Positive serologic results to RHAMM/CD168, mRNA expression, and specific cellular immune responses against RHAMM/CD168 in a 69-year-old AML patient with a normal karyotype. Positive serologic immune responses against RHAMM/CD168 were detected using the phage plaque assay (A): bold arrows point to positive plaque-forming units (PFUs); thin arrows, to negative PFUs from the background of the cDNA library. Pictures of immunocytological stainings were taken with an Axiophot microscope with 25 × objective lens (Zeiss, Oberkochen, Germany) and a digital camera (JVC, Friedberg, Germany) coupled to a commercially avalable imaging system (Diskus, Königswinter, Germany). Positive mRNA expression was assessed by conventional RT-PCR (B). Lane M represents marker VI; lane 1, AML patient; lane 2, K562; and lane 3, healthy volunteer. In real-time RT-PCR, high expression of RHAMM/CD168 could be detected (C). Curve 1 represents 6.19 × 106 copies; curve 2, 6.19 × 105 copies; curve 3, 6.19 × 104 copies; curve 4, 6.19 × 103 copies; curve 5, 6.19 × 102 copies; curve 6, AML blasts; and curve 7, PBMCs of healthy volunteers (HV). The inset demonstrates the regression analysis with a regression coefficient of 0.99. Specific lysis of T2 cells pulsed with the RHAMM/CD168 R3 peptide occurred as demonstrated in panel D: specific CD8+ T cells generated of MLPC with the RHAMM/CD168-derived peptide R3 were able to lyse R3-pulsed T2 cells but not T2 cells alone.
