Figure 2.

RANKL expression on in vitro-derived human proT cells is potentially involved in lymphostromal crosstalk with the thymic stroma. (A) Flow cytometric analysis for RANKL, LTαβ and CD40L expression on day 11 in vitro-derived CD34⁺CD7⁺-gated in vitro-derived proT1 (open, thick line) and proT2 cells (shaded). IgG controls are included as a control (open, thin line). These results are representative of >3 independent experiments for RANKL and LTαβ and 2 independent experiments for CD40L. (B) Flow cytometric analysis of 2 × 10⁵ day 10 in vitro-derived CD34⁺CD7⁺-gated proT cells showing OPG-Fc staining (shaded), IgG control (open, thick line) and unstained cells (open, thin line). OPG-Fc was used at the doses indicated. (C) OPG-Fc was used to block RANK-RANKL interactions in fetal thymic organ culture (FTOC). A schematic of the experimental set-up is shown. (D) Gene expression analyses from day 5 NOD/SCID/γc^null FTOCs. QPCR analysis for the expression of human PTPRC (CD45), and mouse Ccl25, Ccl19, Ccl21, and Krt8 (Cytokeratin-8) for lobes containing no cells (n = 8), proT cells (n = 7) or proT cells with OPG-Fc (n = 7). Transcript levels for all genes were normalized to mouse β-actin. The results shown are representative of at least 3 independent experiments. Statistical analysis was performed using the Kruskal-Wallis one-way analysis of variance with a calculated significance level of p > 0.05, followed by post-hoc Dunn's test. All data are represented as mean ± SEM, with asterisks representing statistical significance as compared to the control (no cells) group (*p < 0.05, **p < 0.01).