Figure 5.

Provision of IL-2 by CD8 T cells is necessary for expansion of DN T cells in vitro. Sorted kidney DN cells and splenic CD8 cells from WT mice (2×10⁴ cells per well used for surface staining or 5×10⁴ cells per well used for intracellular staining) were cultured together or separately and stimulated with immobilized anti-CD3/CD28 for 5 days before cells were harvested and analyzed for surface expression CD25 and intracellular IL-2, and supernatants examined for Th1/Th2/Th17 cytokines using cytometric bead array. (A) Dot plots show the representative flow data of kidney DN T cells and splenic CD8 T cells that were cultured together or separately. Numbers in quadrants indicate percentages. Graph shows absolute cell numbers of each T cell in single and mixed cultures. (B) Graph shows amounts of cytokines in culture supernatants from different cultures. (C) Dot plots show expression of CD25 and intracellular IL-2 by gated DN and CD8 T cells. Numbers in quadrants indicate percentages. Graphs show frequency and absolute cell numbers of CD25⁺ and IL-2⁺ DN or CD8 T cells. (D) Top dot plots show representative flow data of kidney DN T cells and splenic CD8 cells that were cultured together with or without anti-IL-2, or cultured separately. Numbers in quadrants indicate percentages. Top graph shows absolute cell numbers of each T cell in single and mixed cultures. Bottom dot plots show expression of CD25 by gated DN and CD8 T cells. Numbers in quadrants indicate percentages. Graphs show frequency of CD25⁺ DN or CD8 T cells. (E) Graph shows amounts of cytokines in culture supernatants from different cultures in the presence or absence of anti-IL2. Data points are shown for each mouse with mean ±SEM, from at least three independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.001.