Ž . Journal of Immunological Methods 219 1998 69–83 Optimisation of the conditions for generating human DC initiated antigen specific T lymphocyte lines in vitro Stuart I. Mannering, Judith L. McKenzie, Derek N.J. Hart ) Haematology r Immunology r Transfusion Medicine Research Group, Christchurch School of Medicine, Christchurch, New Zealand Received 1 September 1997; revised 16 March 1998; accepted 22 June 1998 Abstract Naive T lymphocytes specific for a given primary antigen occur in low frequencies and require the relevant antigen to be Ž . Ž . presented by specialist antigen presenting cells APC , i.e., dendritic cells DC . For these reasons, the in vitro induction of primary T lymphocyte responses remains a significant technical challenge. We have attempted to improve current strategies Ž. Ž. for generating in vitro responses by optimising i isolation and concomitant activation of DC from peripheral blood, ii Ž . uptake, processing and presentation of antigen by DC and iii antigen driven T lymphocyte proliferation. We established that RPMI 1640 media supplemented with 10% autologous serum resulted in the best yield of CMRF-44 q , CD14 y , CD19 y DC after enrichment over a Nycodenz gradient. Optimal presentation of whole protein and peptide antigen was 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 lymphocyte responses to the Ž . Ž . chronic myeloid leukaemia CML specific bcr–abl b3a2 peptide. Peptide specific T lymphocyte responses were generated with both purified DC and whole PBMC, suggesting that T lymphocyte precursor frequency was the limiting factor in these experiments. These results will aid in the generation of human T lymphocyte lines to primary antigens, for in vitro and therapeutic applications. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Dendritic cells; Primary response; bcr–abl; T lymphocyte lines; Media; Tumour associated antigen Ž. Abbreviations: PHA, phytohemagglutinin; FCS, fetal calf serum; APC, antigen presenting cell s ; PE, phycoerythrin; TAA, tumor associated antigen; TT, tetanus toxoid; IL-2, interleukin-2; SI, stimulation index; SWM, sperm whale myoglobin leukaemia; SD, standard Ž. deviation; SEB, staphylococcal enterotoxin B; DC, dendritic cell; PBMC, peripheral blood mononuclear cell s ; FITC, fluorescein 5-isothiocyanate; PBS, phosphate buffered saline; KLH, keyhole limpet haemocyanin; DMSO, dimethyl sulphoxide; ANOVA, analysis of variance; CML, chronic myeloid ) Corresponding author. Mater Medical Research Institute, Mater Misericordiae Hospitals, Raymond Terrace, South Brisbane, Queens- land 4101, Australia. Tel.: q61-7-3840-2555; Fax: 61-7-3840-2550; E-mail: dhart@mater.org.au 0022-1759r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. Ž . PII: S0022-1759 98 00125-2