Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation

Abstract

Effective B cell-mediated immunity and antibody responses often require help from CD4+ T cells. It is thought that a distinct CD4+ effector T cell subset, called T follicular helper cells (T(FH)), provides this help; however, the molecular requirements for T(FH) differentiation are unknown. We found that expression of the transcription factor Bcl6 in CD4+ T cells is both necessary and sufficient for in vivo T(FH) differentiation and T cell help to B cells in mice. In contrast, the transcription factor Blimp-1, an antagonist of Bcl6, inhibits T(FH) differentiation and help, thereby preventing B cell germinal center and antibody responses. These findings demonstrate that T(FH) cells are required for proper B cell responses in vivo and that Bcl6 and Blimp-1 play central but opposing roles in T(FH) differentiation.

Fig. 1

Bcl6 is a T_FH-specific transcription factor. Naïve SMtg CD4⁺ T cells were transferred into B6 mice. In all panels, splenocytes were analyzed 8 days after infection with LCMV. (A and B) SMtg expression of CXCR5 and PD-1 (A) or SLAM (CD150) (B). SMtg⁺ (CD45.1⁺) CD4⁺ gated cells are shown. CXCR5^high T_FH cells are boxed in fluorescence-activated cell sorter (FACS) plots. Histogram overlays depict T_FH cells (red) as well as naïve CD4⁺ T cells (gray) and CXCR5^low non-T_FH SMtg cells (black). Data are representative of more than 10 independent experiments. (C) IL-21 mRNA in SMtg CD4⁺ T cells, normalized to the β-actin mRNA level (×10^–4). **P = 0.008. (D) In vitro chemotaxis toward CXCL13 (BLC) by ex vivo SMtg CD4⁺ T cells. Results are expressed as percentages of SLAM^low T_FH SMtg (solid circles) and SLAM^high non-T_FH SMtg (open circles) that migrated in a transwell assay. ***P ≤ 0.001. 1 μg, P = 0.001; 2 μg, P = 0.0006; 4 μg, P = 0.0006. Data are representative of three independent experiments; n = 2 per group. (E) Scatterplot of the average signal of biological replicates of T_FH versus non-T_FH SMtg gene expression microarray data. Blue lines indicate changes in gene expression by a factor of 3; 386 gene probes exhibited a factor of >3.0 increase in T_FH. Data from one of two independent experiments are shown; n = 2 per group. (F and G) Quantitative reverse transcription PCR of Bcl6 (F) and Blimp-1 (G) mRNA expression, normalized to β-actin (×10^–4). ***P < 0.0001. Data are representative of four independent experiments; n = 2 per group. Error bars in all graphs are SEM.

Fig. 2

Bcl6 expression is sufficient for T_FH differentiation in vivo. (A to E) Naïve SMtg CD4⁺ T cells were transduced with Bcl6-RV (Bcl6⁺) or left untransduced (control) and transferred into B6 mice subsequently infected with LCMV. (A) Gating of CD45.1⁺ untransduced SMtg (GFP^–) and Bcl6-RV⁺ SMtg (GFP⁺) in the same host. CD4⁺ B220^– gate is shown. (B) T_FH (SLAM^low CXCR5^high, boxed) and non-T_FH (SLAM^high CXCR5^low) differentiation of untransduced SMtg (left) and Bcl6-RV⁺ SMtg (right). (C) Quantitation of SMtg T_FH differentiation. Mice received Bcl6-RV⁺ SMtg and untransduced SMtg, or GFP-RV⁺ and untransduced SMtg. “–,” untransduced; “+,” transduced with indicated RV. ***P < 0.0001. Data are representative of three independent experiments; n = 4 per group. (D and E) CXCR5 expression (D) and PD-1 expression (E) on naïve CD4⁺ T cells (gray), GFP-RV⁺ SMtg (black), and Bcl6-RV⁺ SMtg (red). Bar graphs show mean fluorescence intensity (MFI) of naïve CD4⁺ T cells, GFP-RV⁺ SMtg non-T_FH, GFP-RV⁺ SMtg T_FH, and Bcl6-RV⁺ SMtg. For PD-1 MFI, non-T_FH versus T_FH or Bcl6-RV⁺, P < 0.05; T_FH versus Bcl6-RV⁺, P > 0.05. Data are representative of three independent experiments; n = 4 to 6 per group. (F and G) GFP-RV⁺ or Bcl6-RV⁺ SMtg cells were adoptively transferred separately into B cell–deficient mice (μMT) or HEL-specific BCR transgenic mice (MD4) on a μMT background. Host mice were subsequently infected with LCMV. Each group is a composite of three experiments; n = 2 (GFP-RV⁺ μMT), 6 (Bcl6-RV⁺ MD4), 6 (Bcl6-RV⁺ μMT), or 8 (GFP-RV⁺ MD4) per group. (F) Differentiation of SMtg CD4⁺ T cells in MD4 BCR transgenic mice. T_FH cells (SLAM^low CXCR5^high) are boxed. CD4⁺ B220^– CD45.1⁺ GFP⁺ gate is shown. (G) Quantitation of SMtg T_FH differentiation. GFP-RV⁺ versus Bcl6-RV⁺ in MD4, ***P < 0.0001. GFP-RV⁺ versus Bcl6-RV⁺ in μMT, ***P < 0.0001.

Fig. 3

Bcl6 expression is necessary for inducing T_FH B cell help in vivo. (A) Germinal center B cells (PNA⁺ Fas⁺, gated) in mice that received GFP-RV⁺ or Bcl6-RV⁺ SMtg CD4⁺ T cells and were subsequently infected with LCMV, analyzed at day 8 and gated on activated B cells (B220⁺ IgD^low). (B) Frequency of germinal center B cells of total splenocytes; n = 4 per group. Data are representative of three independent experiments. *P = 0.029. (C) GFP-RV⁺ or Bcl6-RV⁺ OT-II CD4⁺ T cells were transferred into B6 mice subsequently immunized with NP-Ova in alum. Control mice were immunized but received no OT-II cells. NP-Ova enzyme-linked immunosorbent assay (ELISA) was performed at day 15 and day 45; n = 6 per group. Data are representative of two independent experiments. Day 15 endpoint ELISA titers, **P = 0.008; day 45 endpoint ELISA titers, *P = 0.017.(D) Bcl6^+/+ or Bcl6^–/– OT-II CD4⁺ Tcells were transferred into congenically mismatched B6 mice subsequently immunized with Ova in alum. Splenocytes were analyzed 6 days after immunization; n = 4 per group. Data are representative of four independent experiments. OT-II⁺ CD44^high gate is shown. Quantitation of OT-II T_FH differentiation is also shown. ***P < 0.0001. (E to G) Bcl6^+/+ or Bcl6^–/– OT-II CD4⁺ T cells were cotransferred with B1-8 B cells into Icos^–/– mice subsequently immunized with NP-Ova in alum; n = 2 per group. Data are representative of two independent experiments. (E) Germinal center B cells (PNA⁺ GL7⁺, boxed) 7 days after immunization. TCRβ^– IgD^low gate is shown. (F) Quantitation of GC B cells as percent of spleen. **P = 0.0015. (G) Germinal center histology. Spleen sections were stained with IgD (green), PNA (red), and CD4 (blue).

Fig. 4

Blimp-1 and Bcl6 are antagonistic and reciprocal regulators of T_FH differentiation. (A) Immunoblot of Bcl6 protein expression (and β-actin control) in transduced SMtg CD4⁺ T cells in vivo. (B) T_FH (SLAM^low CXCR5^high, boxed) differentiation of untransduced SMtg (left, “Control”) and Blimp1-RV⁺ SMtg (right, “Blimp-1⁺”) cells within a common host 8 days after LCMV infection. Gating is shown in fig. S10B. (C) Quantitation of SMtg T_FH differentiation. “–,” untransduced; “+,” transduced with the indicated RV. ***P < 0.0001; n = 4 per group. Data are representative of two independent experiments. (D) SLAM and CXCR5 expression by naïve CD4⁺ T cells (gray), GFP-RV⁺ SMtg (black), and Blimp1-RV⁺ SMtg (red). (E and F) GFP-RV⁺ or Blimp1-RV⁺ OT-II CD4⁺ T cells were transferred into SAP-deficient mice subsequently immunized with NP-Ova in alum; n = 4 per group. Data are representative of three independent experiments. (E) Germinal center B cells (PNA⁺ Fas⁺, gated) in mice that received GFP-RV⁺ or Blimp1-RV⁺ OT-II CD4⁺ T cells. B220⁺ IgD^low gate is shown. Quantitation of germinal center B cells in the spleen is also shown. ***P < 0.0001. (F) NP-Ova IgG ELISA endpoint titers at day 10. *P = 0.016. (G) Purified naïve B6 and prdm1^fl/fl CD45.2⁺ CD4⁺ T cells were transduced with SMtg-RV, with or without Cre-RV (Cre⁺ or Cre^–), sorted, and transferred into CD45.1⁺ mice subsequently infected with LCMV. FACS plots depict T_FH (CXCR5^high SLAM^low, boxed) differentiation of control Cre⁺ SMtg⁺ B6 cells (left), Blimp-1–sufficient Cre^– SMtg⁺ prdm1^fl/fl cells (center), and Blimp-1–deficient Cre⁺ SMtg⁺ prdm1^fl/fl cells (right). CD4⁺ CD45.1^– CD44^high 7AAD^– gate is shown. Quantitation of T_FH differentiation is also shown. Data are representative of two independent experiments. **P = 0.002, ***P = 0.0006; n = 4 to 5 per group.