CD1d-dependent T-cell reactivity in response to endogenous GPI. (A) (Left) Representative flow cytometry plots showing intracellular IFNγ-producing CD8⁺ T cells from a PNH patient after monocyte-depleted PBMCs were cocultured with C1R, C1R-CD1d/GPI-positive, or C1R-CD1d/GPI-negative B cells for 7 days, followed by the addition of the same number of APCs. Brefeldin A was added 16 hours before harvesting the culture. Cells were stained with mAbs against human CD3, CD8, and intracellular IFNγ. Flow cytometry plots shown are gated on CD3⁺ cells. (Right) Cumulative data from 9 patients showing frequency of CD8⁺ T cells expressing intracellular IFNγ (Wilcoxon signed rank test). (B) IFNγ ELISPOT assays performed with CD8⁺ T cells from 4 PNH patients cultured for 24 hours with C1R-CD1d/GPI-positive cells and its GPI-negative derivative. The APC number was 2 × 10⁴ in each well; E:T ratios as shown. (C) (Left) Representative flow cytometry plots showing IFNγ-producing, patient CD8⁺ T cells after a 3-day coculture of monocyte-depleted PBMCs with autologous, GPI-negative, mature DCs loaded with h-GPI or Veh. Flow cytometry plots shown are gated on CD3⁺ cells. (Right) Cumulative data from 11 PNH patients (Wilcoxon signed rank test).