Th1 cell differentiation is promoted by CD4⁺ T cell activation.

<p>CD4⁺ T cells were cultivated on astrocytes in the presence of 10 µg/mL anti-IL-4 alone (anti-IL-4), with 10 µg/mL anti-IL-4 and 10 ng/mL IL-12 (Th1), or with 10 µg/mL anti-IL-4 and 2.5 ng/mL TGFβ (Treg) for 4 days. Cells were stained with Hoechst and fixed and analyzed using an ArrayScan VTI automated fluorescent microscope. (A) astrocytes alone with nuclei traced (a 20-fold magnification is shown in the small box), (B) astrocytes co-cultured with CD4⁺ T cells with nuclei traced (a 20-fold magnification is shown in the small box), (C) quantitation of percent of CD4⁺ T cells associated per astrocyte (mean ± s.e.m.; n = 3), (D) scatter plot showing the cells identified as astrocytes (red) and CD4⁺ T cells (blue). (E) CD4⁺ T cells were cultivated on astrocytes in the presence of 10 µg/mL anti-IL-4 and 10 ng/mL IL-12 for 4 days, with or without 10 µg/mL anti-CD4 (GK1.5). Cells were washed, and restimulated with PMA/ionomycin/BFA for 4 h and analyzed by flow cytometry. The histogram represents IFNγ-expressing CD4⁺ T cells (n = 2, unpaired t-test *p<0.05). (F) CD4⁺ T cells were cultivated on astrocytes in the presence of 10 µg/mL anti-IL-4 and with 10 ng/mL IL-12 for 4 days, with or without 10 µg/mL anti-CD4 (GK1.5). Cells were stained with Hoechst and fixed and analyzed with an ArrayScan VTI automated fluorescent microscope. Bars represent mean ± s.e.m. (n = 3).