Germinal center (GC) B cells undergo affinity selection, which depends on interactions with CD4+ follicular helper T cells (TFH cells). We found that TFH cells progressed through transcriptionally and functionally distinct stages and provided differential signals for GC regulation. They initially localized proximally to mutating B cells, secreted interleukin 21 (IL-21), induced expression of the transcription factor Bcl-6 and selected high-affinity B cell clones. As the GC response evolved, TFH cells extinguished IL-21 production and switched to IL-4 production, showed robust expression of the co-stimulatory molecule CD40L, and promoted the development of antibody-secreting B cells via upregulation of the transcription factor Blimp-1. Thus, TFH cells in the B cell follicle progressively differentiate through stages of localization, cytokine production and surface ligand expression to 'fine tune' the GC reaction.
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King, C., Tangye, S.G. & Mackay, C.R. T follicular helper (TFH) cells in normal and dysregulated immune responses. Annu. Rev. Immunol. 26, 741–766 (2008).
Xu, J. et al. Mice deficient for the CD40 ligand. Immunity 1, 423–431 (1994).
Glatman Zaretsky, A. et al. T follicular helper cells differentiate from Th2 cells in response to helminth antigens. J. Exp. Med. 206, 991–999 (2009).
Zotos, D. et al. IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism. J. Exp. Med. 207, 365–378 (2010).
Linterman, M.A. et al. IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. J. Exp. Med. 207, 353–363 (2010).
Reinhardt, R.L., Liang, H.E. & Locksley, R.M. Cytokine-secreting follicular T cells shape the antibody repertoire. Nat. Immunol. 10, 385–393 (2009).
King, I.L. & Mohrs, M. IL-4-producing CD4+ T cells in reactive lymph nodes during helminth infection are T follicular helper cells. J. Exp. Med. 206, 1001–1007 (2009).
Yusuf, I. et al. Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150). J. Immunol. 185, 190–202 (2010).
Kühn, R., Rajewsky, K. & Müller, W. Generation and analysis of interleukin-4 deficient mice. Science 254, 707–710 (1991).
Fairfax, K.C. et al. IL-4-secreting secondary T follicular helper (Tfh) cells arise from memory T cells, not persisting Tfh cells, through a B cell-dependent mechanism. J. Immunol. 194, 2999–3010 (2015).
Dedeoglu, F., Horwitz, B., Chaudhuri, J., Alt, F.W. & Geha, R.S. Induction of activation-induced cytidine deaminase gene expression by IL-4 and CD40 ligation is dependent on STAT6 and NFκB. Int. Immunol. 16, 395–404 (2004).
Ozaki, K. et al. A critical role for IL-21 in regulating immunoglobulin production. Science 298, 1630–1634 (2002).
McGuire, H.M. et al. IL-21 and IL-4 collaborate to shape T-dependent antibody responses. J. Immunol. 195, 5123–5135 (2015).
Allen, C.D., Okada, T. & Cyster, J.G. Germinal-center organization and cellular dynamics. Immunity 27, 190–202 (2007).
Oprea, M. & Perelson, A.S. Somatic mutation leads to efficient affinity maturation when centrocytes recycle back to centroblasts. J. Immunol. 158, 5155–5162 (1997).
Kallies, A. et al. Plasma cell ontogeny defined by quantitative changes in blimp-1 expression. J. Exp. Med. 200, 967–977 (2004).
Shulman, Z. et al. Dynamic signaling by T follicular helper cells during germinal center B cell selection. Science 345, 1058–1062 (2014).
Xu, H. et al. Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility. Nature 496, 523–527 (2013).
Liu, D. et al. T-B-cell entanglement and ICOSL-driven feed-forward regulation of germinal center reaction. Nature 517, 214–218 (2015).
Gitlin, A.D., Shulman, Z. & Nussenzweig, M.C. Clonal selection in the germinal center by regulated proliferation and hypermutation. Nature 509, 637–640 (2014).
Kumamoto, Y. et al. CD301b+ dermal dendritic cells drive T helper 2 cell-mediated immunity. Immunity 39, 733–743 (2013).
Ozaki, K. et al. Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6. J. Immunol. 173, 5361–5371 (2004).
Ismail, N. & Bretscher, P.A. The Th1/Th2 nature of concurrent immune responses to unrelated antigens can be independent. J. Immunol. 163, 4842–4850 (1999).
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).
Mohrs, M., Shinkai, K., Mohrs, K. & Locksley, R.M. Analysis of type 2 immunity in vivo with a bicistronic IL-4 reporter. Immunity 15, 303–311 (2001).
Shinkai, K., Mohrs, M. & Locksley, R.M. Helper T cells regulate type-2 innate immunity in vivo. Nature 420, 825–829 (2002).
Pepper, M., Pagán, A.J., Igyártó, B.Z., Taylor, J.J. & Jenkins, M.K. Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells. Immunity 35, 583–595 (2011).
Lawrence, R.A., Gray, C.A., Osborne, J. & Maizels, R.M. Nippostrongylus brasiliensis: cytokine responses and nematode expulsion in normal and IL-4-deficient mice. Exp. Parasitol. 84, 65–73 (1996).
Casola, S. & Rajewsky, K. B cell recruitment and selection in mouse GALT germinal centers. Curr. Top. Microbiol. Immunol. 308, 155–171 (2006).
Karlsson, A.C. et al. Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells. J. Immunol. Methods 283, 141–153 (2003).
Eddahri, F. et al. Interleukin-6/STAT3 signaling regulates the ability of naive T cells to acquire B-cell help capacities. Blood 113, 2426–2433 (2009).
Bauquet, A.T. et al. The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells. Nat. Immunol. 10, 167–175 (2009).
Ise, W. et al. The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells. Nat. Immunol. 12, 536–543 (2011).
Liu, X. et al. Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development. Nature 507, 513–518 (2014).
Allen, C.D. et al. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat. Immunol. 5, 943–952 (2004).
Bannard, O. et al. Germinal center centroblasts transition to a centrocyte phenotype according to a timed program and depend on the dark zone for effective selection. Immunity 39, 912–924 (2013).
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).
Bolduc, A. et al. Constitutive CD40L expression on B cells prematurely terminates germinal center response and leads to augmented plasma cell production in T cell areas. J. Immunol. 185, 220–230 (2010).
Lüthje, K. et al. The development and fate of follicular helper T cells defined by an IL-21 reporter mouse. Nat. Immunol. 13, 491–498 (2012).
Jin, H. & Malek, T.R. Redundant and unique regulation of activated mouse B lymphocytes by IL-4 and IL-21. J. Leukoc. Biol. 80, 1416–1423 (2006).
Hömig-Hölzel, C. et al. Constitutive CD40 signaling in B cells selectively activates the noncanonical NF-kappaB pathway and promotes lymphomagenesis. J. Exp. Med. 205, 1317–1329 (2008).
Weiss, U., Zoebelein, R. & Rajewsky, K. Accumulation of somatic mutants in the B cell compartment after primary immunization with a T cell-dependent antigen. Eur. J. Immunol. 22, 511–517 (1992).
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).
Kawabe, T. et al. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity 1, 167–178 (1994).
Basso, K. et al. Tracking CD40 signaling during germinal center development. Blood 104, 4088–4096 (2004).
Saito, M. et al. A signaling pathway mediating downregulation of BCL6 in germinal center B cells is blocked by BCL6 gene alterations in B cell lymphoma. Cancer Cell 12, 280–292 (2007).
Shapiro-Shelef, M. et al. Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells. Immunity 19, 607–620 (2003).
Minnich, M. et al. Multifunctional role of the transcription factor Blimp-1 in coordinating plasma cell differentiation. Nat. Immunol. 17, 331–343 (2016).
Shaffer, A.L. et al. BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Immunity 13, 199–212 (2000).
Oxenius, A., Bachmann, M.F., Zinkernagel, R.M. & Hengartner, H. Virus-specific MHC-class II-restricted TCR-transgenic mice: effects on humoral and cellular immune responses after viral infection. Eur. J. Immunol. 28, 390–400 (1998).
Poholek, A.C. et al. In vivo regulation of Bcl6 and T follicular helper cell development. J. Immunol. 185, 313–326 (2010).
Odegard, J.M. et al. ICOS-dependent extrafollicular helper T cells elicit IgG production via IL-21 in systemic autoimmunity. J. Exp. Med. 205, 2873–2886 (2008).
Beck, T.C., Gomes, A.C., Cyster, J.G. & Pereira, J.P. CXCR4 and a cell-extrinsic mechanism control immature B lymphocyte egress from bone marrow. J. Exp. Med. 211, 2567–2581 (2014).
McHeyzer-Williams, M.G., McLean, M.J., Lalor, P.A. & Nossal, G.J. Antigen-driven B cell differentiation in vivo. J. Exp. Med. 178, 295–307 (1993).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
We thank members of our departments for critical review of the manuscript; J. Henao-Mejia for assistance in generating the IL-21 reporter construct; L. Evangelisti for generating embryonic stem cells; C. Hughes for generating chimeric mice; J. Stein for help with the screening of knock-in mice; E. Nevius and J. Pereira for assistance with migration assays; and S. Kaech (Yale University) for B6.Tg(TcrLCMV)1Aox (Smarta; Stg) mice. Supported by the Arthritis Foundation (J.S.W.), the US National Institutes of Health (5 T32 AR07107 and K01AR067892 to J.S.W.; T32GM07205 and F30HL120497 to E.I.H.; R37AR40072, P30AR053495 and R21AR063942 (NIAMS) to J.C.), the Alliance for Lupus Research (J.C.) and the Howard Hughes Medical Institute (R.A.F.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Cells expressing cells IL-21–KAT reside in the splenic B cell follicle with accumulation in GCs following SRBC immunization.
(a) Serial analysis of splenic CXCR5hiPD-1hi TFH cells from Il21Kat/KatIl4GFP/GFP mice, with percentages compared to CXCR5loPD-1lo non-TFH cells. Both subsets were gated within CD4+CD44hi activated T cells. (b) Representative splenic sections from 4 (top) and 7 days (bottom) following immunization, stained with anti-IgD (green), -CD4 (blue), and -Turbo635 (red), and PNA (white), analyzed by confocal microscopy. In the absence of PNA (center panels), the dotted white line outlines the IgD follicle (right panels), with the number of Il21-Kat expressing T cells shown (far right). (c) Serial sections following immunization, stained with anti-IgD (green), -CD4 (blue), and -Turbo635 (red), and PNA (white) (top panel), or without PNA (bottom panel), with analysis of GCs on the far right (20 GCs/spleen examined). *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t-test). Error bars represent standard error of the mean (SEM). Data are from one experiment of three independent experiments with similar results, with five mice per group.
Supplementary Figure 2 Cytokine-reporter expression in TFH cells in lymph nodes (LNs) following N. brasiliensis infection.
(a, b) Mediastinal (a) and mesenteric (b) LNs were harvested at days 2, 3, 5, 8, and 15 following infection of Il21Kat/KatIl4GFP/GFP mice. Gating as in Fig. 1a. (c) Measurement of CD4-B220+IgDloCD95hiGL-7hi GC B cells from mediastinal and mesenteric LNs, and spleens before infection (0) and at days 8, 12, and 15 post-infection. *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t-test). Error bars represent SEM. Data are from one experiment of two (a,b) or three independent experiments (c) with similar results, with five mice per group.
Flow cytometry of enriched CD4+CD45.2+ Il21Kat/KatIl4GFP/GFP OT-II cells labeled with Cell Trace Violet (CTV) and transferred into CD45.1 recipients. Spleens were harvested 3 days post-infection with N. brasiliensis and administration of NP-OVA. Data are from one experiment of two independent experiments with similar results, with five mice per group.
Supplementary Figure 4 Cytokine expression in TFH cells following N. brasiliensis infection or after protein immunization.
(a) Intracellular IL-21 and IL-4 staining of TFH cells from 0, 5, and 8 days post infection with N. brasiliensis. (b) Cytokine reporter expression by splenic CD4+CD44hiCXCR5hiPD-1hi TFH cells 8 days following immunization of Il21Kat/KatIl4GFP/GFP mice with NP-KLH and alum i.p. (c) Intracellular staining for IL-4 and IL-21, gating on splenic CD4+CD44hiCXCR5hiPD-1hi TFH cells following NP-KLH and alum immunization of wild type B6 mice. IL-21 deficient and unimmunized mice were used as controls for IL-21 staining (a,c). *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t-test). Error bars represent SEM. Data are from one experiment of three independent experiments with similar results, with three mice per group.
Supplementary Figure 5 Expression of IL-21–Kat and IL-4–GFP defines transcriptionally distinct populations of TFH cells.
(a) Strategy for sorting 3 populations of CXCR5hiPD-1hi TFH cells and for GFP+CXCR5loPD-1lo TH2 cells 8 days post N. brasiliensis infection of Il21Kat/KatIl4GFP/GFP mice, with sorting from B220-CD4+CD44hi cells. (b) Quantification of differentially expressed genes between sorted T cell populations. The areas of the ellipses are proportional to the number of differentially expressed genes in Venn diagram (bottom). (c) Expression of differentially expressed genes in the 4 clusters. Each thin line represents a single gene; thick black lines are the median of expression in that cluster. FPKM: Fragments Per Kilobase of transcript per Million mapped reads. RNA-seq data were collected from two independent experiments with 15-20 mice each (a-c), with threshold for significance FDR q-value < 0.05 (b and c). *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t-test). Error bars represent SEM. Analysis utilizes data from two independent experiments with 15 and 20 mice per group.
Supplementary Figure 6 Tracking of adoptively transferred antigen-specific TFH4, TFH21+4 and TFH4 populations.
(a) Sorted TFH populations from Thy1.2+ (Thy1.1-) Il21Kat/KatIl4GFP/GFP mice infected 8 days prior with N. brasiliensis were transferred into Thy1.1+ B6 recipients bearing an irrelevant TCR transgene specific for the GP66-77 epitope of lymphocytic choriomeningitis virus (LCMV; Smarta TCR transgenics; Stg) which were then re-infected; spleens were harvested 13 days after the secondary infection. (b) Normalized measurement of the distance of each of the TFH donor populations to the center of the DZ at 13 days after transfer (~10 GCs/recipient examined) with CXCR4 expression on each transferred subsets in recipient spleens. (c) Flow cytometry of CD4+Thy1.1- CXCR5hiPD-1hi donor TFH cells in recipient spleens. (d) Numbers of CD4+Thy1.1- CXCR5hiPD-1hi donor TFH cells in recipient spleens. (e) Numbers of CD4+Thy1.1- CXCR5loPDlo donor TFH cell number in recipient spleens. (f) Splenic CD4+Thy1.1- CXCR5hiPD-1hi donor TFH cells 7 days after infection of the recipients. (g) Numbers of CD4+Thy1.1- CXCR5loPD-lo non-TFH cells and CXCR5hiPD-1hi TFH cells in spleens, 7 days after infection of transfer recipients. *P < 0.05; **P < 0.01 (Student’s t-test). Error bars represent SEM. Data are from one experiment of two three independent experiments with similar results, with three or four mice per group.
Supplementary Figure 7 TFH cells induce production of class-switched antibodies in recipient mice after adoptive transfer.
(a) TFH cells from IL4-competent Thy1.2+ Il21Kat/KatIl4GFP/GFP or Thy1.2+ Il21Kat/KatIl4GFP/GFP IL4-/- mice infected 8 days prior with N. brasiliensis were transferred into naïve Thy1.1+ Stg B6 recipient mice which were then infected with the same pathogen. Spleens were harvested 13 days after infection of the recipients and numbers of IgG1+ GC B cells were determined by intracellular staining. (b) Total ELISPOT IgG1 (left) and IgE (right) size among splenic B220+ B cells was determined 13 days after secondary infection. (c) Representative flow cytometry plots and quantification of splenic CD4-B220loCD138hi plasma cells from recipient mice. (d) BrdU uptake by B220+IgDloCD95hiGL-7hi GC B cells in recipient mice. (e) Caspase 3 activity in GC B cells in recipient mice. (f) Il21Kat/KatIl4GFP/GFP OT-II+ TFH cells from mice infected 8 days prior with N. brasiliensis and NP-OVA were transferred into congenic Stg recipients, which were then re-infected with N. brasiliensis and challenged with NP-OVA, with GC B cells from recipient spleens analyzed 13 days later. Boosting injections of NP-OVA were given i.v. at the indicated time points. Experiments were performed 2-3 times with 3-5 mice per group *P < 0.05; **P < 0.01 (Student’s t-test). Error bars represent SEM. Data are from one experiment of two three independent experiments with similar results, with three or four mice per group.
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Weinstein, J., Herman, E., Lainez, B. et al. TFH cells progressively differentiate to regulate the germinal center response. Nat Immunol 17, 1197–1205 (2016). https://doi.org/10.1038/ni.3554
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