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Differences in Type 1 and Type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuppression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients

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Abstract

Recent multicenter, randomized clinical trials have shown that in renal transplant patients tacrolimus (FK506) was more efficient than cyclosporine A (CsA) at preventing acute rejection. In order to try and evaluate whether this difference was related to a different in vivo T-cell suppression we assessed, in a prospective study, the frequencies of interleukin (IL)-2-, IL-4-, IL-5-, IL-6-, IL-10-, interferon-gamma (IFN-Γ)- and double-positive IL-2/IFN-Γ-producing whole T cells, CD4+ and CD8+ T-cell subsets by means of cytokine flow cytometry. This was performed after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol myristate acetate (PMA) and ionomycin, in the presence of monensin, in 14 healthy volunteers (controls) and in 14 renal transplant patients. The immunosuppression of the latter was based either on CsA (n=7) or on FK506 (n=7). Cytokine-expressing T-cell frequencies were assessed immediately pre-transplantation (D0), and subsequently 3 months (M3) and 6 months (M6) afterwards in fasting patients prior to the morning intake of the immunosuppressive drug. We found that at D0 the frequencies of IL-2- (22±2% vs. 22.2±2%), IFN-Γ- (26±3% vs. 29±3.4%) and IL-4- (0.8±0.2% vs. 1.4±0.2%)-expressing T lymphocytes were not significantly different between the controls and the patients, respectively. Conversely, the frequency of IL-2/IFN-Γ double positive cells was higher in the latter (9.3±1.6%) than in the controls (5.6±0.8); p=0.06. Finally, on D0 the frequencies of IL-5-, IL-6-, and IL-10-producing T lymphocytes were lower than 1% in both groups, as well as after grafting, i.e. on M3 and M6. As compared to baseline (D0): (a) chronic immunosuppression significantly decreased the frequencies of IL-2-, IL-4- and IL-2/IFN-Γ-expressing T cells, whereas those of IFN-Γ, IL-5, IL-6, and IL-10 were not significantly affected; (b) the frequencies of cytokine-expressing T cells were not statistically different between M3 and M6; (c) the decrease in the frequencies of IL-2- and IL-2/IFN-Γ-expressing T cells affected CD4+ and CD8+ cells equally; (d) there was a marginal decrease in the frequency of IFN-Γ-expressing cells only in the CD4+ subset but not in the CD8 population; and (e) for CsA, but not for FK506, the frequency of the IL-2-expressing T cells was negatively correlated with the whole blood trough levels. When we compared the frequencies of cytokine-expressing cells in FK506- and CsA-treated patients, we found that the frequency of IL-2-expressing T cells was significantly lower with FK506 (10.9±1.6%) than with CsA (16.3±1.8%; p=0.03), whereas the frequencies of the other cytokine-expressing cells were not statistically different between the two groups. In conclusion, our study clearly demonstrated that studied ex vivo, FK506 and CsA decrease the frequencies of cells expressing IL-2, IL-4 and IL-2/IFN-Γin vivo but do not affect those expressing IFN-Γ. Meanwhile, the frequency of IL-2-producing T cells was more affected with FK506 than with CsA and was negatively correlated with the CsA trough level. Finally, our results regarding IL-2 might explain to some extent the higher efficiency of FK506 in vivo than CsA.

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