Validation of microarray by real-time PCR. (A) Expression regulation of tested genes across 3 patients (patient nos. 4-6). Relative mRNA levels corresponding to representative genes from CD3⁺CD4⁻TCRVβ⁺CD57⁺ and CD3⁺CD4⁻TCRVβ⁺CD57⁻ cells were determined by quantitative real-time PCR. The expression level of each gene was normalized to a mean reference value, which was the average of GAPDH and β-ACTIN. Results are presented as mean ± SD of at least triplicates. (B) Dot plot of the expression values in fold change ratio between microarray and qPCR from all validated genes and patient samples. Linear regression demonstrates a significant correlation (R² = 0.8846; P < .006). (C) Protein expression as demonstrated by flow cytometry assay of CD28, GZMB, and perforin in the CD57⁺ (clonal) and CD57⁻ (polyclonal) TCRVβ3-restricted CD8 cells in a representative patient. (D) Significant up-regulated perforin and granzyme-B expression and down-regulated CD28 expression in the CD57⁺ subpopulation was present in all 11 TCRVβ expansions studied but results have been recorded only when more than 50 cells were able to be analyzed by flow cytometry in each subpopulation.