Induction of cytomegalovirus specific t-lymphocytes from seropositive and -negative donors using dendritic cells grown in autologous plasma

Abstract

We have an induction protocol for CMV-specific cytotoxic T-lymphocytes (CTLs) using the immunodominant pp65 peptide495–503 and dendritic cells (DCs). Monocyte derived DCs were cultured in media supplemented with autologous plasma (AP-DCs), IL4, GM-CSF & TNFα. Use of fetal calf serum (FCS) was avoided since it is immunogenic and may be infectious. The AP-DCs had similar morphology, immunophenotype, and functional activity as FCS-DCs Mature AP-DCs (2×105) were pulsed with pp65495–503 and β2 microglobulin, and added to 2×106 CD8+ cells or 2×106 CD14 depleted PBLs. Cultures were restimulated (R/S) on day 7 & 14 with peptide-pulsed monocytes at a ratio of 1 monocyte to 3 CTLs, and cytotoxicity to peptide-pulsed autologous PHA-blasts and CMV infected fibroblasts was measured. Peptide specific cells were quantitated by pp65 peptide495–503/tetramers. Specific lines were produced in 17 of 19 (89%) cultures derived from seropositive donors. Higher specific lysis was observed for cultures derived from CD8+ cells. In contrast, using the same induction protocol for seronegative donors, we found that 4 R/S were necessary to obtain specific lysis and we observed success only when cultures were initiated with both CD4+ & CD8+ cells. CTL induction for seronegative donors is more difficult presumably due to the absence of primed/memory T-cells. Our data suggests that CMV-specific CTL induction from seronegative donors is not consistent using the protocol developed for seropositive donors. We only generated CMV specific cultures when we did not use purified CD8+ cells as responders indicating that CD4+ help is crucial for CTL induction from CMV seronegative donors.