Identification and Expansion of Myeloma-Specific Cytotoxic T Cells In Vitro.

Multiple myeloma (MM) has been considered as low immunogenic incurable disease. The attempts has been made to invert the immune status to recognize myeloma cells by T cells or other cells of the immune system. Here we studied biology of myeloma-specific T cells in vitro.

Irradiated myeloma cell line ARH 77 has been used as tumor antigen to stimulate peripheral blood mononuclear cells (PBMC) of 8 healthy volunteers. Dendritic Cells loaded by irradiated autologous MM cells has been used to stimulate PBMC of 10 MM patients. Activated responder T cells have been immunomagnetically separated based on surface expression of interferon gamma (Miltenyi Biotech) and expanded in 5 cases by phytohemaglutinin and repeated high-doses of interleukin 2. Cytotoxicity against the original myeloma cells has been tested after the expansion using propidium iodide or 7-amino actinomycin D. Responder T cells were labeled by CFSE to distinguish them from myeloma cells. Third-party PBMC and interferon gamma negative fraction of T cells served as controls.

The percentage of interferon gamma positive cells in healthy donors has been enriched from 2.8±0.9 and 2.6±0.8 to 48.6±23.4 and 73.2±25.9 of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells, respectively, by immunomagnetic separation. Interferon gamma positive T cells have been further expanded in vitro from 0.54x10⁶ ± 0.05x10⁶ to 214.00x10⁶ ± 103.46x10⁶ within 4 weeks. A specific cytotoxicity has been tested after expansion. The killing of myeloma cells by expanded IFN g⁺ T cells reached 68.1±14.2%, while interferon gamma negative fraction killed only 0.8±0.3% of myeloma cells. As another control, killing of third-party PBMC by expanded interferon gamma positive T cells was 6.9±2.5%. Similar observations were made in MM patients and will be presented at the meeting.

These data demonstrate a specific cytotoxic effect of expanded interferon gamma positive T cells against myeloma cell line ARH 77 and open the possibility for clinical use of tumor-specific T cells in MM.