Immune phenotypic analysis of circulating lymphocytes in PNH patients. (A) Expression of IRS members on peripheral-blood T lymphocytes from a patient with PNH. Peripheral-blood mononuclear cells isolated from patient PNH5 (Ai-v) or healthy donor HD3 (Avi-x) were stained with either anti-pan-KIR2D mAb (Aii, Avii), anti-CD158a/h mAb (Aiii, Aviii), anti-CD158b/j mAb (Aiv, Aix), or anti-CLIR (CD94) mAb (Av, Ax) and anti-CD3 mAb (Aii-Av, Avii-Ax) followed by anti-isotype specific goat antimouse antiserum PE-conjugated (for anti-IRS mAbs) or FITC-conjugated (for anti-CD3 mAb). Samples were run on a FACSort and at least 10 000 events were analyzed with the CellQuest computer program (Becton Dickinson). Panels Ai and Avi were stained with an unrelated mAb followed by the second reagent. Results are expressed as Log green fluorescence intensity versus Log red fluorescence intensity in arbitrary units (au). (B) IRS⁺ T cells in patients with PNH have features of cytotoxic T cells. Peripheral-blood mononuclear cells (PNH5) were stained with PE-conjugated anti-CD94 mAb, PerCP-conjugated anti-CD3 (Bi), and either FITC-conjugated anti-CD57 (Bii, Biv) or anti-CD8 (Biv, Biii) mAb. Samples were run on a FACSort and analyzed with the CellQuest program. R1 and R2 regions (Bi), corresponding to CD3⁺CD94⁺ and CD3⁺CD94^- cells respectively, were depicted to analyze the third color (CD57- or CD8-FITC-conjugated; Bii-Bv). Results are expressed as Log PerCP fluorescence intensity versus Log red (PE) fluorescence intensity in arbitrary units (au) (Bi) or Log green (FITC) fluorescence intensity versus number of cells (Bii-Bv). Similar proportions of the different T-cell subsets were found in 3 additional patients with PNH analyzed.