Nanog T cells suppress stromal cell–dependent T-cell development in vitro. (A) Notch signal–dependent growth of Nanog T cells in vitro. Upper panel shows the growth rate of Nanog T cells cocultured with OP9 or OP9-DL1. Middle panel shows the expression of Hes1 at day 5 of coculture. Lower panel shows the Western blotting for cleaved active Notch1. WT and Nanog T cells were cocultured on OP-DL1 with or without DAPT for 2 days. Cells (5 × 105) were used for Western blotting. (B) Growth advantage of Nanog T cells on OP9-DL1. Wild-type (wt) DN thymocytes and Nanog T cells were mixed at varying ratios, and a total of 2 × 104 cells were cultured on OP9-DL1. Five days later, all cells were harvested and GFP+ (filled column) and GFP− (open column) lymphocytes were counted. The ratio of wild-type DN cells to Nanog T cells are (1) 1:0, (2) 3:1, (3) 1:1, (4) 1:3, and (5) 0:1. (C) Nanog T cells inhibit the differentiation of wild-type thymocytes in vitro. FACS analysis of GFP− cells in cultures containing only wt cells or a 1:1 mixture of wt and Nanog thymocytes. In the absence of Nanog T cells, all stages of T-cell development from DN to SP cells were generated on OP9-DL1. However, differentiation of DP cells was markedly suppressed by coculture with Nanog T cells. DN gated means CD4−CD8− gated. (D) Stromal cell damage induced by Nanog T cells. WT or Nanog thymocytes (104, 2.5 × 104, or 5.0 × 104) were cultured on either OP9 or OP9-DL1 cells for 5 days. The proportion of live to dead stromal cells was determined by FACS of PI-stained cells. Stromal cells were distinguished from lymphocytes by their expression of DsRED.
