IFN-γ requires CD99 to up-regulate HLA class I expression. (A) Kinetics of HLA class I surface expression in IFN-γ–stimulated cells. After treatment with IFN-γ, cells were incubated at 37°C for the indicated time and were then stained with the FITC-HLA class I “W6/32” mAb. The levels of HLA class I molecules in CD99_wt, CD99_neg, and CD99_pos Jurkat T cells were then measured by flow cytometry. (B) CD99_wt, CD99_neg, and CD99_pos Jurkat cells were grown in medium alone or stimulated with IFN-γ. Cells were permeabilized and HLA class I molecules were detected using the FITC-HLA class I “W6/32” mAb, and analyzed by deconvolution fluorescence microscopy. (C) Surface expression of the IFN-γ receptor α-chain in CD99_wt, CD99_neg, and CD99_pos Jurkat cells were analyzed by flow cytometry using a PE-STAT1 mAb. (D) STAT1 phosphorylation was tested by Western blotting. Cell lysates were prepared from untreated (−) or 20-minute IFN-γ–treated (+) CD99_neg andCD99_pos Jurkat cells. Cell lysates were immunoblotted for phospho-Tyr 701 STAT1 (top panel). The membrane was stripped and reprobed with an anti-STAT1 Ab (bottom panel).