title: Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture.
abstract: Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently high anti-recipient IL-4 producing HTLp frequencies have been reported and associated with a decreased risk of GVHD. The aim of the present study was to define the optimal conditions for combined determination of IL-2 and IL-4 producing anti-recipient HTLp frequencies. We have optimised the CT.h4S bioassay with regards to specificity, sensitivity, detection limit, and reproducibility. We have found the optimal assay conditions to be 1 x 10 (4) CT.h4S cells/well deprived of IL-4 for 24 h and preincubated for 7 h followed by 18 h of incubation with tritiated methyl-thymidine. In this setting the CT.h4S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack of IL-4 detection was not due to high amounts of soluble IL-4 receptor. With the use of 1x10(6) responder cells/well in HLA-mismatched MLC, we found limited IL-4 accumulation still increasing at day 12. We conclude that the CT.h4S bioassay is a reliable and specific method for quantification of IL-4 accumulation in cultures of human MNC. The difference in optimal timing for IL-2 (day 3) and IL-4 (>/=day 12) detection and evidence of very low IL-4 producing HTLp frequencies makes the relevance of a combined IL-2/IL-4 HTLp assay questionable.
authors.affiliation: Department of Haematology, Rigshospitalet, Blegdamsvej 9, DK-2100, Copenhagen, Denmark. s.l.peterson@dadlnet.dk
authors.forename: Søren Lykke
authors.initials: SL
authors.lastname: Petersen
authors.forename: Charlotte Astrid
authors.initials: CA
authors.lastname: Russell
authors.forename: Klaus
authors.initials: K
authors.lastname: Bendtzen
authors.forename: Lars Lindhardt
authors.initials: LL
authors.lastname: Vindeløv
mesh_terms: D001681:Biological Assay / Q000379:methods* / Q000706:statistics & numerical data; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006680:HLA Antigens; D006801:Humans; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000032:analysis / Q000096:biosynthesis; D015847:Interleukin-4 / Q000032:analysis* / Q000096:biosynthesis; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology; D007959:Lymphocyte Culture Test, Mixed; D010835:Phytohemagglutinins / Q000494:pharmacology; D019948:Receptors, Interleukin-4 / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D006680:HLA Antigens; D007376:Interleukin-2; D010835:Phytohemagglutinins; D019948:Receptors, Interleukin-4; D011994:Recombinant Proteins; D015847:Interleukin-4