Because of advances in immunological technology for detecting a very small number of blood CTL cells, clinicians have been able to monitor cellular immunity against CMV and evaluate the status of CMV infections in highly advanced cancer patients or transplant recipients. Our previous study using healthy volunteer PBLs revealed a significant increase in CMV HLA-A24 tetramer+ CTLs after stimulation in vitro with autologous DCs. However, the efficiency of CMV A24 peptide-specific CTL expansion in highly advanced cancer patients has yet to be studied in detail. In the present study, we tried to characterize and expand HLA-A*2402 CMVpp65 peptide (QYDPVAALF)-specific tetramer+ CTLs from HLA-A*2402+ metastatic melanoma patients, and eventually demonstrated that expansion efficiency was closely related to both post-stimulation CMV tetramer frequency and anti-CMV IgG titer. This is a novel finding regarding in vitro CMVpp65-A24 peptide-specific CTL expansion based on metastatic cancer patient-derived PBLs. Interestingly, the current results using metastatic melanoma PBLs showed a much higher frequency of CMVpp65-A24 tetramer+ CTLs and expansion efficiency than in healthy volunteers. Finally, we were successful in cloning CMVpp65 HLA-A24 peptide-specific TCR cDNAs from in vitro expanded CTL lines derived from melanoma patients. Additionally, CMVpp65 HLA-A24 peptide-specific TCR cDNA was transduced into naive T cells from patients and functionally reconstructed. The results showed that cloned CMV-specific TCR genes were efficient in reconstituting specific anti-CMV activity and might be good tools for adoptive immunotherapy against CMV infections.

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