

    a, Gel shows PCR products after amplification of human DBH mRNA in T cells, total tonsil, and B cells. GSα (also known as GNAS) was used as the housekeeping gene. For gel source data, see Supplementary Fig. 1. b, RNA sequencing showing expression of DBH mRNA in human Tnaive, TFH, and TFR cells extracted from three tonsils, expressed as counts per million. c, Immunofluorescence images showing green fluorescent protein (GFP) expression in adrenal medulla of DBHgfp/w mice. d, FACS plot showing GFP expression in splenocytes of DBHgfp/w mice. e, Quantification of DBH–GFP expression in mouse splenocytes. Bars, median values; each dot represents a mouse (n = 10). f, FACS plot showing DBH–GFP expression in B cells localizing outside germinal centres of mice immunized with sheep red blood cells (n = 10).

  10. Mouse endogenous and induced dopamine content.
    Extended Data Fig. 4: Mouse endogenous and induced dopamine content.

    a, b, Quantification and representative FACS plot of dopamine content in mouse naive and follicular T (TFO) cells differentiated by the expression of IL-21. T-cell subsets were FACS sorted into Tnaive (CD4+CD44lo), TFO IL-21+ (CD4+CD44hiIL-21gfp/w), and TFO IL-21 (CD4+CD44hiIL-21w/w), and dopamine content was analysed by flow cytometry before and after treatment with FSK for 24 h. Bars, median values; each dot represents a mouse (n = 5). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; non-parametric Mann–Whitney U-test.

  11. Dopamine release from human TFH cells.
    Extended Data Fig. 5: Dopamine release from human TFH cells.

    a, Dopamine release from TFH cells after stimulation for 30 min with autologous B cells (1:2) alone or with anti-CD3/CD28 beads (1:1). TFH cells were pre-stimulated with FSK before inducing dopamine release. Bars, median; each dot represent a single experiment conducted in triplicates (n = 4). b, Dopamine release from TFH cells after stimulation for 30 min with allogeneic germinal centre B cells (1:2) alone or in the presence of ICOSL blocking antibody (10 μg ml−1). TFH cells were pre-stimulated with FSK before inducing dopamine release and B cells were pre-stimulated with 10 μM dopamine to increase ICOSL surface levels before incubation with TFH cells. Bar, median of dopamine level in TFH cells (n = 3); each triangle is the allogeneic B cells from a single donor paired with its control (square, n = 11). *P ≤ 0.05; paired t-test.

  12. Dopamine receptor (DRD) expression in human B-cell subsets.
    Extended Data Fig. 6: Dopamine receptor (DRD) expression in human B-cell subsets.

    a, Relative expression of DRD mRNA in human B-cell subsets normalized to naive B cells. B2M was used as the housekeeping gene (n = 3). Error bars, s.d. b, c, Representative images of DRD1+ cell (green) localization in human germinal centre (dashed line), showing close proximity to CgB+ (b) or CD3+ (c) cells (red) (n = 3).

  13. Regulation of ICOSL upregulation in mouse and human B cells.
    Extended Data Fig. 7: Regulation of ICOSL upregulation in mouse and human B cells.

    a, Fold changes of surface ICOSL expression on mouse germinal centre B cells treated with anti-CD40 (10 μg ml−1) and dopamine (0.5, 1, 5, 10 μM) for 30 min, with medium control set as unit 1 (n = 5). b, Representative histogram and quantification of surface and intracellular ICOSL on germinal centre and non-germinal centre B cells (n = 5). **P ≤ 0.01; non-parametric Mann–Whitney U-test. c, RNA counts per million of ICOSL, CD40, BCL6, IL-21R, CD86, BAFFR, and FAS mRNA in human memory B cells stimulated with or without dopamine (5 μM) for 2 h (n = 3). d, Fold changes of surface ICOSL expression on mouse germinal centre B cells treated with cycloheximide (CHX, 10 μg ml−1) for 4 h, with medium control set as unit 1. Bars, median values; each dot represents a single mouse. e, Fold changes of surface ICOSL expression on mouse germinal centre B cells stimulated with BAFF (100 ng ml−1), lipopolysaccharides (1 or 10 μg ml−1), anti-CD40 (10 μg ml−1), and anti-IgM (1 or 10 μg ml−1) for 30 min and 4 h. Unit 1 set on medium control. f, Fold changes of surface ICOSL expression on mouse germinal centre B cells treated with actinomycin D (ActD, 5 μg ml−1), anti-CD40 (10 μg ml−1) for 4 h, with medium control set as unit 1. Bars, median; each dot represents a single mouse (n = 5). df, NS, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; two-tailed Student’s t-test. g, Representative histogram of surface ICOSL expression on human germinal centre B cells stimulated with dopamine (10 μM) or anti-CD40 (1 μg ml−1) for 30 min. h, Fold changes of surface ICOSL expression on human germinal centre B cells stimulated with several concentrations of anti-CD40 for 4 h and 8 h, with medium control set as unit 1 (n = 3). i, Survival of germinal centre B cells in the presence of anti-CD40 (1 μg ml−1) after stimulation for 4 h or 8 h (n = 4). *P ≤ 0.05, ***P ≤ 0.001; non-parametric Mann–Whitney U-test.

  14. Effect of ICOSL on CD40L presentation and reception in SLB model for TFH–germinal-centre B-cell interaction.
    Extended Data Fig. 8: Effect of ICOSL on CD40L presentation and reception in SLB model for TFH–germinal-centre B-cell interaction.

    a, Activated human T cells expressing ICOS and CD40L were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3) as a basal condition with a ring of ICAM-1 surrounding a central cluster in T-cell antigen receptor (TCR)-enriched extracellular vesicles for 15 min (ref. 26). This condition resulted in low presentation of CD40L in punctate structures detected by anti-CD40L monoclonal antibody that accumulated in the same central synapse as the TCR-enriched extracellular vesicles. Addition of ICOSL to the SLB resulted in strong central accumulation of fluorescent ICOSL with the TCR-enriched extracellular vesicles, but no increase in CD40L presentation. Addition of CD40 to the SLB resulted in a significant increase in CD40L accumulation, which we refer to as reception because it is receptor dependent. When ICOSL and CD40 were added, the reception of CD40L was further significantly enhanced over the level observed with CD40 alone. Thus, ICOSL ligation in the centre of the immunological synapse increases CD40L reception. All levels are shown in grey scale except CD40L panels, for which the pseudo-colour scale is indicated. Scale bar, 5 μm. b, Human TFH cells were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3). Addition of ICOSL resulted in increased accumulation of CgB at the synapse centre. Addition of CD40 did not further increase CgB accumulation.

  15. Effect of ICOSL upregulation speed in the published and extended germinal centre LEDA and classic recycling models.
    Extended Data Fig. 9: Effect of ICOSL upregulation speed in the published and extended germinal centre LEDA and classic recycling models.

    a, Characteristics of germinal centre reactions in simulations with short (black) and long (colours) search phases for TFH help using the previously published LEDA model (see text). All tested variants (see legend box and text for details on the quantities) exhibit reduced and retarded output production while keeping affinity maturation unchanged. Mean (full lines) and s.d. (shades) of 100 simulations. b, The LEDA model in a was extended to allow for multiple short contacts between B and T cells and to explicitly represent ICOSL dynamics in B cells (see text for details). Characteristics of germinal centre reactions in simulations with fast (black, grey) and slow (colours) ICOSL upregulation. All tested variants (see legend box and text for details on the quantities) exhibit reduced and retarded output production while keeping germinal centre B-cell affinity unchanged. Output affinity is enhanced in a subset of settings. Mean (full lines) and s.d. (shades) of 100 simulations. c, The simulations in b were repeated using the classic textbook recycling model, with 80% of the selected B cells doing recycling and 20% of the selected B cells differentiating to output cells42. This replaced the LEDA model in b. The simulations with short search periods for TFH help were repeated. Note that the overall output production is smaller in the classic recycling model43. The relative reduction of output in simulations with slow ICOSL upregulation is unchanged. Mean (full lines) and s.d. (shades) of 100 simulations.

  16. Dopamine derivative structure.
    Extended Data Fig. 10: Dopamine derivative structure.

    Chemical structure of dopamine derivative after sample reconstitution with trifluoroacetic anhydride (TFAA) and trifluoroethanol (TFE).

Videos

  1. Live cell in vitro imaging
    Video 1: Live cell in vitro imaging
    FSK-treated TFH cells (blue), untreated TFH cells (green) and allogeneic GC B cells (red) were mixed together with a 1:2=T:B ratio and visualised for at least 30 minutes (See Methods for more details).

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Author information

  1. These authors contributed equally to this work.

    • David Saliba &
    • Maurilio Ponzoni

Affiliations

  1. Department of Immunology and Infectious Disease, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia

    • Ilenia Papa,
    • Pablo F. Canete,
    • Paula Gonzalez-Figueroa,
    • Hayley A. McNamara,
    • Rebecca A. Sweet,
    • Ian A. Cockburn &
    • Carola G. Vinuesa
  2. Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7LD, UK

    • David Saliba,
    • Salvatore Valvo &
    • Michael L. Dustin
  3. Ateneo Vita-Salute, Department of Pathology, IRCCS Scientific Institute San Raffaele, Milan 20132, Italy

    • Maurilio Ponzoni &
    • Claudio Doglioni
  4. Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, University of New South Wales, Sydney, New South Wales 2052, Australia

    • Sonia Bustamante
  5. Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, South Australia 5000, Australia

    • Michele Grimbaldeston
  6. OMNI-Biomarker Development, Genentech Inc., South San Francisco, California 94080, USA

    • Michele Grimbaldeston
  7. Imaging and Cytometry Facility, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia

    • Harpreet Vohra
  8. Department of Systems Immunology and Braunschweig Integrated Centre of Systems Biology, Helmholtz Centre for Infection Research, Braunschweig 38124, Germany

    • Michael Meyer-Hermann
  9. China-Australia Centre for Personalised Immunology, Shanghai Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200085, China

    • Carola G. Vinuesa

Contributions

C.D. and C.G.V. contributed equally to this work. I.P. performed most of the experiments and analysed the data. P.C., P.G., and H.V. helped with the experiments. M.P. contributed to data analysis. D.S. and S.V. performed SLB experiments and contributed to interpretation together with M.L.D. S.B. performed GC/MS/MS experiments. M.M.-H. performed in silico modelling. H.M. performed two-photon experiments and contributed to data analysis together with I.C. M.G., M.L.D., M.M.-H., M.P., and R.A.S. provided intellectual input, expertise, and reading of the manuscript. I.P. and C.G.V. wrote the manuscript. C.G.V. supervised the project with D.C.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Reviewer Information Nature thanks S. Crotty, J. Cyster, H. Qi, and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: CgB+ cells in human germinal centre. (941 KB)

    a, b, Representative immunohistochemistry for CgB (brown) of human lymph node (a) and spleen (b) (n = 10). c, Quantification of CD3+CgB+ cells in human tonsils, lymph nodes (n = 10), and spleens (n = 5). d, Percentage of CgB+ T cells in human reactive and neoplastic conditions. c, d, NS, not significant; *P ≤ 0.05 and **P ≤ 0.01; non-parametric Mann–Whitney U-test. e, Representative double immunohistochemistry for CgB (left) and CD3 (middle) after colour deconvolution. Pseudo-colour image (right) showing signal co-localization. Original magnification ×40. Scale bar, 100 μm (n = 3). f, Representative immunofluorescence images for CD3 (green) and ICOS (red) in human germinal centres.

  2. Extended Data Figure 2: Mouse CgB expression. (1,200 KB)

    ai, Immunohistochemistry staining shows no CgB reactivity in mouse germinal centres of immunized wild-type (WT) or Sanroque spleens and Peyer’s patches (n = 3). j, Immunohistochemistry control staining for CgB in mouse pancreas islets. aj, Scale bar, 100 μm; n = 3. k, Relative mouse Chgb mRNA expression in different T-cell subsets with adrenal gland as positive control. T cells were FACS sorted as follows: Tnaive (CD4+CD44loCD25); T effector memory (TEM, CD4+CD44hiCD25PD-1−/lo CXCR5−/lo); TFH (CD4+CD44hiPD-1hiCXCR5hi); Treg (CD4+CD25+CD44int). Gapdh was used as the housekeeping gene (n = 3).

  3. Extended Data Figure 3: Dopamine β-hydroxylase expression in human and mouse lymphocytes. (720 KB)

    a, Gel shows PCR products after amplification of human DBH mRNA in T cells, total tonsil, and B cells. GSα (also known as GNAS) was used as the housekeeping gene. For gel source data, see Supplementary Fig. 1. b, RNA sequencing showing expression of DBH mRNA in human Tnaive, TFH, and TFR cells extracted from three tonsils, expressed as counts per million. c, Immunofluorescence images showing green fluorescent protein (GFP) expression in adrenal medulla of DBHgfp/w mice. d, FACS plot showing GFP expression in splenocytes of DBHgfp/w mice. e, Quantification of DBH–GFP expression in mouse splenocytes. Bars, median values; each dot represents a mouse (n = 10). f, FACS plot showing DBH–GFP expression in B cells localizing outside germinal centres of mice immunized with sheep red blood cells (n = 10).

  4. Extended Data Figure 4: Mouse endogenous and induced dopamine content. (171 KB)

    a, b, Quantification and representative FACS plot of dopamine content in mouse naive and follicular T (TFO) cells differentiated by the expression of IL-21. T-cell subsets were FACS sorted into Tnaive (CD4+CD44lo), TFO IL-21+ (CD4+CD44hiIL-21gfp/w), and TFO IL-21 (CD4+CD44hiIL-21w/w), and dopamine content was analysed by flow cytometry before and after treatment with FSK for 24 h. Bars, median values; each dot represents a mouse (n = 5). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; non-parametric Mann–Whitney U-test.

  5. Extended Data Figure 5: Dopamine release from human TFH cells. (160 KB)

    a, Dopamine release from TFH cells after stimulation for 30 min with autologous B cells (1:2) alone or with anti-CD3/CD28 beads (1:1). TFH cells were pre-stimulated with FSK before inducing dopamine release. Bars, median; each dot represent a single experiment conducted in triplicates (n = 4). b, Dopamine release from TFH cells after stimulation for 30 min with allogeneic germinal centre B cells (1:2) alone or in the presence of ICOSL blocking antibody (10 μg ml−1). TFH cells were pre-stimulated with FSK before inducing dopamine release and B cells were pre-stimulated with 10 μM dopamine to increase ICOSL surface levels before incubation with TFH cells. Bar, median of dopamine level in TFH cells (n = 3); each triangle is the allogeneic B cells from a single donor paired with its control (square, n = 11). *P ≤ 0.05; paired t-test.

  6. Extended Data Figure 6: Dopamine receptor (DRD) expression in human B-cell subsets. (333 KB)

    a, Relative expression of DRD mRNA in human B-cell subsets normalized to naive B cells. B2M was used as the housekeeping gene (n = 3). Error bars, s.d. b, c, Representative images of DRD1+ cell (green) localization in human germinal centre (dashed line), showing close proximity to CgB+ (b) or CD3+ (c) cells (red) (n = 3).

  7. Extended Data Figure 7: Regulation of ICOSL upregulation in mouse and human B cells. (225 KB)

    a, Fold changes of surface ICOSL expression on mouse germinal centre B cells treated with anti-CD40 (10 μg ml−1) and dopamine (0.5, 1, 5, 10 μM) for 30 min, with medium control set as unit 1 (n = 5). b, Representative histogram and quantification of surface and intracellular ICOSL on germinal centre and non-germinal centre B cells (n = 5). **P ≤ 0.01; non-parametric Mann–Whitney U-test. c, RNA counts per million of ICOSL, CD40, BCL6, IL-21R, CD86, BAFFR, and FAS mRNA in human memory B cells stimulated with or without dopamine (5 μM) for 2 h (n = 3). d, Fold changes of surface ICOSL expression on mouse germinal centre B cells treated with cycloheximide (CHX, 10 μg ml−1) for 4 h, with medium control set as unit 1. Bars, median values; each dot represents a single mouse. e, Fold changes of surface ICOSL expression on mouse germinal centre B cells stimulated with BAFF (100 ng ml−1), lipopolysaccharides (1 or 10 μg ml−1), anti-CD40 (10 μg ml−1), and anti-IgM (1 or 10 μg ml−1) for 30 min and 4 h. Unit 1 set on medium control. f, Fold changes of surface ICOSL expression on mouse germinal centre B cells treated with actinomycin D (ActD, 5 μg ml−1), anti-CD40 (10 μg ml−1) for 4 h, with medium control set as unit 1. Bars, median; each dot represents a single mouse (n = 5). df, NS, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; two-tailed Student’s t-test. g, Representative histogram of surface ICOSL expression on human germinal centre B cells stimulated with dopamine (10 μM) or anti-CD40 (1 μg ml−1) for 30 min. h, Fold changes of surface ICOSL expression on human germinal centre B cells stimulated with several concentrations of anti-CD40 for 4 h and 8 h, with medium control set as unit 1 (n = 3). i, Survival of germinal centre B cells in the presence of anti-CD40 (1 μg ml−1) after stimulation for 4 h or 8 h (n = 4). *P ≤ 0.05, ***P ≤ 0.001; non-parametric Mann–Whitney U-test.

  8. Extended Data Figure 8: Effect of ICOSL on CD40L presentation and reception in SLB model for TFH–germinal-centre B-cell interaction. (798 KB)

    a, Activated human T cells expressing ICOS and CD40L were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3) as a basal condition with a ring of ICAM-1 surrounding a central cluster in T-cell antigen receptor (TCR)-enriched extracellular vesicles for 15 min (ref. 26). This condition resulted in low presentation of CD40L in punctate structures detected by anti-CD40L monoclonal antibody that accumulated in the same central synapse as the TCR-enriched extracellular vesicles. Addition of ICOSL to the SLB resulted in strong central accumulation of fluorescent ICOSL with the TCR-enriched extracellular vesicles, but no increase in CD40L presentation. Addition of CD40 to the SLB resulted in a significant increase in CD40L accumulation, which we refer to as reception because it is receptor dependent. When ICOSL and CD40 were added, the reception of CD40L was further significantly enhanced over the level observed with CD40 alone. Thus, ICOSL ligation in the centre of the immunological synapse increases CD40L reception. All levels are shown in grey scale except CD40L panels, for which the pseudo-colour scale is indicated. Scale bar, 5 μm. b, Human TFH cells were incubated with SLB containing ICAM-1 and UCHT1 (anti-CD3). Addition of ICOSL resulted in increased accumulation of CgB at the synapse centre. Addition of CD40 did not further increase CgB accumulation.

  9. Extended Data Figure 9: Effect of ICOSL upregulation speed in the published and extended germinal centre LEDA and classic recycling models. (619 KB)

    a, Characteristics of germinal centre reactions in simulations with short (black) and long (colours) search phases for TFH help using the previously published LEDA model (see text). All tested variants (see legend box and text for details on the quantities) exhibit reduced and retarded output production while keeping affinity maturation unchanged. Mean (full lines) and s.d. (shades) of 100 simulations. b, The LEDA model in a was extended to allow for multiple short contacts between B and T cells and to explicitly represent ICOSL dynamics in B cells (see text for details). Characteristics of germinal centre reactions in simulations with fast (black, grey) and slow (colours) ICOSL upregulation. All tested variants (see legend box and text for details on the quantities) exhibit reduced and retarded output production while keeping germinal centre B-cell affinity unchanged. Output affinity is enhanced in a subset of settings. Mean (full lines) and s.d. (shades) of 100 simulations. c, The simulations in b were repeated using the classic textbook recycling model, with 80% of the selected B cells doing recycling and 20% of the selected B cells differentiating to output cells42. This replaced the LEDA model in b. The simulations with short search periods for TFH help were repeated. Note that the overall output production is smaller in the classic recycling model43. The relative reduction of output in simulations with slow ICOSL upregulation is unchanged. Mean (full lines) and s.d. (shades) of 100 simulations.

  10. Extended Data Figure 10: Dopamine derivative structure. (166 KB)

    Chemical structure of dopamine derivative after sample reconstitution with trifluoroacetic anhydride (TFAA) and trifluoroethanol (TFE).

Supplementary information

Video

  1. Video 1: Live cell in vitro imaging (2.71 MB, Download)
    FSK-treated TFH cells (blue), untreated TFH cells (green) and allogeneic GC B cells (red) were mixed together with a 1:2=T:B ratio and visualised for at least 30 minutes (See Methods for more details).

PDF files

  1. Supplementary Information (328 KB)

    This file contains Supplementary Figure 1 (the uncropped gels) and Supplementary Tables 1-2.

Additional data