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We have previously characterised the WT-1 HLA-A2.1 restricted peptide epitope p 126-134 and shown that allorestricted CTLs recognising specifically the p 126-134 epitope were able efficiently and selectively to lyse CML progenitor cells. The aim of the present study is to investigate whether an immunoresponse to the WT-1 p126 epitope is present in CML patients. Tetramers specific for the p 126 peptide epitope were used to quantify the frequency of WT-1 specific CDS T cells ex vivo from freshly separated PB MC of CML patients and normal donors. CTL cell lines raised against the p 126 peptides stained brightly with the fluorescent p 126 tetramers and no staining was seen when irrelevant tetramers were used instead. This indicates that the tetrameric complexes selectively stain WT-1 p 126 specific CTLs. CML patients and normal donors were selected for the study on the basis of the expression of the HLA-A2.1 allele. PBMC from 10 HLA-A2.1 positive CML patients(4 IFN, 3 post-BMT, 2 post-DLI and 1 HU) and 7 HLA-A2 negative CML controls (4 HU, 2 post-BMT and 1 IFN) were triple stained with anti-CD8, anti-CD4 mAbs and with the p126 tetrameric complexes. In two out of 10 HLA-A2.1 positive CML cells 0.04% of CD8+ cells stained with the tetramer, equivalent to a frequency of 1:2500 CD8+ T cells. This exceeded the background level of 0.03% in circulating CD8+ cells observed in the control group of HLA-A2 negative CML patients and in HLA-A2 positive PBMC in normal donors (5 donors examined). The present tetramer data are compatible with a lack of CTL responses to the WT-1 p 126 epitope or with a low frequency of specific CTLs in CML patients. Ongoing functional experiments will show whether p 126 responsive CTLs can be isolated from HLA-A2.1 positive CML patients. These experiments will determine whether the p 126 epitope can be used for vaccination strategies aimed at expanding autologous CTLs, or whether strategies based on allo-restricted CTLs will be required to overcome poor autologous T cell responses.


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