Figure 7: Antigen activation initiates the expression of IL-23R on CD4^hiCD8^hiDP thymocytes.

(a) Flow cytometry analysis of the expression of αβTCR and CD3∈ on IL-23R⁺CD4⁺CD8⁺ or IL-23R⁻ CD4⁺CD8⁺ thymocytes from naïve C57L/B6J mice. The percentage of αβTCR⁺ or CD3∈ cells in the indicated population is shown. (b–e) Female D^b/H-Y TCR Tg mice and female C57L/B6J mice were injected with either AdIL-23 or AdLacZ. Mice were killed at day 9. (b) Left: Gross anatomic images showing the size of a representative thymus of the indicated mouse strains. Right: The weight and cellularity are shown. Scale bars, 3.0 mm. (c) In situ hematoxylin and eosin (H&E) (left) and in situ TUNEL staining (dark brown) (right) of a representative thymus section from the indicated group. The magnification of objective lens is × 10 × . Scale bars, 300 μm. (d) BioQuant quantification of the frequency of thymic cortical area (left) or the frequency of TUNEL-positive area (right) from 10 randomly chosen areas. The results represent mean±s.e.m. (N=2–3 per group for two independent experiments, *P<0.05, **P<0.01, ***P<0.005 for the indicated sample compared with AdLacZ injected group). (e) Left: Flow cytometry analysis of CD4 and CD8 expression on Thy1.2⁺ cells in the indicated group. The percent of CD4⁺CD8⁺ cells is shown. Right: Flow cytometry analysis of IL-23R on CD4^loCD8^lo and CD4^hiCD8^hi thymocytes. The percentage of IL-23R⁺ cells is indicated. (f) Thymocytes from female D^b/H-Y mice were co-cultured in vitro with irradiated female or male splenocytes from Thy1.1 C57BL/6 in the presence or absence of exogenous mouse recombinant IL-23. Top: Flow cytometry analysis of CD4 and CD8 expression on Thy1.2⁺ cells in the indicated group. The percent of CD4^hiCD8^hi cells is shown; Middle: The expression of IL-23R and RORγt on gated Thy1.2⁺CD4⁺CD8⁺ cells is shown; bottom: analysis of apoptosis (7-amino actinomycin D (7-AAD)) in Thy1.2⁺CD4⁺CD8⁺ cells. The percent of apoptotic cells is shown (N=3–4 mice per group for two independent experiments; *P<0.05, **P<0.01, ***P<0.005 between thymocytes stimulated with female spleen cells in the absence of IL-23 compared with the indicated group).