PMC

Role of Bcl6 in initial IL-4 production by MPT cells to induce NAT_H2 cells in vitro. (A,B) Bcl6-WT KJ1-26⁺ naïve CD4⁺ T cells were cocultured with KJ1-26⁻ MPT cells (Bcl6-TG, Bcl6-WT, or Bcl6-KO) in the presence of soluble anti-CD3 and CD28 mAbs and irradiated CD11c⁺ DCs as the T_H0 condition with or without anti-IFN-γ Abs. (A) FACS analysis of intracellular cytokines in each effector T cell type derived from KJ1-26⁺ naïve CD4⁺ T cells are presented as a representative figure among three independent experiments after restimulation with anti-CD3 mAbs. Numbers in the corners denote the percentages of gated KJ1-26⁺ CD4⁺ T cells. (B) Frequency of the populations of IL-4⁺ and IFN-γ⁺ KJ1-26⁺ T cells after reactivation. All results are representative of three independent experiments with similar outcomes. Data are presented as the mean ± SEM (n = 8–9). *P < 0.05; **P < 0.01. Ab, antibody; Bcl6, B-cell lymphoma 6; DC, dendritic cell; KO, knockout; mAb, monoclonal antibody; MPT cell, memory phenotype CD4⁺ T cell; NAT_H2 cell; naïve CD4⁺ T cell-derived T_H2 cell; TG, transgenic; WT, wild-type.

IL-4⁺ MPT cells in mice with varying genetic Bcl6 expression. (A,B) FACS analysis of intracellular IL-4⁺ [(A) Bcl6-TG, Bcl6-WT, and Bcl6-KO] and CNS2-activation-related GFP⁺ [(B) Bcl6-TG and Bcl6-WT] MPT cells in a CD44^high population by gating CD4⁺ CD49b⁻ T splenocytes at rest. The presented data are representative of four independent experiments. The numbers in the corners represent the percentages of gated T cells. (C,D) Frequency of GFP⁺ [(C) Bcl6-TG and Bcl6-WT] and IL-4⁺ [(D) Bcl6-TG, Bcl6-WT, and Bcl6-KO] MPT cells. (E) MFI of GFP in MPT cells from Bcl6-TG and Bcl6-WT mice. (F) qRT-PCR analysis of the relative expression of Il4 in GFP⁻ and GFP⁺ MPT cells from Bcl6-TG and Bcl6-WT spleens. (G–I) Absolute cell numbers of populations of IL-4⁺ [(G) Bcl6-TG, Bcl6-WT, and Bcl6-KO], GFP⁺ [(H) Bcl6-TG and Bcl6-WT], and total (I) MPT cells in one spleen. Data are presented as the mean ± SEM (n = 7–9). *P < 0.05; **P < 0.01, comparison between two groups as indicated. Bcl6, B-cell lymphoma 6; CNS, conserved noncoding sequence; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; KO, knockout; MFI, mean fluorescence intensity; MPT cell, memory phenotype CD4⁺ T cell; NS, not significant; TG, transgenic; WT, wild-type.

Role of Bcl6 in the CNS2 enhancer activity of MPT_H2 cells. (A) A conserved sequence (positions +12805 to +13151 relative to the transcription start site; Mouse Genome Informatics accession no. 96556) in the CNS2 region of mice is shown with human CNS2, including BS7 (1) and (2). Conserved sequences between mice and humans are indicated by shaded boxes. (B,C) Splenic Bcl6-KO and Bcl6-WT MPT cells were cultured under T_H2 conditions, and a retrovirus containing the d2EGFP reporter gene, with CNS2-WT BS7 (B), CNS2-Mut BS7 (1), or CNS2-Mut BS7 (2), was introduced into T_H2 cells on day 2 of culture. After 7 days of culture, cells were restimulated with anti-CD3 monoclonal antibodies for 8 h and subjected to FACS analysis of the intracellular MFI of d2EGFP. (C) Histograms of FACS analysis are representative of eight to nine independent experiments. Numbers in each column represent the MFI of d2EGFP. (D) Mean values of the MFI of d2EGFP are indicated. Data are presented as the mean ± SEM (n = 8–9). *P < 0.05, comparison between two groups is indicated; ^†P < 0.05, compared with CNS2-WT. Bcl6, B-cell lymphoma 6; CNS, conserved noncoding sequence; d2EGFP, d2-enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; MFI, mean fluorescence intensity; MPT, memory phenotype CD4⁺ T; MPT_H2 cell, MPT cell-derived T_H2 cell; Mut, mutant; KO, knockout; WT, wild-type.

Regulatory role of Bcl6 in the differentiation of T_H2 cells. (A–E) KJ1-26⁺ MPT and NA T cells (CD4⁺ CD44^l°^w CD62L⁺) among splenocytes from Bcl6-TG, Bcl6-WT, and Bcl6-KO DO11.10 mice cultured with ovalbumin peptides and antigen-presenting cells in vitro for 7 days to produce T_H0, T_H1, and T_H2 cells. Cells were restimulated with anti-CD3 monoclonal antibodies. After 8 h, IL-4- and IFN-γ-producing cells among gated KJ1-26⁺ CD4⁺ T cells were analyzed by FACS. (C–E) Analysis of cytokine production by Bcl6-WT MPT_H2 cells treated with a Bcl6 inhibitor (inh.) for 12 h prior to restimulation. (A,C) Numbers in the corners represent percentages among gated T cells. (B,D) Percentage of IL-4⁺ (Bcl6-TG, Bcl6-WT, and Bcl6-KO) cells for each T_H cell type (B) and IL-4⁺ and IFN-γ⁺ Bcl6-WT MPT_H2 cells cultured with or without Bcl6 inhibitor (D). (E) qRT-PCR analysis of the relative expression of Gata3, Il4, Il5, and Il13 in restimulated Bcl6-WT MPT_H2 cells treated with or without a Bcl6 inhibitor. Data are presented as the mean ± SEM (n = 7–8). *P < 0.05, **P < 0.01, comparison between two groups is indicated; ^†P < 0.05, compared with Bcl6-WT. All results are representative of five independent experiments with similar outcomes, excluding (C), for which four experiments were conducted. Bcl6, B-cell lymphoma 6; Cont., control; FACS, fluorescence-activated cell sorting; KO, knockout; MPT cell, memory phenotype CD4⁺ T cell; MPT_H2 cell, MPT cell-derived T_H2 cell; NA, naïve; TG, transgenic; WT, wild-type.

Role of Bcl6-mediated MPT cell functions in NAT_H2 differentiation in an allergic murine model. [(A) top] Mixture of purified KJ1-26⁻ MPT cells (Bcl6-WT or Bcl6-KO) and KJ1-26⁺ WT naïve CD4⁺ T cells were transferred into BALB/c nu/nu mice intravenously (day 0). These mice were immunized with alum-conjugated OVA and then intratracheally challenged with OVA. [(A) bottom] Absolute cell numbers of Neu, Eos, AM, and Lym in BALF, (B) hematoxylin and eosin-stained, formalin-fixed lung sections (magnification: 200×), and (C) T_H2 cytokine levels in the BALF of recipient mice 48 h after the last OVA challenge. (D) Relative Il4, Il5, and Il13 expression mRNA in splenic KJ1-26⁺ T cells restimulated with anti-CD3 monoclonal antibodies 5 days after the last challenge. (E) OVA-specific IgE antibody titers in sera from each recipient of Bcl6-WT NAT_H2 cells, plus MPT_H2 cells transferred from Bcl6-TG, Bcl6-WT, or Bcl6-KO mice 2 days after the last challenge. All results are representative of four independent experiments with similar outcomes. Data are presented as the mean ± SEM (n = 5–7). *P < 0.05, **P < 0.01, comparison between two groups is indicated. AM, alveolar macrophages; BALF, bronchoalveolar lavage fluid; Bcl6, B-cell lymphoma 6; Eos, eosinophils; KJ⁺, KJ1-26-positive; KO, knockout; Lym, lymphocytes; MPT cell, memory phenotype CD4⁺ T cell; MPT_H2 cell, MPT cell-derived T_H2 cell; NAT_H2 cell; naïve CD4⁺ T cell-derived T_H2 cell; Neu, neutrophils; NS, not significant; OVA, ovalbumin; TG, transgenic; WT, wild-type.

IL-33 reinforces IL-4 production by MPT cells through functional competition against the suppressor activity of Bcl6. (A,B) FACS analysis of splenic CNS2-GFP-TG MPT cells from Bcl6-TG and Bcl6-WT mice at rest. (A) Data show the expression of GFP and ST2 gated cells among all CD4⁺ CD44⁺ cells (representative of six independent experiments). (B) Percentages of ST2⁺ cells among GFP⁺ and GFP⁻ MPT cells. (C,D) IL-33 was added to the culture of MPT cells from Bcl6-TG and Bcl6-WT mice three times in the presence of IL-7. [(C) top] Six hours after the last IL-33 dose, MPT cells were analyzed for intracellular IL-4 levels. Numbers indicate the percentage of IL-4⁺ cells among all MPT cells. [(C) bottom] FACS analysis data are representative of four independent experiments. (D) Absolute numbers of IL-4⁺ MPT cells 8 h after the last IL-33 dose. (E) ChIP analysis of Bcl6 and STAT5 binding and Ac-H3 at each BS in CNS2-GFP⁺ MPT cells from Bcl6-TG (T) and Bcl6-WT mice(W). Cells were primed with or without IL-33 three times in the presence of IL-7. Analysis was performed 8 h after the last IL-33 dose. All results are representative of three (A–D) or four (E) independent experiments with similar outcomes. Data are presented as the mean ± SEM (n = 6–7). *P < 0.05, **P < 0.01, comparison between two groups is indicated; ^†P < 0.05, ^††P < 0.01, compared with WT. Ac-H3, acetylated histone H3; Bcl6, B-cell lymphoma 6; BS, binding sequence; ChIP, chromatin immunoprecipitation; CNS, conserved noncoding sequence; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; MPT cell, memory phenotype CD4⁺ T cell; TG, transgenic; WT, wild-type.

Role of Bcl6 in hcIE activity in MPT_H2 cells. (A–D) KJ1-26⁺ cells among splenic MPT cells were cultured with ovalbumin peptides and antigen-presenting cells in vitro under T_H0 or T_H2 conditions. [(A) top] Diagram of the HS2 region in Il4 intron 2, indicating regulatory regions. The shaded square indicates the hcIE region including the GATA3-binding site (G3) and BS3 within HS2. (A) Gata3 mRNA levels in GFP⁺ and GFP⁻ MPT, MPT_H0, and MPT_H2 cells derived from Bcl6-TG and Bcl6-WT mice on a CNS2-GFP-TG background. (B) GATA3 binding to G3 analyzed by ChIP assays for GFP⁺ MPT and MPT_H2 cells on a CNS2-GFP-TG background. (C,D) Analysis of splenic MPT cells or MPT_H2 cells derived from hcIE-KO or hcIE-WT mice. (A) FACS analysis of intracellular cytokine populations of MPT cells by gating CD4⁺ CD49b⁻ T cells in the resting phase and MPT_H2 cells restimulated with anti-CD3 monoclonal antibodies. The numbers in the corners represent the percentages among the gated T cells. (D) Gata3 mRNA levels were measured by qRT-PCR for MPT and MPT_H2 cells derived from hcIE-KO and hcIE-WT mice. (E) Bcl6 levels and STAT5 binding to each BS were analyzed by ChIP assay for GFP⁺ MPT_H2 cells from hcIE-KO or hcIE-WT mice on a CNS2-GFP-TG background. All results are representative of three (A,B) or five (C–E) independent experiments with similar outcomes. Data are means ± SEMs (n = 9–10). *P < 0.05, comparison between two groups as indicated; ^†P < 0.05, ^††P < 0.01, compared with the MPT cells. Bcl6, B-cell lymphoma 6; BS, binding sequence; ChIP, chromatin immunoprecipitation; CNS, conserved noncoding sequence; FACS, fluorescence-activated cell sorting; hcIE, highly conserved intron enhancer; HS, DNase hypersensitive site; KO, knockout; MPT cell, memory phenotype CD4⁺ T cell; MPT_H2 cell, MPT cell-derived T_H2 cell; ND, not detected; NS, not significant; TG, transgenic; WT, wild-type.

Role of Bcl6 in interactions between MPT_H2 and naïve NAT_H2 cells in allergy pathogenesis. (A–E) KJ1-26⁺ MPT_H2 cells and NAT_H2 cells were differentiated from the spleens of Bcl6-TG (T), Bcl6-WT (W), and Bcl6-KO (K) mice in the presence of OVA peptides and antigen-presenting cells in T_H2 conditions. (A) Il4 mRNA levels in each T_H2 cell type were measured by qRT-PCR at rest and at 1 and 8 h after restimulation with anti-CD3 monoclonal antibodies. (B–E) Bcl6-WT BALB/c nu/nu mice were administered KJ1-26⁺ MPT_H2 cells (3 × 10⁷), KJ1-26⁺ NAT_H2 cells (3 × 10⁷), or combinations of MPT_H2 (1.5 × 10⁷) and NAT_H2 cells (1.5 × 10⁷) via adoptive transfer (day 0). (B) Representative FACS data for donor cells in circles with their percentages among total CD4⁺ T cells in whole lungs from recipients at 24 and 48 h after the last intratracheal OVA challenge. (C) Absolute numbers of KJ1-26⁺ cells in the lungs, (D) T_H2 cytokine levels, and (E) cell types in the bronchoalveolar lavage fluid 48 h after the last challenge. All results are representative of four independent experiments with similar outcomes. Data are presented as the mean ± SEM (n = 8–10). *P < 0.05, **P < 0.01, comparison between two groups is indicated (A,B,D,E); ^†P < 0.05, compared with MPT_H2 cells. AM, alveolar macrophages; Bcl6, B-cell lymphoma 6; Eos, eosinophils; FACS, fluorescence-activated cell sorting; KO, knockout; Lym, lymphocytes; MPT cell, memory phenotype CD4⁺ T cell; MPT_H2 cell, MPT cell-derived T_H2 cell; NAT_H2 cell; naïve CD4⁺ T cell-derived T_H2 cell; Neu, neutrophils; NS, not significant; OVA, ovalbumin; TCR, T cell receptor; TG, transgenic; WT, wild-type.

Role of Bcl6 and signal transducer and activator of transcription (STAT) binding to the Il4 locus MPT cells. (A) Bcl6 mRNA levels in GFP⁺ and GFP⁻ MPT cells, GFP⁺ and GFP⁻ MPT_H2 cells, and NA T_H2 cells, as measured by qRT-PCR. (B) Western blot analysis of Bcl6 protein in GFP⁺ and GFP⁻ MPT cells MPT (Bcl6-WT) and MPT cells (Bcl6-KO) in the spleen and GFP⁺ and GFP⁻ MPT_H2 cells (Bcl6-WT). Data are representative of three independent experiments. (C,D) KJ1-26⁺ cells among MPT cells from the spleens of Bcl6-WT-CNS2-GFP-TG DO11.10 mice were cultured with ovalbumin peptides and antigen-presenting cells in vitro for 7 days under T_H2 conditions. Cells were restimulated with anti-CD3 and anti-CD28 monoclonal antibodies. After 48 h, IL-4, IL-5, IL-13, and IFN-γ levels in culture supernatants were measured by ELISA (C). After 8 h, the mRNA levels of Il4, Il5, Il13, and Ifn-γ were measured by qRT-PCR (D). [(E), top] Diagram of T_H2 cytokine gene loci, with regulatory regions indicated by arrows [CNS, gene promoter regions (p), and Bcl6/STAT (BSs): IL5BS in Il5; IL13BS in Il13 intron 1; BS1 and BS7 (1) (2) in CNS1 and CNS2, respectively; BS2 in Il4p; and BS3, BS4, and BS5 in Il4 intron 2]. (E,F) Bcl6 levels [(E) bottom], STAT5 and STAT6 binding, and Ac-H3 (F) at each BS were analyzed by chromatin immunoprecipitation assay for CNS2-active (GFP⁺) (closed bar) and CNS2-inactive (GFP⁻) (open bar) MPT_H2 cells. All results are representative of three (A,C,D) or four (E,F) independent experiments with similar outcomes. Data are presented as the mean ± SEM (n = 7–9). *P < 0.05, **P < 0.01, comparison between two groups is indicated. Ac-H3, acetylated histone H3; CNS, conserved noncoding sequence; BS, binding sequence; Bcl6, B-cell lymphoma 6; GFP, green fluorescent protein; KO, knockout; ND, not detected; MPT cell, memory phenotype CD4⁺ T cell; MPT_H2 cell, MPT cell-derived T_H2 cell; NA, naïve; TG, transgenic; WT, wild-type.