TFH cells progressively differentiate to regulate the germinal center response

Abstract

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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Figure 1: IL-21–Kat+IL-4–GFP TFH cells transition to IL-21–KatIL-4–GFP+ TFH cells following infection of Il21Kat/KatIl4GFP/GFP mice with N. brasiliensis.
Figure 2: Expression of Il21 and Il4 defines transcriptionally distinct populations of TFH cells.
Figure 3: TFH21 and TFH4 cells localize in the GC differentially, with distinct expression of CXCR4 and CD40L.
Figure 4: TFH cells modulate cytokine production after helminth infection.
Figure 5: TFH cells transferred to Stg recipients 5 d after N. brasiliensis infection of the donor mice become TFH4 cells after re-infection of the host mice.
Figure 6: Distinct TFH cell populations regulate the GC response differentially.
Figure 7: TFH21 cells promote the development of GC B cells of higher affinity than those generated by TFH4 cells.

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Acknowledgements

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.).

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Contributions

J.S.W. and E.I.H. designed and performed experiments, and wrote the manuscript; B.L. designed and constructed the Il21Kat/Kat mouse and performed experiments; P.L.-L. contributed to the design of experiments and assisted with N. brasiliensis infections; R.F. and E.E. assisted with the design and construction of the Il21Kat/Kat mouse and helped to write the manuscript; and J.C. helped design experiments and wrote the manuscript.

Corresponding author

Correspondence to Joe Craft.

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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.

Supplementary Figure 3 Cell-division-dependent expression of IL-21–Kat and IL-4GFP in TFH cells.

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-21Kat and IL-4GFP 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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