Deficiency in T follicular regulatory cells promotes autoimmunity

Abstract

T follicular regulatory (Tfr) cells are a new subset of regulatory T (T reg) cells localized in the germinal center to limit the humoral response. Until now, the physiological function of Tfr cells has been largely unknown. In this study, we developed a Bcl6^fl/flFoxp3Cre mouse to analyze the function of Tfr cells in immune and autoimmune responses. These mice exhibited enhanced immunity to influenza virus; moreover, Bcl6^fl/flFoxp3Cre/Cre mice developed late-onset spontaneous autoimmune diseases, affecting the salivary glands with lymphocyte infiltration and antibody deposition. In a mouse experimental Sjögren's syndrome model, ablation of Bcl6 in T reg cells greatly enhanced disease development. Conversely, Bcl6^fl/flCd4Cre mice were protected in the model. Thus, our study indicates that Tfr cells control autoimmune diseases and can be targeted in infectious and autoimmune disease.

Figure 1.

Loss of Bcl6 in T reg cells enhances protection to influenza virus infection. (A) Body weights of control and KO mice were monitored daily after infection. n = 5 or 7 per group. (B) Mice were sacrificed at day 9 after infection, and viral titers in the lungs were assessed by quantitative RT-PCR measurement of active HA gene expression. n = 4. (C) FACS staining (left), frequency quantitation (middle), and cell number (right) analysis of CXCR5⁺Bcl6⁺ Tfr cells in CD4⁺Foxp3⁺ cells in lung dLN from influenza-infected control and KO mice. n = 5–6 per group. (D and E) FACS staining (left), frequency quantitation (middle), and cell number (right) analysis of CXCR5⁺Bcl6⁺ Tfh cells in CD4⁺Foxp3⁻ cells (D) and GL-7⁺Fas⁺ GC B cells in B220⁺ cells (E) in lung dLNs from infected control and KO mice. n = 5 or 7 per group in D; 4 or 5 per group in E. (F and G) After restimulation with PMA and ionomycin for 5 h, IFN-γ, IL-4, and IL-17A expression in CD4⁺ T cells (F) and FACS staining (top row) and frequency quantitation (bottom row) of IFN-γ⁺ cells in CD4⁺Foxp3^-Bcl6⁺ cells (G) in lung dLNs from the influenza-infected control and KO mice were measured by flow cytometric analysis. n = 5­–7 per group. (H) At day 9 after infection, sera levels of Ig specific for virus were measured by ELISA. n = 4–7 per group. Bcl6^fl/flFoxp3Cre/Cre mice were KO, and Bcl6^fl/flFoxp3WT/WT mice from the Bcl6^fl/flFoxp3Cre/WTxBcl6^fl/flFoxp3WT breeder were used as control. All data are representative of two independent experiments. Data shown are mean ± SEM; two-tailed t test; p-values in A and G were analyzed by two-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., no significance.

Figure 2.

Loss of Bcl6 in T reg cells leads to humoral autoimmunity. (A) Histopathology analysis of SG, lung, and pancreas from WT and KO mice at 30 wk of age. Black arrows indicate the immune cell infiltrates in KO mice. Bars, 100 µm. n = 6 per group. (B) Anti-IgG immunofluorescent staining of SG and kidney. WT, n = 3; KO, n = 6 per group. Bars, 50 µm. (C) Anti-dsDNA autoantibodies in the sera of WT and KO mice were measured by ELISA. n = 9 per group. (D) Saliva flow rates were measured in WT and KO mice. n = 5–6 per group. (E) FACS staining (left), frequency quantitation (middle), and cell number (right) analysis of CXCR5⁺Bcl6⁺ Tfr cells in CD4⁺Foxp3⁺ cells in different organs from the old steady-state control and KO mice. n = 6 per group. (F) FACS staining (left), frequency quantification (middle), and cell number (right) analysis of Bcl6⁺CXCR5⁺ Tfh cells in CD4⁺Foxp3⁻ T cells from different organs of WT and KO mice. (G) FACS staining (left), frequency quantification (middle), and cell number (right) analysis of GL7⁺Fas⁺ GC B cells in B220⁺ cells from different organs of WT and KO mice. (H–J) After restimulation with PMA and ionomycin for 5 h, IFN-γ (H), IL-4 (I), and IL-17A (J) expression in CD4⁺ T cells from different organs was measured by flow cytometric analysis. All mice were 30-wk-old unmanipulated mice. n = 6 per group in F–J. SP, spleen. All experimental data were verified in at least two independent experiments. Bcl6^fl/flFoxp3Cre/Cre mice were KO, and Bcl6^fl/flFoxp3WT/WT mice from the Bcl6^fl/flFoxp3Cre/WTxBcl6^fl/flFoxp3WT breeder were used as control. Data shown are mean ± SEM; two-tailed t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., no significance.

Figure 3.

Ablation of Bcl6 in T reg cells enhances ESS development. (A) ESS model was set up and analyzed after 15 wk. The kinetics of the saliva flow rates were measured during the 15 wk after SG protein immunization (ESS) in both WT and KO mice and adjuvant immunization (control). n = 5–10 per group. *, statistics analysis between control and ESS-WT group; ^#, analysis between ESS-WT and ESS-KO group. (B) IgG autoantibodies against SG antigens were detected in the serum samples from ESS mice and controls 15 wk after first immunization by ELISA. n = 4–5 per group. (C) Histological evaluation of glandular destruction in WT and KO mice after ESS induction was performed on tissue sections of submandibular glands with H&E staining. Bars, 100 µm. (D) Infiltrated lymphocytes in the SGs were assessed for histological scores. WT, n = 12; KO, n = 16. (E) Anti-IgG immunofluorescent staining of SGs. WT, n = 4; KO, n = 6. Bars, 50 µm. (F) Histological evaluation of glandular destruction in WT and KO mice at 5 wk after ESS induction was performed on tissue sections of submandibular glands with H&E staining. Bars, 100 µm. n = 5 per group. (G–I) FACS staining (left), frequency quantification (middle), and cell number (right) analysis of CXCR5⁺PD1⁺ Tfr cells in CD4⁺Foxp3⁺ cells (G), CXCR5⁺PD1⁺ Tfh cells in CD4⁺Foxp3⁻ cells (H), and Fas⁺GL7⁺ GCB in B220⁺ cells (I) in CLNs of WT and KO mice at 15 wk after first immunization. n = 9 or 13. (J and K) After restimulation with PMA and ionomycin for 5 h, FACS staining (left), frequencies (middle), and cell number (right) analysis of IFN-γ and IL-17-producing Th1 (J) and Th17 cells (K) among CD4⁺ T cells in CLN of WT and KO mice was performed by flow cytometry. n = 9 or 17 in J and K. (L and M) Serum IFN-γ (L) and IL-17 (M) levels were detected by ELISA. WT, n = 3 or 4; KO, n = 9 or 11. Data in G–K were pooled from two independent experiments. All experimental data were verified in at least two independent experiments. Bcl6^fl/flFoxp3Cre/Cre mice were KO, and Bcl6^fl/flFoxp3WT/WT mice from the Bcl6^fl/flFoxp3Cre/WTxBcl6^fl/flFoxp3WT breeder were used as control. Data shown are mean ± SEM; two-tailed t test; p-values in B were analyzed by two-way ANOVA; * or ^#, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., no significance.

Figure 4.

GC response plays a critical role in the development of ESS. (A) Saliva flow rates were measured 15 wk after SG protein-immunization (ESS) in both WT and KO mice and adjuvant immunization (control). n = 3 or 5. (B) Autoantibodies against SG antigens were detected in the serum samples from ESS WT and KO mice and controls 15 wk after first immunization by ELISA. n = 3 or 5. (C) Histological evaluation of glandular destruction in WT and KO mice was performed on tissue sections of submandibular glands with H&E staining. Bars, 100 µm. (D) Infiltrated lymphocytes in the SGs were assessed for histological scores. n = 11. (E) Anti-IgG immunofluorescent staining of SG. Bars, 50 µm. n = 3 or 4 per group. (F and G) FACS staining (left) and frequency quantification (right) of the CXCR5⁺PD1⁺ Tfh cells among CD4⁺Foxp3⁻ (F) and Fas⁺GL7⁺ GCB cells among B220⁺ cells (G) in CLN of WT and KO mice immunized for ESS induction at 15 wk after first immunization. n = 7–9 per group. (H and I) After restimulation with PMA and ionomycin for 5 h, frequencies of IFN-γ- and IL-17–producing Th1 (H) and Th17 (I) cells among the CD4⁺ T cells in the CLN of WT and KO mice were examined by flow cytometry analysis. n = 10–16 per group. (J and K) The serum IFN-γ (J) and IL-17 (K) levels were detected by ELISA. n = 5 or 7 per group. Data in D and F–I were pooled from two independent experiments. All experimental data were verified in at least two independent experiments. Bcl6^fl/flCd4^Cre were KO, and Bcl6^fl/fl littermates from the Bcl6^fl/flCd4^Cre were used as control. Data shown are mean ± SEM; two-tailed t test; p-values in B were analyzed by two-way ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., no significance.