Figure 3.

Soluble factors, such as interleukin-7 (IL-7) and transforming growth factor-β (TGF-β), do not contribute to stromal cell support for regulatory T (Treg) cells. (a) IL-7, or (b) TGF-β₁ and TGF-β₂ expression in stromal cells was assessed by RT-PCR. Total RNA prepared from stromal cells and ST-2 cells was used. (c) Culture supernatants of stromal cells and ST-2 cells (1·2 × 10⁴ cells, for 3 days culture) were harvested. TGF-β₁ concentrations in the supernatants were measured by ELISA. (d) Cells from lymph nodes were cultured with stromal cells or ST-2 cells for 5 days. Expression levels of CD4 and CD25 were assessed by flow cytometry. TGF-β neutralizing monoclonal antibody (mAb), clone 1D11, was added into the co-culture of lymphocytes and stromal cells. Proportions of CD4⁺ CD25⁺ cells and CD4⁺ CD25⁻ cells are indicated. (e) Culture supernatants of stromal cells, CD4⁺ T cells, and CD4⁺ T cells with stromal cells (stromal cells: 5 × 10³, CD4⁺ T cells: 5 × 10⁵, for 5 days culture) were harvested. IL-2 concentrations in the supernatants were measured by ELISA. ND, not detected. (f) Supernatants from stromal cell culture or co-culture of stromal cells with CD4⁺ T cells were added into CD4⁺ T-cell culture at a final concentration of 50%. Five days later, proportions of CD4⁺CD25⁺ cells among the cultures were assessed. Indexes of proportions of CD4⁺ CD25⁺ cells are indicated. (NS indicates ‘not significant’, n = 4). (g) Lymphocytes, prepared from lymph nodes and spleens, and stromal cells were co-cultured in the same culture well. Lymphocytes were allowed to interact with the stromal cells directly (middle panel) or they were co-cultured separately using a cell culture insert system (bottom panels). As a control culture, lymphocytes were cultured alone (top panels). Five days after culture, expression levels of CD4, CD25 and Foxp3 were assessed. Indexes of proportions of CD4⁺ CD25⁺ cells in the cultures are summarized by bar graphs. (*P < 0·0001, **P < 0. 01, NS indicates ‘not significant’, n = 5) The data shown represent one of two (a–c) or three (e) experiments.