High-Fold Expansion of Human Cytotoxic T-Lymphocytes Specific for Autologous Melanoma Cells for Use in Immunotherapy

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Abstract
We set up a culture protocol that consistently allows high-fold expansion of tumor-specific T-lymphocytes from most melanoma-invaded biopsies with low doses of recombinant interleukin-2 (rIL-2). Between 2-60 x 10(6) T-lymphocytes could be obtained and cryopreserved from 12 out of 13 patients, by culturing only 50 mm3 tumor tissue with rIL-2. Thawed lymphocytes from 11 of these patients could then be expanded by a median factor of 32,800 by culturing them successively in microplates on irradiated feeder cells with rIL-2 for approximately 2 weeks and then in culture bags or flasks with only rIL-2 for 1-2 additional weeks. Dead feeder cells disappeared during the last phase of the lymphocyte culture with rIL-2. Interestingly, each time they were expanded under these conditions, tumor-infiltrating lymphocytes (TIL) or lymph-node lymphocytes developed a lytic activity apparently restricted to the autologous melanoma line. Tumor-specific lysis, which was maximum at around the end of T-lymphocyte expansion, ranged between 31-63% lysis at an effector:target (E:T) ratio of 20:1. This culture method would thus appear to be suitable for reliable production of over 10(10) T-lymphocytes with good tumor-specific lytic activity from most melanoma-invaded biopsy. It should permit analysis of the immunotherapeutic potential of these populations reinjected into cancer patients.

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