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.
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Extended data figures and tables
Extended Data Figures
- 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.
- Extended Data Figure 2: Mouse CgB expression. (1,200 KB)
a–i, 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. a–j, 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+CD44hiCD25−PD-1−/lo CXCR5−/lo); TFH (CD4+CD44hiPD-1hiCXCR5hi); Treg (CD4+CD25+CD44int). Gapdh was used as the housekeeping gene (n = 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 TFΗ 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).
- 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.
- 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.
- 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).
- 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). d–f, 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.
- 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.
- 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.
- 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 (328 KB)
This file contains Supplementary Figure 1 (the uncropped gels) and Supplementary Tables 1-2.