Figure 4. EBV-induced CD8⁺ NKT cell differentiation depends upon thymic dendritic cells.

(A) The experimental and analysis scheme for detecting co-receptor-expressing NKT cells and T cells in various reaggregated fetal thymic organ cultures (RTOCs). After 14-days of culture, the various cell types were identified by flow cytometry as in Figure 2. (B–D) Frequency of total NKT cells and total T cells (B), co-receptor-expressing NKT cells (C) and T cells (D), in the different RTOCs. The protocols for the establishment of the RTOCs were described in the leftmost panels in each sub-figure: DP thymocytes were reaggregated with either total thymic stromal cells (DC-included), purified thymic dendritic cells, or BM-derived dendritic cells. The stimuli were added as indicated. Nil, no stimulus; EBV-epitopes, HLA-A2-restricted, derived from the lytic cycle protein BMLF1 and HLA-DRB1-restricted, derived from nuclear antigen EBNA1 (10 µg/ml each); EBV, infectious EBV (10⁷ pfu); Solvent, 0.005% polysorbate 20; α-GalCer (0.1 µg/ml). The RTOCs were harvested, and assessed by flow cytometry. Only the data for CD4⁺ and CD8⁺ NKT cells, and CD4 SP and CD8 SP T cells were shown. VL, very low level (below detectable level). Data were mean ± s.d. (n  =  10). *, p<0.001, EBV-challenged RTOCs vs. non-challenged RTOCs.