234. Treatment of melanoma patients with genetically modified tumor vaccines (GMTV) does not decrease the spontaneous apoptosis of CD4+ and CD8+ T cells

In this study, the spontaneous apoptosis of CD4+ and CD8+ T-cells in patients with melanoma were examined. Fifty-seven patients enrolled in the study were participating in an experimental immunotherapy study and were administered vaccines consisting of irradiated, gene modified allogenic melanoma cells: MICH I H6/ GMCSF + MICH II H6/GMCSF. The control group consisted of 20 healthy volunteers. Venous blood was obtained on the day of the administration of the first vaccine, and then approximately once a month. Blood was obtained in EDTA tubes, after which the Iymphocytes were isolated by Ficoll-Hypaque gradient centrifugation. The Iymphocytes were then incubated for 24hrs in medium containing fetal calf serum at 37°C in 5% CO2 to allow for spontaneous apoptosis to take place. The Iymphocytes were then stained with PE-Iabeled CD4 or CD8 and next, stained for apoptosis using the FITC-labeled Annexin V binding method. A two color flow cytometry was used to measure the proportion of CD4+ and CD8+ Iymphocytes that underwent apoptosis. SD) of CD4+ T cells were Annexin±6.3% (mean ±In patients with melanoma 21 6.3% in healthy controls (P±V+ compared with 15 < 0.0001). For CD8+ T cells, 4.5%±8.1% of T cells were Annexin V+ and in healthy controls 19±in patients 31 of T cells were Annexin V+(P < 0.0001). There were no significant changes with time in the proportion of CD4+ and CD8+ Iymphocytes undergoing spontaneous apoptosis after the start of the gene modified tumor vaccine therapy