CD1d-dependent T-cell reactivity in response to exogenous GPI. (A) IFNγ spot formation by CD8⁺ T cells from PNH patients and normal donor controls assessed in ELISPOT assays against t-GPI– or Veh-loaded C1R-CD1d cells. Assays were performed on days 1, 7, or 14 at a fixed E:T ratio 3:1 (number of plated T cells varied between 5 × 10⁴ and 10⁵ per well) as described in “Patients, materials, and methods.” The median frequency of IFNγ spot-forming CD8⁺ T cells as a percentage of total CD8⁺ T cells was derived after the mean number of spots of triplicate or quadruplicate assays was calculated. In the box-and-whisker plots, the horizontal line inside the box represents the median, the top and bottom of the box represent the interquartile range, and the vertical bars the range. Number of samples used in each time point and group are also shown. Significance P values were estimated by Mann-Whitney test. (B) IFNγ ELISPOT assay performed with CD8⁺ T cells from 6 PNH patients (P) and 3 normal controls (N) cultured for 24 hours with the parental CD1d-negative C1R cell line or its C1R-CD1d derivative loaded with Veh or t-GPI (E:T ratio of 1:1; 5 × 10⁴ T cells per well plated in triplicate assays). Data are shown as mean ± SEM. (C) IFNγ ELISPOT assay performed with CD8⁺ T cells from 9 PNH patients and 3 controls cultured for 24 hours with the GPI-negative K562 cell line KC or its derivative KC-CD1d loaded with GPI or Veh (E:T ratio of 1:1; 5 × 10⁴ T cells per well plated in triplicate assays). Data are shown as mean ± SEM. *Not performed.