Follicular regulatory T cells repress cytokine production by follicular helper T cells and optimize IgG responses in mice

Abstract

Follicular helper T (Tfh) cells provide crucial help to germinal center B (GCB) cells for proper antibody production, and a specialized subset of regulatory T cells, follicular regulatory T (Tfr) cells, modulate this process. However, Tfr-cell function in the GC is not well understood. Here, we define Tfr cells as a CD4(+) Foxp3(+) CXCR5(hi) PD-1(hi) CD25(low) TIGIT(high) T-cell population. Furthermore, we have used a novel mouse model ("Bcl6FC") to delete the Bcl6 gene in Foxp3(+) T cells and thus specifically deplete Tfr cells. Following immunization, Bcl6FC mice develop normal Tfh- and GCB-cell populations. However, Bcl6FC mice produce altered antigen-specific antibody responses, with reduced titers of IgG and significantly increased IgA. Bcl6FC mice also developed IgG antibodies with significantly decreased avidity to antigen in an HIV-1 gp120 "prime-boost" vaccine model. In an autoimmune lupus model, we observed strongly elevated anti-DNA IgA titers in Bcl6FC mice. Additionally, Tfh cells from Bcl6FC mice consistently produce higher levels of Interferon-γ, IL-10 and IL-21. Loss of Tfr cells therefore leads to highly abnormal Tfh-cell and GCB-cell responses. Overall, our study has uncovered unique regulatory roles for Tfr cells in the GC response.

Keywords: Antibody; Bcl6; Follicular T cells; Germinal Center Response; Regulatory T cells.

Figure 1. Bcl6, CD25 and TIGIT levels in various conventional and regulatory T cell subsets

Splenic CD4+ T cells from WT mice immunized with Sheep Red Blood Cells (SRBC) 7 d before were studied by flow cytometry. (A) Foxp3+ and Foxp3- CD4+ T cells are gated as in the flow plot (left). In Foxp3- conventional CD4+ T (Tconv) cells, Tfh cells, PD-1+TH cells and PD-1- TH cells are defined as CXCR5^hiPD-1^hi, CXCR5^negPD-1⁺ and CXCR5^negPD-1^neg, respectively. In Foxp3+ regulatory CD4+ (Treg) cells, Tfr, PD-1+Treg and PD-1- Treg cells are gated on CXCR5^hiPD-1^hi, CXCR5^-PD-1⁺ and CXCR5^-PD-1^-, respectively (right). Bcl6 (B), CD25 (C) and TIGIT (D) mean fluorescent intensity (MFI) of Tfh, PD-1+TH, PD-1- TH, Tfr, PD-1+Treg and PD-1- Treg cells. Data shown as mean +/− SEM, n = 4-5. *p < 0.05, **p < 0.01, *** p < 0.001 (ANOVA). Data are representative of two independent experiments with similar results.

Figure 2. Loss of Tfr cells in Bcl6FC mice does not affect the size of Tfh cell population following SRBC immunization

Control and Bcl6FC KO mice were immunized with SRBC by i.p. injection. 10 day post immunization (dpi), spleens were isolated for flow cytometric analysis. (A) Tfr cells are defined as Foxp3+CXCR5^hiPD-1^hi. (B) Tfr cell percentage in CD4+Foxp3+ T cells and Tfr cell number. (C) Tfh cells are defined as Foxp3-CXCR5^hiPD-1^hi. (D) Tfh cell percentage in CD4+Foxp3- T cells and Tfh cell number. Data shown as mean +/− SEM, n = 11. ** p < 0.01 (student t test). Data are pooled from three independent experiments.

Figure 3. Tfr cells do not regulate the size of GCB cell population, but contribute to the generation of antigen specific IgG antibodies by restraining antigen specific IgA antibodies

Control and Bcl6FC KO mice were immunized with SRBC by i.p. injection. Spleens were isolated for flow cytometric analysis at 10 dpi. (A) GCB cells are defined as B220+CD38⁻ GL7⁺ in flow plot. (B) GCB cell percentage in B220+ cells and cell number. Serum samples from control and Bcl6FC mice were also collected. (C) Anti-SRBC IgG, (D) anti-SRBC IgA titers. The X-axis shows the dilution factors. (E, F) Control and Bcl6FC mice were immunized with NP-KLH in Alum. Serum samples were collected at 25 dpi. (E) Anti-NPKLH IgG, (F) anti-NPKLH IgA titers. The X-axis shows the dilution factors. Graphs show mean +/− SEM, (A-B) n = 11, (C-D) n = 4, (E-F) n = 4-6. **p < 0.01, *** p < 0.001 (student t test for B, two-way ANOVA for C-F). For A-B, data are pooled from three independent experiments. For C-F, data are representative of three independent experiments with similar results.

Figure 4. Decreased antibody avidity in the absence of Tfr cells in the DNA prime-protein boost gp120 vaccine model

(A) Experimental setup of gp120 DNA prime and protein boost immunization. Control and Bcl6FC mice were primed i.m. with gp120-encoding DNA 3 times, 2 wk apart. 4 wk later they were given 2 booster injections of gp120 protein, 2 wk apart. Spleen and serum samples from control and Bcl6FC mice were collected one week after the final booster. (B) Serum anti-gp120 specific IgG titers measured by ELISA and (C) avidity of gp120- specific IgG antibodies measured by NaSCN displacement method are shown. Data shown as mean +/− SEM, n = 9-11. **p < 0.01 (student t test). Data are pooled from two independent experiments.

Figure 5. Tfr cells regulate the induction of anti-dsDNA IgA antibodies in Pristane-induced lupus model

Four months after single 0.5 ml pristane i.p. injection, serum samples were collected from control and Bcl6FC mice for anti-dsDNA antibody detection by ELISA. (A) Anti-dsDNA IgM, (B) anti-dsDNA IgG and (C) anti-dsDNA IgA titers are shown. The X-axis labels are dilution factors. Data shown as mean +/− SEM, n = 6-7. *p < 0.05, **p < 0.01, *** p < 0.001 (two-way ANOVA). Data are pooled from two independent experiments.

Figure 6. Tfr cells regulate IFN-γ, IL-10 and IL-21 production in Tfh cells

(A) PD-1^hi, PD-1^int and PD-1^neg CD4+ T cells gates. Tfh cells are defined as CD4+PD-1^hi cells. Percentages of cytokine-producing (IFN- γ, IL-4, IL-10 and IL-21) in CD4+PD-1^hiTfh (B), CD4+PD-1^intT (C) and CD4+PD-1^negT cells (D) from control and Bcl6FC mice 10 days after SRBC immunization measured by ICS are shown. Data shown as mean +/− SEM, n = 11. *p < 0.05, *** p < 0.001 (student t test). Data are pooled from three independent experiments.