CD8⁺ T cells within expanded Vβ subfamilies respond functionally to WT1 peptides and suppress trisomy 8 colony growth. Functional and tetrameric analyses of CD8⁺ T cells within expanded Vβ subfamilies were performed using samples from patient 1. (A) Expanded CD8⁺ T-cell subfamilies were identified by flow cytometry using directly conjugated Vβ-specific mAbs (top left panel). PBMCs from patient 1 were stimulated with a comprehensive WT1 peptide library and stained as described in “Flow cytometry for intracellular cytokine production.” CD8⁺ T cells within the expanded Vβ subfamilies showed substantial intracellular cytokine production compared with unstimulated controls (top right panel). Expanded and control, nonexpanded Vβ subfamilies were subsequently sorted by flow cytometry and cultured for up to 3 weeks. These T-cell lines were then tested for clonogenic hematopoietic progenitor cell lysis (bottom left panel): Autologous BMMNCs were incubated for 4 hours with the (25 Gy-irradiated) expanded T cells (BM + T INC) before plating in semisolid medium containing cytokines to support hematopoietic progenitor cell growth.²⁶  WT1 specificity of the T-cell lines was assessed in this assay by pulsing the BMMNCs with the WT1 peptide library before this 4-hour preincubation (BM + T + WT1 INC). As a negative control, we incubated T cells and BMMNCs separately before mixing and plating (BM + T NO INC). Suppression of trisomy 8 cell growth was observed, relative to both controls in the absence of CD8⁺ T cells and in the presence of 3 times the number of CD8⁺ T cells from Vβ subfamilies that were not expanded (bottom panel). (B) Cognate WT1_126-134/HLA-A*0201 tetramer-binding memory CD8⁺ T cells were present within the expanded Vβ3⁺ subfamily.