Quantitative expression of CD99 and HLA class I molecules is closely related. (A) CD99 and HLA class I surface expression was measured by flow cytometry for wild-type Jurkat T cells, CD99_neg and CD99_pos cells. CD49d (alpha 4 integrin), CD29 (beta 1 integrin), CD47, and CD45 were measured as controls using “BU49,” “K20,” “B6H12,” and “MEM-28” mAbs, respectively. (B) Effects of a CD99 shRNA on HLA class I expression measured by Western blot analysis with the anti–HLA class I “W6/32” mAb; CD99 expression was measured using the “12E7” mAb. (C) Flow cytometric analysis was performed with a PE-CD99 “3B2/TA8” mAb and with an FITC-HLA class I “W6/32” mAb; analysis was performed 72 hours after transfection with CD99-shRNA or control vector. Results are reported as dot-plot profiles and are representative of at least 3 independent experiments. For controls we included staining with an Ig control isotype and FITC-CD47 (B6H12). (D) Surface expression of HLA class I on BLA2 cells, a lung adenocarcinoma cell line, forced to express CD99. Two gates (R1 and R2) were defined on CD99 histograms (top left panel) and HLA class I expression was measured on cells from each gate (bottom panel). CD44 expression was used as control. Results are representative of at least 3 independent experiments. (E) Correlation between CD99 and HLA class I molecules at the surface of Jurkat CD99_neg cells stably transfected with various amounts of CD99 cDNA. Cells were costained with the FITC-CD99 “3B2/TA8” mAb and a PE-HLA class I “W6/32” mAb (Pearson correlation coefficient r = 0.89; P < 10⁻⁴).