Intracytoplasmic cytokines can be measured following mitogen stimulation of immune cells, addition OSI-906 manufacturer of a monoclonal antibody directed against the cytokine of interest, and then FACS analysis. Positive cells are expressed as percentage of cytokine-producing cells within the T-cell population. An advantage of this technique is the potential to simultaneously distinguish lymphocyte phenotype.5 A study of 14 kidney transplant patients treated with a CNI, azathioprine and prednisolone demonstrated significantly lower frequencies of IL-2 secreting CD4+ and CD8+ T

cells and IFN-γ and double positive IL-2/IFN-γ secreting CD4+ T cells at 3 and 6 months post-transplantation compared with pre-transplantation.5 This study also showed that the frequency of IL-2 secreting GSI-IX molecular weight T cells was more

affected by tacrolimus than cyclosporine, again suggesting increased immunosuppressive potency of the former drug. In a study of 41 kidney transplant recipients treated with a CNI, azathioprine (n = 4) or MMF (n = 37) and corticosteroids, a reduction in the frequencies of IL-2, IFN-γ and TNF-α secreting CD4+ and CD8+ T cells was seen in the first 14 days post-transplantation compared with at baseline.8 However, in contrast to the previous study, variable increases in most of these T-cell frequencies were seen thereafter, with IFN-γ secreting T-cell frequencies increasing all the way Interleukin-3 receptor back to baseline levels by 1 year. This raises concern that this measure of cytokine secretion may not be sufficiently sensitive to quantify immunosuppression in patients some time from transplantation. Consistent with data from studies using ELISA, studies using flow cytometry have failed to detect an effect of MMF monotherapy on cytokine secretion (IL-2 and TNF-α).10 The ELISPOT identifies and enumerates cytokine-producing cells at the single-cell level. It has increased sensitivity compared with conventional ELISA and flow cytometry, being able to detect as few as 10 cytokine secreting T cells per 1 million peripheral blood mononuclear cells (PBMCs).50 However, it has a lower dynamic range, making it less able to quantify the magnitude of

a response.51 Although multiple studies have shown an association between ELISPOT reactivity to donor antigens and clinical outcomes, only one study has investigated ELISPOT reactivity following non-specific mitogen stimulation. Following PHA stimulation of PBMC, no difference was found in the number of IFN-γ (as a surrogate for Th1 immunity) or IL-5 (as a surrogate for Th2 immunity) secreting cells between dialysis patients, kidney transplant recipients and healthy controls.16 However, in a subset of 23 kidney transplant recipients with acute graft dysfunction, 8 of 12 cases with rejection had PBMC IFN-γ/IL-5 ratios >15, whereas 10 of 11 cases of graft dysfunction from other causes were associated with ratios of <15 (P = 0.07).