e. expressing at least one of the markers). This clearly confirmed that degranulation became increasingly dominant after transplantation, with a median of 92% of CD8+ pp65-specific T cells and 85% of IE-specific CD8+ T cells expressing this marker (alone or in combination) after transplantation compared with 84% and 71%, respectively, in controls (not shown). However, because of their likely protective role, we were primarily interested in the effect of immunosuppression on the T cells producing IFN-γ, TNF-α and IL-2 simultaneously, or any two
of them.9 For this purpose, the analyses shown in Fig. 1(b) disregard degranulation and focus on IFN-γ, TNF-α and IL-2 alone. They show that
the most dominant CMV-specific CD8+ subset Kinase Inhibitor Library order (as defined by these functions) in healthy donors produces just IFN-γ and TNF-α, while the subset producing all three measured cytokines is the only other sizeable subset. Both are strongly reduced in transplant patients. A similar distribution was observed for pp65-specific CD8− T cells. When studying each of 15 non-overlapping functional subsets individually (Boolean gating) it became apparent that T cells exhibiting degranulation as a single function were dramatically increased in transplant patients (Fig. 1c). As all patients received calcineurin inhibitors (but only one-third each received everolimus or mycophenolate mofetil), we attempted to reproduce this effect in vitro by incubating donor-derived cells overnight with the KPT-330 in vivo calcineurin inhibitors cyclosporin A or tacrolimus before stimulation, because these were the most likely
drugs to cause this change. This resulted in a dose-dependent reduction of polyfunctionality; the subsets producing IFN-γ, TNF-α and IL-2, or IFN-γ and TNF-α decreased (Fig. 2a,b) whereas subsets displaying only single functions emerged and increased (Fig. 2c,d). Dot plots in Fig. 2(e) show a dose-dependent decrease in TNF-α, IFN-γ and IL-2 production, but little effect on degranulation. Our results show that immunosuppression induces marked changes in the CMV-specific T-cell response after heart and lung transplantation. These are reflected in response quality (i.e. the functional response profile) Farnesyltransferase rather than quantity (i.e. the number of inducible cells). The most obvious effects were reduction of IL-2 and TNF-α production, IFN-γ seemed somewhat less affected and degranulation not at all. This predominantly translated into the generation of T-cell subsets with one single function, most frequently degranulation, at the expense of subsets displaying IFN-γ, TNF-α and IL-2 at the same time. Degranulation was the most inclusive marker of total response size but not the most informative with regard to the effect of immunosuppression.