title: The development of in vitro culture methods to characterize primary T-cell responses to drugs.
abstract: Adverse drug reactions represent a major stumbling block to drug development and those with an immune etiology are the most difficult to predict. We have developed an in vitro T-cell priming culture method using peripheral blood from healthy volunteers to assess the allergenic potential of drugs. The drug metabolite nitroso sulfamethoxazole (SMX-NO) was used as a model drug allergen to establish optimum assay conditions. Naive T cells were cocultured with monocyte-derived dendritic cells at a ratio of 25:1 in the presence of the drug for a period of 8 days, to expand the number of drug-responsive T cells. The T cells were then incubated with fresh dendritic cells, and drug and their antigen responsiveness analyzed using readouts for proliferation, cytokine secretion, and cell phenotype. All five volunteers showed dose-dependent proliferation as measured by 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester content and by (3)H-thymidine uptake. CD4 T cells that had divided in the presence of SMX-NO had changed from a naive phenotype (CD45RA+) to a memory phenotype (CD45RO+). These memory T cells expressed the chemokine receptors CCR2, CCR4, and CXCR3 suggesting a mixture of T(H)1 and T(H)2 cells in the responding population, with a propensity for homing to the skin. Drug stimulation was also associated with the secretion of a mixture of T(H)1 cytokines (interferon γ) and T(H)2 cytokines (interleukin [IL]-5 and IL-13) as detected by ELISpot. We are currently developing this approach to investigate the allergenic potential of other drugs, including those where an association between specific human leucocyte antigen alleles and susceptibility to an immunological reaction has been established.
authors.affiliation: Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool L69 3GE, UK.
authors.forename: Lee
authors.initials: L
authors.lastname: Faulkner
authors.forename: Klara
authors.initials: K
authors.lastname: Martinsson
authors.forename: Anahi
authors.initials: A
authors.lastname: Santoyo-Castelazo
authors.forename: Karin
authors.initials: K
authors.lastname: Cederbrant
authors.forename: Ina
authors.initials: I
authors.lastname: Schuppe-Koistinen
authors.forename: Helen
authors.initials: H
authors.lastname: Powell
authors.forename: Jonathan
authors.initials: J
authors.lastname: Tugwood
authors.forename: Dean J
authors.initials: DJ
authors.lastname: Naisbitt
authors.forename: B Kevin
authors.initials: BK
authors.lastname: Park
mesh_terms: D000485:Allergens / Q000633:toxicity*; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D007156:Immunologic Memory / Q000276:immunology; D017493:Leukocyte Common Antigens / Q000378:metabolism; D009603:Nitroso Compounds / Q000633:toxicity; D019707:Receptors, Chemokine / Q000378:metabolism; D013420:Sulfamethoxazole / Q000633:toxicity; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D006377:T-Lymphocytes, Helper-Inducer / Q000187:drug effects / Q000276:immunology
chemical_list: D000485:Allergens; D016207:Cytokines; D009603:Nitroso Compounds; D019707:Receptors, Chemokine; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human; D013420:Sulfamethoxazole