Staining for Melan-A (antibody M27C10), NY-ESO-1/LAGE-1 (antibody D8.38, a gift from G. Spagnoli, Basel, Switzerland), and MAGE-A proteins (detected either by clone 57B, top 2 panels, or clone 6C1, bottom 2 panels) was performed on paraffin sections of metastases removed at the times indicated. Subcutaneous metastases (Apr. 03, Mar. 04), LN (Oct. 03) and the last brain metastasis (June 07) are shown. Insets show CT antigen negative areas in the last metastasis.

(A) Liver and multiple lung metastases seen before the start of chemotherapy in 04.2004. Complete remission was seen since 10.2004 (computed tomography scan not shown), persisting in 09.2005. Massive relapse of a right temporal brain metastasis was observed in 07.2007, with no extracranial evidence of disease. (B) Percentage of in vivo circulating HLA-A2/Melan-A tetramer positive T cells in CD8 T cells over time. The time period for immuno- and chemotherapy are indicated. Abbreviations: pp, peptide; IFA, incomplete Freund's adjuvant; CpG, CPG 7909 oligonucleotides. (C) Flow cytometry of PBMCs (two time-points) showing relatively high percentages of HLA-A2/Melan-A tetramer positive T cells. Gating on these cells reveals that they are mostly CCR7- and CD45RA- (so called effector-memory cells), with a progressively increasing proportion of CD28- cells. (D) Spectratyping of PCR products amplified with primers specific for the TCR b-chain variable region BV7 shows a single peak with a unique CDR3 length (207 bp). Subsequent sequencing confirms the presence of a single dominant T cell clonotype, with a TCR composed of BV7, BJ1.5 and the CDR3b variable region sequence indicated.