GPI-specific and IFN-γ-producing T cells in patients with IAA. (A) Representative FACS density plots. Monocyte-depleted peripheral blood mononuclear cells were cocultured using as APCs a C1R-CD1d/GPI-positive lymphoblastoid B-cell lines (E:T ratio 2:1). On day 6, the same number of APCs added initially were added once again. On day 7, cells were stained with mAbs against human CD3 and CD8, as well as with h-GPI-loaded CD1d dimer. The CD8⁺ CD1d/GPI dimer⁺ T cells (GPI-specific T cells) are shown here as percentage of the total T (CD3⁺) cells. By this technique, we are able to detect GPI-specific T cells when they are >1:25.000 CD3⁺ T cells. (B) Frequency of CD1d/GPI dimer⁺ CD8⁺ T cells (GPI-specific T cells) from healthy control patients, patients with PNH, and patients with IAA tested at the time of diagnosis. On the ordinate, T cells counted as percentage of CD3⁺ T cells. P values by Mann Whitney test are shown. (C) Frequency of GPI-specific T cells measured on sequential samples taken at diagnosis and at different times (1-84 months) from the start of immunosuppressive treatment. In these 6 patients, the average frequency decreased significantly from 1.4 ± 1% at diagnosis to 0.4 ± 0.3% at the last follow-up point (P = .03, Wilcoxon signed rank test). (D) Representative FACS density plots. Monocyte-depleted peripheral blood mononuclear cells were cocultured, using as APCs the C1R-CD1d/GPI-negative (right) as well as the C1R-CD1d/GPI-positive (left) lymphoblastoid B-cell lines (E:T ratio, 2:1). On day 6, the same number of APCs added initially were added once again. On day 7, cells were stained with mAbs against human CD3 and CD8, as well as intracellular IFN-γ. The IFN-γ⁺Cd8⁺ T cells (IFN-γ-producing T cells) are shown here as percentage of the total T (CD3⁺) cells. By this technique, we are able to detect IFN-γ-producing T cells when they are >1:25.000 CD3⁺ T cells. (E) Frequency of IFN-γ⁺CD8⁺ T cells (IFN-γ-producing T cells) obtained after coculture with C1R-CD1d/GPI-negative APC (empty symbols) or C1R-CD1d/GPI-positive APC (full symbols) from 43 healthy control patients, 28 patients with PNH, and 17 patients with IAA studied at the time of diagnosis. On the ordinate, IFN-γ-producing T cells are shown as percentage of the total T (CD3⁺) cells. P values by Mann-Whitney test are shown. (F) The frequency of T cells that specifically produce IFN-γ in response to GPI (GPI-reactive IFN-γ-producing T cells) has been calculated as difference (∆IFN-γ⁺ T cells) between the frequency of IFN-γ-producing T cells after culture on GPI-positive APC and the frequency of IFN-γ-producing T cells after culture on GPI-negative APC. Here GPI-reactive IFN-γ-producing T cells (∆IFNγ⁺ T cells) are shown separately for the 13 patients with IAA who had detectable GPI-specific T cells and for the 4 patients with IAA who did not have detectable GPI-specific T cells. P values by Mann Whitney test are shown.