Functional WT1-specific T cells are present in IST-responsive MDS patients. (A) PBMCs from 38 patients with MDS were stimulated with a comprehensive WT1 peptide library as described in “Flow cytometry for intracellular cytokine production.” Cells cultured in R10 alone were used as negative controls. Live T cells were discriminated from dead (ViViD⁺) cells, B cells, and monocytes in a dump versus CD3 bivariate plot. Next, single cells were selected in a FSC-A versus FSC-H plot, and inact lymphocytes in a FSC-A versus side scatter-area (SSC-A) plot. Fluorochrome aggregates were then excluded (not shown) before selection of the CD4⁺ and CD8⁺ T cells. (B) Cytokine production after stimulation for 6 hours with the WT1 peptide library or positive control (staphylococcal enterotoxin B); unstimulated cells were used as a negative control. The percentages of cytokine-producing CD4⁺ (bottom) and CD8⁺ (top) T cells are shown for IST-responsive patients 45 and 50, respectively. (C) A composite figure for cytokine expression in response to stimulation with the comprehensive WT1 peptide library in IST responders and nonresponders. Horizontal bars represent mean values. (D) WT1-specific TNF-α production by CD4⁺ and CD8⁺ T cells was correlated with the percentage of trisomy 8 cells in the bone marrow. (E) WT1-specific TNF-α production by CD4⁺ T cells was correlated with the presence of HLA DR15.