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Originally published in Science Express on 23 July 2009
Science 21 August 2009:
Vol. 325. no. 5943, pp. 1001 - 1005
DOI: 10.1126/science.1176676
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Reports

Bcl6 Mediates the Development of T Follicular Helper Cells

Roza I. Nurieva*, Yeonseok Chung, Gustavo J. Martinez, Xuexian O. Yang, Shinya Tanaka, Tatyana D. Matskevitch, Yi-Hong Wang and Chen Dong*

Department of Immunology, M. D. Anderson Cancer Center, Houston, TX 77030, USA.


Figure 1
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  		Fig. 1. Bcl6 regulates TFH-specific gene expression. (A) Naïve T cells were activated with antibodies to CD3 and CD28, and with or without indicated cytokines for 1 or 2 days. Bcl6 mRNA expression was analyzed by real-time RT-PCR. The graph shows means ± SD. *P < 0.005; **P < 0.001 when comparing IL-6 and IL-6 plus TGFβ to nontreated sample, analysis of variance (ANOVA) test. ++P < 0.001 when comparing IL-21 and IL-21 plus TGFβ to nontreated sample, ANOVA test. (B) Naïve OT-II T cells were activated with antibodies to CD3 and CD28 alone or together with IL-6 or IL-21 and with antibodies to IL-4, IFN{gamma}, and TGFβ, and infected with a bicistronic retrovirus containing internal ribosomal entry site green fluorescent protein (GFP) expressing Bcl6 or a vector control virus. mRNA expression of indicated genes was assessed by real-time RT-PCR. (C) Naïve OT-II T cells were activated with antibodies to CD3 and CD28 alone and infected with retroviruses expressing Bcl6, Bcl6 mutants (ZF3 and ZF5), or vector alone. GFP+ cells were sorted from (B) and (C) and restimulated for 4 hours with antibody to CD3. mRNA expression of indicated genes was analyzed by real-time RT-PCR. The data shown were normalized to the expression of a reference gene, Actb. The graph shows means ± SD. *P < 0.005; **P < 0.001, t test. The data represent at least three independent experiments with consistent results.

 

Figure 2
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  		Fig. 2. Bcl6 suppresses TH17 and TH1 differentiation. (A and B) Naïve OT-II T cells were activated under TH17 conditions and infected with retroviruses expressing Bcl6, Bcl6 mutants, or vector alone. GFP+ cells sorted by fluorescence-activated cell sorting (FACS) were restimulated with antibody to CD3, and mRNA expression of indicated genes was analyzed by real-time RT-PCR (A). The data shown were normalized to the expression of reference gene Actb. The graph shows means ± SD. *P < 0.005; **P < 0.001 when comparing Bcl6 and ZF3 to vector alone, ANOVA test. ++P < 0.001 when comparing Bcl6 and ZF5 to vector alone, ANOVA test. IL-17–expressing cells were measured by intracellular staining of the sorted GFP+ population (B). Numbers in dot-plot quadrants represent the percentages. (C) EL-4 cells were transfected with a vector containing the firefly luciferase gene under the control of the IL-17 promoter and the CNS2 region, a vector expressing Renilla luciferase, and vectors expressing ROR{gamma}t, Bcl6 wild-type, various Bcl6 mutants, or vector alone. The firefly luciferase activity was determined and normalized to Renilla luciferase. Values were also normalized to vector alone. The graph shows means ± SD. (D and E) Naïve OT-II T cells were activated under TH1 conditions and infected with the indicated viruses. GFP+ cells were restimulated with antibody to CD3, and mRNA expression of the indicated genes was analyzed by real-time RT-PCR (D). The data shown were normalized to the expression of reference gene Actb. The graph shows means ± SD. *P < 0.005; **P < 0.001, t test. IFN{gamma} expression was analyzed by intracellular staining on FACS-sorted GFP+ cells (E). Numbers in dot-plot quadrants represent the percentages. The data represent at least three independent experiments with consistent results.

 

Figure 3
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  		Fig. 3. Bcl6 deficiency causes defective T cell differentiation in vitro. Naïve CD4+ T cells from Bcl6–/– mice and their littermate control mice carrying the OT-II TcR transgene were activated under TFH (A), TH0 and TH1 (B), TH17 (C), and Treg (TGFβ or TGFβ with antibodies to IL-4 and IFN{gamma}) (D) conditions. Five days later, cells were assessed for IFN{gamma} and IL-17 production [(A) to (C)] or Foxp3 expression (D) using intracellular staining. Numbers in FACS plot quadrants represent the percentages. mRNA expression of various genes was analyzed by real-time RT-PCR, and the data shown were normalized to the expression of reference gene β-actin. The graph shows means ± SD. P values were calculated using a t test comparing wild-type and Bcl6–/– CD4+ T cells and are indicated as follows: *P < 0.005; **P < 0.001. The experiments were repeated three times with consistent results.

 

Figure 4
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  		Fig. 4. Bcl6 is necessary for the generation of TFH cells in vivo. (A and B) Naïve CD4+ cells from wild-type (WT) or Bcl6–/– mice were mixed with WT B cells and transferred into Rag1–/– mice (3 mice per group). The recipient mice were immunized subcutaneously with KLH emulsified in Freund’s complete adjuvant (FCA). In (B), recipient mice were also treated with antibody to IL-4. Seven days after the immunization, germinal center B cells were determined by staining with GL-7, FAS and B220 antibodies, and TFH cells by CD4 and CXCR5 antibodies. Numbers in dot-plot quadrants represent the percentages in gated T and B cells, respectively. Spleen cells from immunized mice were stimulated with the indicated concentration of KLH. Effector cytokines were measured after 4 days of treatment. The graph shows means ± SD. P values were calculated using a t test comparing CXCR5 expression on T cells or GL7/Fas expression on B cells between mice transferred with WT or Bcl6-deficient T and B cells, respectively, and are indicated as follows: *P < 0.005; **P < 0.001. The results are representative of multiple mice (n = 6 per group) from two independent experiments with similar results. (C) Mixed–bone marrow chimeric mice (n = 15) were generated and immunized with KLH in FCA. Spleen cells were stained with antibody to CD45.2 to distinguish WT and Bcl6 knockout compartments and then analyzed for the germinal center B cells and TFH cells. P values were calculated using a t test comparing CXCR5 and GL7/Fas expression between WT and Bcl6-deficient T and B cells, respectively, and are indicated as follows: *P < 0.005; **P < 0.001. The results are a representative of multiple mice (n = 15) from three independent experiments with similar results.

 

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