OPTIMIZATION OF THE CONDITIONS FOR GENERATING HUMAN DC INITIATED ANTIGEN-SPECIFIC T-LYMPHOCYTE LINES IN-VITRO

Naive T lymphocytes specific for a given primary antigen occur in low frequencies and require the relevant antigen to be presented by specia list antigen presenting cells (APC), i.e., dendritic cells (DC). For t hese reasons, the in vitro induction of primary T lymphocyte responses remains a significant technical challenge. We have attempted to impro ve current strategies for generating in vitro responses by optimising (i) isolation and concomitant activation of DC from peripheral blood, (ii) uptake, processing and presentation of antigen by DC and (iii) an tigen driven T lymphocyte proliferation. We established that RPMI 1640 media supplemented with 10% autologous serum resulted in the best yie ld of CMRF-44(+), CD14(-), CD19(-)DC after enrichment over a Nycodenz gradient. Optimal presentation of whole protein and peptide antigen wa s achieved by addition after the purification of the APC, i.e., at the start of the T lymphocyte proliferation assay. RPMI 1640 supplemented with 10% autologous serum or plasma supported the best antigen driven specific T lymphocyte responses. Using these optimised conditions, we compared the efficacy of PBMC and purified blood DC for priming T lym phocyte responses to the chronic myeloid leukaemia (CML) specific bcr- abl (b3a2) peptide. Peptide specific T lymphocyte responses were gener ated with both purified DC and whole PBMC, suggesting that T lymphocyt e precursor frequency was the limiting factor in these experiments. Th ese results will aid in the generation of human T lymphocyte lines to primary antigens, for in vitro and therapeutic applications. (C) 1998 Elsevier Science B.V. All rights reserved.