Figure 3: Androgen enhances Aire transcription, TSA expression and negative selection of self-reactive T cells.

(a) Relative AIRE mRNA expression levels in male and female thymic stromal cells incubated with 10 nM DHT or vehicle control (−) for 6 h. Cells were from <1-week-old paired human subjects. Data shown are representative of at least three representative experiments. (b) Relative Aire mRNA expression levels in thymic stroma of 6-week-old male and female mice incubated with 10 nM DHT or vehicle control (−) for 6 h. Four mice were pooled per group, and data shown are representative of at least two independent experiments. (c) Relative Aire mRNA expression in LNCaP cells pretreated with indicated concentrations of 5-Aza and 100 ng ml⁻¹ trichostatin in the presence of 10 nM DHT (gray bars) or vehicle control (black bars) for 8 h. Data shown are representative of two independent experiments. (d,e) Relative Aire (d) and TSA (e) (Spt-1 and TRP-1) mRNA expression in isolated thymic stroma from mice treated with DHT pellets or sham implant procedure. (f) Two-week-old TRP-1 TCR Tg RAG^−/− male mice were treated with 20 mg kg⁻¹ acyline to minimize endogenous androgen production and implanted with DHT pellets or sham treated for 4 weeks total. Representative flow cytometry plots of thymocytes with frequencies of CD4 single-positive (CD4SP), double-positive (DP), CD8 single-positive (CD8SP) and double-negative (DN; clockwise, starting from left) populations shown in DHT-treated versus sham-operated male mice. (g) Average frequencies (top) and absolute numbers (bottom) of thymocyte subpopualtions as indicated for DHT-treated (n=4) and sham-operated (n=6) male mice. (h) Flow cytometric analysis of annexin V levels in CD4 single-positive thymocytes. Representative flow plot (left) and cumulative data (right) are shown. For all data sets, unpaired Student’s t-test was used for comparisons. Error bars represent s.e.m. *P<0.05. NS, not significant.