Early Th1 cell differentiation is marked by a Tfh cell-like transition - PubMed (original) (raw)

. 2011 Dec 23;35(6):919-31.

doi: 10.1016/j.immuni.2011.11.012.

Yuka Kanno, Hayato Takahashi, Dragana Jankovic, Kristina T Lu, Thomas A Johnson, Hong-wei Sun, Golnaz Vahedi, Ofir Hakim, Robin Handon, Pamela L Schwartzberg, Gordon L Hager, John J O'Shea

Affiliations

Early Th1 cell differentiation is marked by a Tfh cell-like transition

Shingo Nakayamada et al. Immunity. 2011.

Abstract

Follicular helper T (Tfh) cells comprise an important subset of helper T cells; however, their relationship with other helper lineages is incompletely understood. Herein, we showed interleukin-12 acting via the transcription factor STAT4 induced both Il21 and Bcl6 genes, generating cells with features of both Tfh and Th1 cells. However, STAT4 also induced the transcription factor T-bet. With ChIP-seq, we defined the genome-wide targets of T-bet and found that it repressed Bcl6 and other markers of Tfh cells, thereby attenuating the nascent Tfh cell-like phenotype in the late phase of Th1 cell specification. Tfh-like cells were rapidly generated after Toxoplasma gondii infection in mice, but T-bet constrained Tfh cell expansion and consequent germinal center formation and antibody production. Our data argue that Tfh and Th1 cells share a transitional stage through the signal mediated by STAT4, which promotes both phenotypes. However, T-bet represses Tfh cell functionalities, promoting full Th1 cell differentiation.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. IL-12 Induces Genes that Contribute to Tfh and Th1 Phenotypes

(A–I) Sorted naïve CD4+ T cells were cultured under neutral, IL-12-, and IL-6-stimulated conditions for 5 days. The figure depicts FACS plots gated on CD4+ cells and percentage or mean MFI of CD4+ cells positive for intracellular IL-21, IFN-γ, and T-bet (A, F, I), and cell surface expression of CXCR5, PD-1 (B, G), and ICOS (C, H). Staining with an isotype control antibody was used to set gates (dotted lines). (D) Relative mRNA expression of Bcl6 was evaluated by qPCR. (E) Bcl6 and T-bet levels in whole cell lysates were analyzed by immunoblotting. A human Burkitt’s lymphoma cell line, Ramos, was used as a reference control. (F–I) The percentages or MFI levels of the indicated markers gated on IL-21 or IFN-γ positive cells (red) are compared to that of IL-21- or IFN-γ-negative cells (blue) or CD4+ T cells cultured under neutral conditions (gray). Representative data of three or more independent experiments are shown (mean ± SD). *, p<0.05, **, p<0.01.

Figure 2

Figure 2. Induction of Tfh and Th1 Phenotypes by IL-12 Is Mediated by STAT4

(A–E) Sorted naïve CD4+ T cells from WT, Stat3−/−, Stat4−/−, or Stat6−/− mice were cultured under neutral, IL-12-, and IL-6- conditions for 5 days. Shown are flow cytometry plots gated on CD4+ cells and percentage or mean MFI of CD4+ cells positive for expression of IL-21 and IFN-γ (A), CXCR5 and PD-1 (B), and ICOS (C). (D, E) Levels of Bcl6, T-bet, phosho-STAT3, total STAT3, phospho-STAT4, or total STAT4 in whole cell lysates were analyzed by immunoblotting. Representative data of three or more independent experiments are shown (mean ± SD). *, p<0.05, **, p<0.01.

Figure 3

Figure 3. Transient Nature of Tfh-like Phenotype during Th1 Differentiation

(A–C) Sorted naïve CD4+ T cells were cultured under IL-12 conditions for up to 8 days. Cells were analyzed by flow cytometry for expression of IL-21 and IFN-γ (A, C), Bcl6 and T-bet (B, C) at the indicated days. (A, B) Time course of IL-21, IFN-γ, Bcl6, and T-bet expression. The figure shows representative FACS plots gated on CD4+ cells (top) and percentage of CD4+ cells positive for the indicated molecules (bottom). (C) Cells were gated based on T-bet and Bcl6 expression and expression of IFN-γ, IL-21 and CXCR5 at day 5 in developing Th1 cells were compared. Representative data from one of three independent experiments are shown (mean ± SD).

Figure 4

Figure 4. T-bet Represses Tfh-like Phenotype in Th1 Cells

(A–C) Sorted naïve CD4+ T cells from WT or Tbx21−/− mice were cultured under neutral or IL-12 conditions for 5 days. Shown are FACS plots gated on CD4+ cells and percentage of CD4+ cells positive for expression of IL-21 and IFN-γ (A), CXCR5 and PD-1 (B), Bcl6 and T-bet (C). Levels of Bcl6 and T-bet in whole cell lysates were also analyzed by immunoblotting (C). (D) Sorted naïve CD4+ T cells from Tbx21−/− mice were infected with either control retrovirus (mock-GRV) or T-bet-expressing retrovirus (T-bet-GRV) for 3 days. GFP+ cells were sorted by FACS and lysed for Western blotting to evaluate Bcl6 and T-bet expression. Shown are FACS plots gated on GFP+CD4+ cells and percentage of GFP+CD4+ T cells positive for expression of Bcl6 and T-bet. Representative data of three or more experiments are shown (mean ± SD). *, p<0.05, **, p<0.01.

Figure 5

Figure 5. Opposing Effects of STAT4 and T-bet on Epigenetic Profiles of Tfh Genes in Th1 Cells

(A–F) Sorted naïve CD4+ T cells from WT, Stat4−/−, or Tbx21−/− mice were cultured under Th1 conditions. (A–E) Cells were restimulated with anti-CD3 and anti-CD28 and IL-12 for 2 h. (A–C) DNase-seq and ChIP-seq were performed using naïve CD4+ T cells and Th1 cells polarized for 7 days to map DHS, histone epigenetic marks, and binding of STAT4 and T-bet. The genome browser view of Ifng (chr10: 117,865,988–117,895,063, DHS Y axis: 0–3, H3K4me3 Y axis: 0–8, H3K36me3 Y axis: 0–3, binding of STAT4 and T-bet Y axis: 0–300), Il21 (chr3: 37,091,490–37,164,309, DHS Y axis: 0–2, H3K4me3 Y axis: 0–4, H3K36me3 Y axis: 0–2, binding of STAT4 and T-bet Y axis: 0–150), and Bcl6 (chr16: 23,951,786–24,014,614, DHS Y axis: 0–3, H3K4me3 Y axis: 0–6, H3K36me3 Y axis: 0–2, binding of STAT4 and T-bet Y axis: 0–60) loci are shown. Y axis shows the normalized tag number which is the tag count per one million of total tag count (tag-per-million). (D, E) Relative mRNA expression of Il21 or Bcl6 at day 5 was compared in cells from WT (black) or Tbx21−/− mice (green). (F) FACS analysis for IL-21, IFN-γ, Bcl6, T-bet, CXCR5, and PD-1 gated on CD4+ cells were performed at day 0, 3, 5, and 8. Representative data of two or more independent experiments are shown (mean ± SD).

Figure 6

Figure 6. Effect of Modulating the Levels of IFN-γand IL-21 during the Course of In Vitro Th1 Differentiation

(A–F) Sorted naïve CD4+ T cells were cultured under IL-12 conditions for 3 to 5 days. Combinations of IFN-γ, anti- IFN-γ antibody, IL-21, or soluble IL-21 receptor-Fc were added as indicated to enhance or deplete the levels of IFN-γ or IL-21 in the culture. (A, B, D, E) Levels of IL-21 and IFN-γ (A, D), and CXCR5 and PD-1 (B, E) on CD4+ cells were determined by FACS at day 3 and/or 5. (C, F) Levels of Bcl6 and T-bet on CD4+ cells were determined by FACS and by Western blotting at day 5. Representative data of three independent experiments are shown (mean ± SD). *, p<0.05, **, p<0.01.

Figure 7

Figure 7. T-bet Negatively Regulates Tfh-related Functionalities of CD4+ T Cells during Infection with T. gondii

(A–C) WT or Tbx21−/− mice were infected with 20 cysts of the avirulent T. gondii strain ME49. Splenocytes were analyzed by FACS 7 days after infection for expression of IL-21, IFN-γ, and T-bet (A) gated on CD4+ cells, and CXCR5 and PD-1 gated on CD4+CD44+ cells (B). Data indicate mean ± SD from at least four mice per group in two separate infections. Bcl6 and T-bet levels in whole cell lysates from sorted CD4+ cells were determined by Western blotting (C). (D–H) Rag2 −/− mice were reconstituted with 1:1 mixture of WT (CD45.1+) and Tbx21−/− (CD45.2+) naive CD4+ T cells. Seven days later mice were infected with T. gondii and followed for 7 to 15 days (D). Expression of IL-21 and IFN-γ (E, F), Bcl6 and T-bet (E, G), and CXCR5 and PD-1 in CD4+CD44+ cells (E, H) was determined by FACS. The levels of expression of the indicated markers on CD45.1 positive cells (WT) are compared to that of CD45.1 negative cells (Tbx21−/−). n=5. (I, J) WT or Tbx21−/− mice were immunized with 20 μg/mL STAg i.p. every 5 days for 3 times. Splenic GC B cells (B220+IgDloGL7hiFashi) were analysed by FACS 20 days post-immunization (I). Serum levels of STAg-specific total IgG were measured by ELISA (J). n=7 each. Representative data from one of two independent experiments are shown (mean ± SD). *, p<0.05, **, p<0.01.

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