Figure 9.
Dox dose-dependent T cell development and thymic selection in Tet-on ZAP mice. (A) CD4 and CD8 expression by thymocytes from Tet-on ZAP mice treated with Dox 2.0 or Dox 0.5 food for 9 wk. Total thymocyte numbers above each plot. (B) GFP expression in thymocyte subsets from Dox-treated Tet-on ZAP mice as in A. Shaded area indicates negative control. Representative of more than five experiments. (C) Numbers of thymic CD4SP, CD8SP, Foxp3+CD4SP, and total thymocytes (left), and splenic CD4+, CD8+ T cells, or Foxp3+CD4+ Treg cells (right) from Dox 2.0 or 0.5 Tet-on ZAP mice Dox-treated for 10 wk (n = 10 each). (D) Correlation between GFP expression (MFI) and frequency of CD4SP, CD8SP, or Foxp3+/CD4SP TCRβhi thymocytes in individual Dox 2.0 or 0.5 Tet-on ZAP mice (n = 10 and 12–16, respectively). (E) Correlation between GFP MFIs and frequency of indicated TCR Vβ+ CD4SP thymocytes in Tet-on ZAP mice. Each circle indicates individual mice and average ratios in adult BALB/c mice (horizontal bar). (F) TCR Vβ subfamily usage by splenic Foxp3− or Foxp3+CD4+ T cells (n = 4). (G) In vitro proliferation of splenic CD4+ T cells from Dox 0.5 or 2.0 Tet-on ZAP mice in the presence of αCD3 antibody and indicated Dox concentrations for 3 d (n = 6). (H) Numbers of splenic CD4+ or CD8+ T cells in RAG−/− mice 8 wk after the transfer of 5 × 105 splenic T cells from Tet-on ZAP mice treated with Dox 2.0 or 0.5 food for 8 wk (n = 5). The recipient RAG−/− mice were maintained with Dox 0.5 food. (I) In vitro suppressive activity of Tet-on ZAP or BALB/c CD25hiCD4+ Treg cells on the proliferation of BALB/c CD25−CD4+ T cells (Tconv) at various ratios in the presence (black) or absence (white) of Dox (1 μg/ml). Proliferation of cocultured cells was shown as a relative percent of CD25−CD4+ T cells alone at various Dox concentrations. Representative of three independent experiments. Two-tailed unpaired Student’s t test (C, F, and H); Spearman rank correlation test (D and E). Mean ± SD in C, and F–H.