T-box 21 transcription factor is responsible for distorted TH2 differentiation in human peripheral CD4+ T cells Osamu Kaminuma, DVM, PhD, Fujiko Kitamura,

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T-box 21 transcription factor is responsible for distorted TH2 differentiation in human peripheral CD4+ T cells Osamu Kaminuma, DVM, PhD, Fujiko Kitamura, BSc, Shoichiro Miyatake, MD, PhD, Kazuko Yamaoka, PhD, Hiroyuki Miyoshi, PhD, Shigeko Inokuma, MD, PhD, Hideki Tatsumi, MD, Soichi Nemoto, MD, Noriko Kitamura, MSc, Akio Mori, MD, PhD, Takachika Hiroi, DDS, PhD Journal of Allergy and Clinical Immunology Volume 123, Issue 4, Pages 813-823.e3 (April 2009) DOI: 10.1016/j.jaci.2009.01.055 Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Expression of TH1/TH2 cytokines and transcription factors in CD4+ T cells of asthmatic patients. mRNA expression in freshly isolated peripheral CD4+ T cells of asthmatic patients and healthy donors was determined by using the quantitative real-time RT-PCR method. Each dataset (plot) and mean (bar) is expressed as mRNA abundance normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression (n = 16-38). P values were determined by using the unpaired t test with the Welch correction. Journal of Allergy and Clinical Immunology 2009 123, 813-823.e3DOI: (10.1016/j.jaci.2009.01.055) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 TH1 and TH2 cell differentiation from peripheral and cord blood naive T cells. CD45RA+CD4+ cells were purified from mononuclear cells in peripheral and cord blood of healthy subjects and stained for CD4 and CD45RA (A). Cell-surface markers on the peripheral (plain line) and cord (bold line) blood CD4+CD45RA+ cells were stained with specific or isotype control antibodies (gray area; B). After 7 to 10 days of stimulation culture under TH1- and TH2-skewing conditions, peripheral and cord blood CD4+ T cells were stimulated with 5 nmol/L phorbol 12-myristate 13-acetate plus 1 μmol/L ionomycin for 6 hours and stained for intracellular IL-4 and IFN-γ (C). The representative plots and means ± SEMs of cytokine-positive cell percentages from 4 separate experiments are shown. Journal of Allergy and Clinical Immunology 2009 123, 813-823.e3DOI: (10.1016/j.jaci.2009.01.055) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Expression of transcription factors in T cells. Purified naive CD4+ T cells and differentiated TH1 and TH2 cells from peripheral and cord blood of healthy subjects were left unstimulated or stimulated with 0.1 μg/mL anti-CD3 plus 1 μg/mL anti-CD28 for 24 hours. Expression of transcription factors and actin in the resulting cells was analyzed by means of Western blotting. Protein samples from 106 cells of individual groups were loaded and blotted on the same membrane (A). GATA-3 and T-bet expression in TH2 cells from peripheral CD4+ T cells was examined by means of intracellular staining (B). Whole cell lysates (WLC) of TH1 and TH2 cells from peripheral blood CD4+ T cells were immunoprecipitated with anti–T-bet antibody. The resulting immunoprecipitation (IP) samples or WCL were blotted with anti-phosphotyrosine, anti–T-bet, and anti–GATA-3 antibodies (C). The results shown are representative of 4 separate experiments. REA assay against HaeIII sites at the TBX21 gene was performed on unstimulated naive and TH1- and TH2-differentiated peripheral and cord blood T cells (D). Data are expressed as means ± SEMs of percentage intact DNA compared with HaeIII-untreated control values (n = 4-6). MW, Molecular weight. Journal of Allergy and Clinical Immunology 2009 123, 813-823.e3DOI: (10.1016/j.jaci.2009.01.055) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Effect of T-bet on cytokine synthesis in T cells. T-bet–internal ribosomal entry site sequence–Venus (T-bet) and empty internal ribosomal entry site sequence–Venus (Vector) expression cassettes were introduced into cord blood naive CD4+ T cells of healthy subjects by using a lentivirus infection system during 7 to 10 days of stimulation culture under TH2 conditions. The resulting cells were stimulated with 5 nmol/L phorbol 12-myristate 13-acetate plus 1 μmol/L ionomycin for 6 hours and stained for intracellular IL-4 and IFN-γ (A). The representative plots and means ± SEMs of cytokine-positive cell percentages in the Venus-positive population from 4 separate experiments are shown. Internal ribosomal entry site sequence–Venus–combined GATA-3, c-Maf, STAT4, and T-bet expression cassettes were introduced into peripheral naive CD4+ T cells during stimulation culture under neutral conditions. The resulting Venus-positive cells (M1 population; B) were purified and left unstimulated or stimulated with 5 nmol/L phorbol 12-myristate 13-acetate plus 1 μmol/L ionomycin for 6 hours, and the expression of cytokine and transcription factor mRNA was measured by means of quantitative real-time RT-PCR (C). Data are expressed as means ± SEMs of mRNA abundance normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression (n = 4). Journal of Allergy and Clinical Immunology 2009 123, 813-823.e3DOI: (10.1016/j.jaci.2009.01.055) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Knockdown effect of T-bet on human T-cell differentiation. A serial expression cassette containing U6 promoter–driven shRNA and EF promoter–driven Venus (A) was introduced into peripheral naive CD4+ T cells of healthy subjects with lentivirus during 7 to 10 days of stimulation culture under neutral conditions. The resulting Venus-positive population (M1 population; B) in T-bet (bold line) and control (plain line) shRNA–introduced cells was purified. The gray area indicates untransfected background. Expression of T-bet, GATA-3, or both in the purified cells was determined by means of quantitative real-time RT-PCR (C) and Western blotting (D). The purified cells were left unstimulated or stimulated with 0.1 μg/mL anti-CD3 plus 1 μg/mL anti-CD28 for 24 hours, and the concentrations of cytokines in the culture supernatant were measured by means of ELISA (E). Journal of Allergy and Clinical Immunology 2009 123, 813-823.e3DOI: (10.1016/j.jaci.2009.01.055) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Effect of T-bet on cytokine mRNA expression and promoter activity in human T cells. A, Jurkat Tag cells were transfected with pMACS–T-bet or empty vector (10 μg each). After 48 hours, H-2k–positive cells purified with a magnetic cell sorting system were left unstimulated or stimulated with phorbol 12-myristate 13-acetate (5 nmol/L) plus ionomycin (1 μmol/L) in the presence of 40 μg/mL anti–IFN-γ for 6 hours, and the expression of cytokine and transcription factor mRNA was determined by using the quantitative real-time RT-PCR method. Data are expressed as means ± SEMs of mRNA abundance normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression (n = 4). B, Cells were transfected with pEF–T-bet or empty vector in the presence of IFN-γ–EGFP, IL-4–EGFP, or IL-13–EGFP (10 μg each). At 24 hours after transfection, cells were stimulated in the presence of 40 μg/mL anti–IFN-γ for 16 hours, and the promoter activity was detected as the fluorescence of synthetic EGFP measured by means of flow cytometry (n = 4). Journal of Allergy and Clinical Immunology 2009 123, 813-823.e3DOI: (10.1016/j.jaci.2009.01.055) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

mRNA expression of GATA-3 and T-bet in peripheral and cord blood CD4+ T cells. Purified naive CD4+ T cells and differentiated TH1 and TH2 cells from peripheral and cord blood of healthy subjects was determined by means of quantitative real-time RT-PCR method. Data are expressed as means ± SEMs of mRNA abundance normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression (n = 3-6). Journal of Allergy and Clinical Immunology 2009 123, 813-823.e3DOI: (10.1016/j.jaci.2009.01.055) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions