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.

Figure 1

Il21-Kat⁺Il4-GFP⁻ T_FH cells transition to Il21-Kat⁻Il4-GFP⁺ T_FH cells upon N. brasiliensis infection of Il21^Kat/+Il4^GFP/+ mice. (a) Flow cytometry of CD4⁺CD44^hi splenocytes from Il21^Kat/+Il4^GFP/+ mice, showing CXCR5^hiPD-1^hi T_FH (green), CXCR5^intPD-1^int (blue), and CXCR5^loPD-1^lo T_H2 (red) cells. (b) Flow cytometry of GFP (Il4) and Kat (Il21) reporter-expressing cells, gated as a. (c) Quantification of T_FH cells. (d) Confocal microscopy of GCs from Il21^Kat/+Il4^GFP/+ mice 7 days post-infection, stained with anti-GFP (green), -CD4 (blue), -Turbo635 (red), and -IgD (white) (left 2 images); anti-GFP (green), -CD4 (blue), and -Turbo635 (red) (third image from left); and anti-CD4 (blue) and -Turbo635 (red) (far right image). Green, yellow, and red arrows indicate Il21⁻Il4⁺ (Kat⁻GFP⁺), Il21⁺Il4⁺ (Kat⁺GFP⁺), and Il21⁺Il4⁻ (Kat⁺GFP⁻) CD4⁺ cells, respectively. (e) Flow cytometry of CellTrace Violet labeled donor CD4⁺Thy1.2⁺ Il21^Kat/+Il4^GFP/+ OT-II cells in Thy1.1 recipient spleens at 5 days post-infection and NP-OVA administration. Histogram showing dividing (shaded) and undivided (open; unimmunized control) donor cells (upper left) and summary of fraction of cytokine⁺ CXCR5⁺ cells among total T_FH cells (lower left). (f) Dual-color ELISPOTs of sorted reporter⁺ T_FH cells 8 days post-infection (as in b). Representative ELISPOTs (left) and total number of counted IL-21⁺ (blue), IL-4⁺ (red), and IL-21⁺IL-4⁺ spots (purple) for each T_FH cell population (right). *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 representative of three independent experiments with similar results, with three or four (a,b), three or five (c), or three (f) mice per group.

Figure 2

Il21 and Il4 expression define transcriptionally distinct populations of T_FH cells. (a) Heatmap of significantly (q < 0.05) differentially expressed genes (rows) in the four populations of cytokine expressing T_FH cells (columns). Light blue: cluster 1; magenta: cluster 2; light brown: cluster 3; green: cluster 4. (b) Heatmap of selected T cell-related genes. (c) Quantitative RT-PCR validation of selected genes, normalized to Hprt. (d) Flow cytometric quantification of geometric mean fluorescence intensity (gMFI) for CXCR5 and PD-1 expression from each T_FH cell population. * q < 0.05 (False discovery rate (FDR)-adjusted, comparing T_FH21 and T_FH4 cells, b) and *P < 0.05 (c, d); **P < 0.01; ***P < 0.001 (Student's t-test, c-d). Error bars represent SEM. FPKM: Fragments Per Kilobase of transcript per Million mapped reads. Analysis utilizes data from two independent experiment with 25 and 30 mice each (a-b), is representative of two independent (of each other and of a-b) experiments performed in technical triplicates with 15 and 20 mice each (c), or is from one experiment representative of three independent experiments with four mice per group (d).

Figure 3

T_FH21 and T_FH4 cells differentially localize in the GC with distinctive expression of CXCR4 and CD40L. (a) Flow cytometric quantification of CXCR4 expression in T_FH cell populations and naïve CD4⁺ T cells. (b) Confocal microscopy of splenic GCs from Il21^Kat/+Il4^GFP/+ mice 10 days post-infection with N. brasiliensis, stained with anti-IgD (white), -CD35 (blue), -Turbo635 (red) and -GFP (green). (c) Migration assay measuring ratio of migrating to total T_FH cell subsets from Il21^Kat/+Il4^GFP/+ mice 8 days post-infection, co-cultured with CXCL12, CXCL13, or media, (d) Flow cytometric quantification of CD40L in T_FH cell subsets. *P< 0.05, **P < 0.01; ***P < 0.001 (Student's t-test, a,c,d). Error bars represent SEM. Data are from one experiment representative of three independent experiments (b-d), with seven (a), four (b), six (c), or eight (d) mice per group.

Figure 4

T_FH cells modulate cytokine production after helminth infection. Flow cytometry (a , b) and dual-color IL-21 and IL-4 ELISPOT assays (c) of donor Thy1.2⁺ T_FH cells from Il21^Kat/+Il4^GFP/+ mice at 8 days post-infection with N. brasiliensis, transferred into Thy1.1⁺ Stg recipient mice and re-infected. *P < 0.05 **P < 0.01 (Student's t-test, b,c). Error bars represent SEM. N.D.: not detected. Data are from one experiment representative of three independent experiments, with three mice per group.

Figure 5

T_FH cells transferred to Stg recipients five days after N. brasiliensis infection primarily become T_FH4 cells upon reinfection. (a, b) Flow cytometry of splenic donor Thy1.2⁺ T_FH cells at 5 days after infection of Thy1.2⁺ (Thy1.1⁻) Il21^Kat/+Il4^GFP/+ mice, before transfer into Thy1.1⁺ Stg recipients. (c) Flow cytometry of donor CD4⁺Thy1.1⁻ T_FH cells at 13 days after secondary infection of Thy1.1⁺ Stg recipients. (d) Flow cytometry measuring GFP (Il4) and Kat (Il21) expression in donor T_FH cells from recipient spleens 13 days following infection (left) with percentages among donor cells (right). **P< 0.01; ***P < 0.001 (Student's t-test, d). Error bars represent SEM. Data are from one experiment representative of three independent experiments, with four mice per group.

Figure 6

Distinct T_FH cell populations differentially regulate the GC response. (a) Flow cytometry of splenic CD4⁻B220⁺IgD^loCD95⁺GL-7⁺ GC B cells in Stg recipients at 13 days after transfer of indicated T_FH cells from mice infected 8 days prior with N. brasiliensis. (b) Confocal microscopy of representative splenic follicles stained with anti-IgD (white) and PNA (blue). (c) Intracellular flow cytometry of IgG1 in GC B cells as in (a). (d) ELISPOT assays of splenic IgG1 (top left) or IgE antibody-secreting cells (ASCs, top right) among sorted B220⁺ B cells as in (a). (e) ELISA of Anti-N. brasiliensis IgG1 (left), IgE (middle), and IgM (right) in sera of recipient mice. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's t-test). Error bars represent SEM. Data are from one experiment representative of three independent experiments, with four mice per group.

Figure 7

T_FH21 cells promote development of higher affinity GC B cells compared to T_FH4 cells. (a) Total mutations (framework and complementary determining regions), and (b) tryptophan (W) to leucine (L) point mutations at position 33, of the V_H186.2 gene in sequences cloned from sorted and pooled GC B cells 13 days after administration of N. brasiliensis and NP-OVA with boosting to mice receiving transferred Il21^Kat/+Il4^GFP/+ OT-II⁺ TCR transgenic T_FH cells (from mice infected 8 days prior with N. brasiliensis and NP-OVA; refer to Supplementary Fig. 7f) (c) ELISPOT assays of low (top) and high (bottom) affinity anti-NP⁺ IgG1⁺, IgE⁺, or IgG2a⁺ ASCs. (e) Intracellular Bcl-6 staining and (f) quantitative RT-PCR for Prdm1 expression in recipient CD4−B220⁺IgD^loCD95⁺GL-7⁺ GC B cells after T_FH-cell transfer and secondary infection (as in a). *P < 0.05; **P < 0.01; ***P < 0.001 (Fisher's exact test (b) and Student's t-test (c-e). Error bars represent SEM. Data are from one experiment representative of three independent experiments, with four mice per group.