Transcriptional differences between circulating and IVS CD8⁺ T cells. (A) Kinetics of response to repeated subcutaneous immunizations with 209-2M. PBMCs obtained before treatment and 3 weeks after 8, 16, and 24 immunizations were simultaneously thawed and tested. Staining was performed with FITC-conjugated anti-CD8 mAb and PE-labeled tHLA/209-2M. In each experiment 200 000 events were analyzed per sample. The first column shows patient 2 (P2) as representative of most patients; the second column portrays the unusual case of patient 6 (P6). T-cell precursor frequency is presented as percent of CD8⁺ T cells in each histogram. The lower panels show the expression of CD45RA and CD27 or tHLA/209-2M and perforin in the 24i samples from the 2 patients. In addition, the level of perforin mRNA expression is shown in color code (green less than and red more than the average expression of perforin mRNA in pooled PBMCs). (B) Enrichment of 209-2M– or Tax-specific CD8⁺ T cells from PBMCs or IVS. In all experiments, the purity of tHLA^– CD8⁺ T cells was above 95%. (C) Eisen hierarchical clustering of all samples applied to a data set of 7580 genes allowed by high-stringency filtering (Cy5/Cy3 ratios with at least a 3-fold change, signal intensity > 500 unless the other channel > 3000 in at least 80% of the sample tested). Samples from individual patients are color coded. 2M indicates 209-2M–specific T cells from patients with melanoma; tax, HTLV-1 Tax-specific T cells from patients with HAM; +, tHLA positive; –, tHLA negative. Horizontal bars underline clusters enriched with circulating (blue) and IVS (orange) CD8⁺ T cells. Blue and orange vertical bars underline functional signatures specific for the 2 respective clusters.