Figure 2: Functional characterization of 2D2-IEL populations.

(a) Sorted populations of 2D2-IEL (CD4⁺ and CD4⁻, 2D2-IEL-T_HIGH and 2D2-IEL-T_LOW) or spleen CD4⁺ T (CD4⁺2D2-Sp-T) cells from 2D2 mice were stimulated with anti-CD3 and anti-CD28 mAb for 3 days in vitro. Cell proliferation was measured by incorporation of [³H] thymidine (c.p.m.) (data represent mean and s.e.m. of triplicates). (b) Supernatants from cultures used in a were analysed for cytokine production (mean±s.e.m. of triplicates). Representative data of two experiments are shown in a and b. (c) Gene expression of the indicated transcription factors. mRNA obtained from 2D2-IEL (CD4⁺- or CD4⁻-T_HIGH and -T_LOW) or 2D2-spleen-CD4⁺ T cells was analysed by quantitative RT–PCR (n=3) (mean and s.e.m.). The expression levels were normalized to that of Gapdh (glyceraldehyde phosphate dehydrogenase). (d) Intracellular Foxp3 staining of 2D2-IEL and 2D2-spleen CD4⁺ T cells. (e) Intracellular staining of IL-17A and IL-10 in CD4⁺2D2-IEL-T_HIGH cells and 2D2-spleen-CD4⁺ T cells. Cells were stimulated with PMA (500 ng ml⁻¹) plus ionomycin (500 ng ml⁻¹) (d,e). Data are representative of two experiments. (f) Expression of chemokine receptors analysed by quantitative RT–PCR as in c (mean and s.e.m.). c.p.m., counts per minute; PMA, phorbol 12-myristate 13-acetate; RT–PCR, reverse transcription PCR.