Figure 1.
Figure 1. Expression of α4 integrin mRNA and protein are down-regulated in Th17 cells. (A) Purified naive T cells (CD4+CD44−Foxp3−) isolated from Foxp3gfp.KI mice were cultured under Th0 (no cytokines), Th1 (IL-12 and anti–IL-4), or Th17 (TGF-β plus IL-6 ± IL-23) polarizing conditions with polyclonal TCR stimulation. On day 3, expression of α4 integrin was determined by surface staining (top row: tinted line, isotype control; black line, α-α4 integrin). The differentiation status was confirmed by intracellular cytokine staining (bottom row: numbers indicate percentages of cytokine-positive cells). Shown are representatives of more than five independent experiments. (B) Time course of α4 integrin expression during Th1 and Th17 differentiation as determined by surface staining (means + SD, n = 4, Student’s t test). (C) RNA was isolated from T cells that had been stimulated under Th1 or Th17 polarizing conditions at indicated time points. Relative expression of α4 integrin was determined by quantitative RT-PCR analysis. Bars indicate fold change in relative expression of α4 integrin in Th1 versus Th17 cells. Shown are representatives of four independent experiments. (D) Naive T cells (CD4+CD44−Foxp3−) from 2D2 × Foxp3gfp.KI MOG35-55-specific TCR transgenic mice were cultivated 1:5 with irradiated syngeneic splenocytes as APCs and 20 µg/ml MOG35-55 peptide under Th1 or Th17 polarizing conditions, respectively. α4 integrin expression and cytokine status were determined on day 4 after the start of differentiation by flow cytometry. Shown are representatives of two independent experiments. (E) Naive (CD4+CD44−CD25−) T cells were FACS sorted and polarized under Th1 or Th17 conditions (R1). After a resting phase, T cells were restimulated by plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence of IL-12 or IL-23 (R2) and allowed to proliferate for another 3 d before analyzing the expression levels of surface α4 integrin and intracellular IL-17 and IFN-γ by flow cytometry (numbers indicate percentages of cytokine-positive cells). In the histogram plots, numbers indicate mean fluorescence intensity (MFI) of α4 integrin surface expression in Th1 (black numbers) or Th17 cells (gray numbers), respectively. Shown are representatives of two independent experiments.

Expression of α4 integrin mRNA and protein are down-regulated in Th17 cells. (A) Purified naive T cells (CD4+CD44−Foxp3−) isolated from Foxp3gfp.KI mice were cultured under Th0 (no cytokines), Th1 (IL-12 and anti–IL-4), or Th17 (TGF-β plus IL-6 ± IL-23) polarizing conditions with polyclonal TCR stimulation. On day 3, expression of α4 integrin was determined by surface staining (top row: tinted line, isotype control; black line, α-α4 integrin). The differentiation status was confirmed by intracellular cytokine staining (bottom row: numbers indicate percentages of cytokine-positive cells). Shown are representatives of more than five independent experiments. (B) Time course of α4 integrin expression during Th1 and Th17 differentiation as determined by surface staining (means + SD, n = 4, Student’s t test). (C) RNA was isolated from T cells that had been stimulated under Th1 or Th17 polarizing conditions at indicated time points. Relative expression of α4 integrin was determined by quantitative RT-PCR analysis. Bars indicate fold change in relative expression of α4 integrin in Th1 versus Th17 cells. Shown are representatives of four independent experiments. (D) Naive T cells (CD4+CD44−Foxp3−) from 2D2 × Foxp3gfp.KI MOG35-55-specific TCR transgenic mice were cultivated 1:5 with irradiated syngeneic splenocytes as APCs and 20 µg/ml MOG35-55 peptide under Th1 or Th17 polarizing conditions, respectively. α4 integrin expression and cytokine status were determined on day 4 after the start of differentiation by flow cytometry. Shown are representatives of two independent experiments. (E) Naive (CD4+CD44−CD25−) T cells were FACS sorted and polarized under Th1 or Th17 conditions (R1). After a resting phase, T cells were restimulated by plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence of IL-12 or IL-23 (R2) and allowed to proliferate for another 3 d before analyzing the expression levels of surface α4 integrin and intracellular IL-17 and IFN-γ by flow cytometry (numbers indicate percentages of cytokine-positive cells). In the histogram plots, numbers indicate mean fluorescence intensity (MFI) of α4 integrin surface expression in Th1 (black numbers) or Th17 cells (gray numbers), respectively. Shown are representatives of two independent experiments.

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