title: Quantitation of immunosuppression by tacrolimus using flow cytometric analysis of interleukin-2 and interferon-gamma inhibition in CD8(-) and CD8(+) peripheral blood T cells.
abstract: The authors have determined the frequency of intracellular interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) synthesis by T-cell subsets in whole blood (WB) and isolated lymphocytes in 16 transplant recipients treated with tacrolimus and 10 control patients who were not transplant recipients. The authors also determined the impact of varying amounts of red blood cells (RBC) on immunosuppression by tacrolimus. Samples were analyzed by two-color flow cytometry, and the results were expressed as a ratio of whole blood to isolated lymphocytes. In healthy subjects who were not transplant recipients, the frequency of IL-2--producing CD8(-) and CD8(+) cells was higher in WB than in isolated lymphocytes (mean +/- SD of whole blood to lymphocytes ratio: 1.24 +/- 0.5 and 1.67 +/- 0.62, respectively). Adding varying amounts of RBC had no significant impact on IL-2 production by CD8(-) and CD8(+) T cells. Adding tacrolimus (10 ng/mL) to lymphocyte cultures inhibited (90%) IL-2 production in isolated T cells but not in the whole-blood assay. The dose of tacrolimus required for a 50% inhibition of IL-2 release in T cells was 10-fold higher in cultures with RBC than without. Peripheral blood mononuclear cells (PBMC) isolated from tacrolimus-treated whole blood (WB) showed less IL-2 inhibition than did lymphocytes in the WB. The authors also tested cytokine production in WB and PBMCs in 16 transplant recipients and observed various patterns of reactivity. The frequency of IL-2--producing CD8(-) and CD8(+) cells was similar using two different methods in 10 of 16 patients tested. By contrast, in the remaining six patients the authors observed a significant inhibition of IL-2 production in both CD8(-) and CD8(+) T-cell subsets in the whole-blood assay but not in the isolated lymphocytes. The frequency of CD8(-) IFN-gamma--producing cells was significantly lower in 9 of 16 patients, but the same individuals showed no inhibition of their CD8(+) IFN-gamma T cells. The trough levels of tacrolimus did not predict the level of cytokine inhibition in the whole-blood assay in these patients. The authors' results show that the whole-blood assay for cytokine production can be used for monitoring the in vivo effect of tacrolimus in transplant recipients.
authors.affiliation: Departments of Pathology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: M
authors.initials: M
authors.lastname: Ahmed
authors.forename: R
authors.initials: R
authors.lastname: Venkataraman
authors.forename: A J
authors.initials: AJ
authors.lastname: Logar
authors.forename: A S
authors.initials: AS
authors.lastname: Rao
authors.forename: G P
authors.initials: GP
authors.lastname: Bartley
authors.forename: K
authors.initials: K
authors.lastname: Robert
authors.forename: F S
authors.initials: FS
authors.lastname: Dodson
authors.forename: R
authors.initials: R
authors.lastname: Shapiro
authors.forename: J J
authors.initials: JJ
authors.lastname: Fung
authors.forename: A
authors.initials: A
authors.lastname: Zeevi
mesh_terms: D000328:Adult; D016827:CD8 Antigens / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007165:Immunosuppression Therapy; D007166:Immunosuppressive Agents / Q000493:pharmacokinetics / Q000627:therapeutic use*; D007371:Interferon-gamma / Q000096:biosynthesis / Q000097:blood*; D007376:Interleukin-2 / Q000096:biosynthesis / Q000097:blood*; D008213:Lymphocyte Activation / Q000187:drug effects; D008297:Male; D008875:Middle Aged; D015166:Monitoring, Immunologic / Q000379:methods; D016377:Organ Transplantation; D016559:Tacrolimus / Q000493:pharmacokinetics / Q000627:therapeutic use*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D016827:CD8 Antigens; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate; D016559:Tacrolimus