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Summary: Blockade of the CD80/86‐CD28 pathway by belatacept after kidney transplantation is associated with an increased risk of rejection compared with standard, calcineurin inhibitor (CNI)‐based therapy. CD28– T cells, which express CD57, are not susceptible to belatacept treatment. High numbers of CD4+CD57+programmed death 1 (PD‐1)– T cells pretransplantation have been associated with a higher chance of rejection, although conflicting data have been reported. To investigate the working mechanism behind this possible higher chance of rejection, we studied the expression of co‐inhibitory molecules (CD223, CD244 and PD‐1), proliferative capacity and cytotoxic potential of fluorescence activated cell sorted (FACS) CD4+CD57+PD‐1– and CD8+CD57+PD‐1– T cells, and their CD57– control populations, after alloantigen stimulation. The effect of belatacept on the cytotoxic capacity of pretransplantation peripheral blood mononuclear cells from 20 patients who received belatacept post‐transplantation was also tested. Expression of co‐inhibitory molecule CD223 increased by approximately 10‐fold after allogeneic stimulation in all four T cell subsets. Proliferation and up‐regulation of CD244 and PD‐1 was observed for CD4+CD57‐PD‐1– T cells after allogeneic stimulation, but no up‐regulation of these markers occurred on CD8+ T cells or CD4+CD57+PD‐1– T cells. However, CD4+CD57+PD‐1– T cells and, to a lesser extent, CD8+CD57+PD‐1– T cells displayed higher cytotoxicity as indicated by granzyme B expression. Belatacept inhibited the cytotoxic potential of CD4+CD57+PD‐1– T cells (median of inhibition 31%, P < 0·01) and CD8+CD57+PD‐1– T cells (median of inhibition 10%, P < 0·05). In conclusion, alloantigen‐activated CD4+CD57+PD‐1– T cells exhibited a less proliferative but more cytotoxic profile than their CD57– counterparts. Their cytotoxic capacity can be inhibited partly by belatacept and was not associated with development of rejection after kidney transplantation. [ABSTRACT FROM AUTHOR]

The novel immunosuppressant sotrastaurin is a selective inhibitor of protein kinase C isoforms that are critical in signalling pathways downstream of the T cell receptor. Sotrastaurin inhibits nuclear factor ( NF)-κB, which directly promotes the transcription of forkhead box protein 3 ( Fox P3), the key regulator for the development and function of regulatory T cells (Tregs). Our center participated in a randomized trial comparing sotrastaurin ( n = 14) and the calcineurin inhibitor Neoral ( n = 7) in renal transplant recipients. We conducted ex vivo mixed lymphocyte reaction (MLR) and flow cytometry studies on these patient samples, as well as in vitro studies on samples of blood bank volunteers ( n = 38). Treg numbers remained stable after transplantation and correlated with higher trough levels of sotrastaurin ( r = 0·68, P = 0·03). A dose-dependent effect of sotrastaurin on alloresponsiveness was observed: the half maximal inhibitory concentration ( IC50) to inhibit alloactivated T cell proliferation was 45 ng/ml (90 nM). In contrast, Treg function was not affected by sotrastaurin: in the presence of in vitro-added sotrastaurin (50 ng/ml) Tregs suppressed the proliferation of alloactivated T effector cells at a 1:5 ratio by 35 versus 47% in the absence of the drug ( P = 0·33). Signal transducer and activator of transcription 5 ( STAT)-5 phosphorylation in Tregs remained intact after incubation with sotrastaurin. This potent Treg function was also found in cells of patients treated with sotrastaurin: Tregs inhibited the anti-donor response in MLR by 67% at month 6, which was comparable to pretransplantation (82%). Sotrastaurin is a potent inhibitor of alloreactivity in vitro, while it did not affect Treg function in patients after kidney transplantation. [ABSTRACT FROM AUTHOR]

Rabbit anti-thymocyte globulins (rATG) induce CD4+CD25+forkhead box P3 (FoxP3+) regulatory T cells that control alloreactivity. In the present study, we investigated whether rATG convert T cells into functional CD4+CD25+FoxP3+CD127−/low regulatory T cells in the presence of drugs that may hamper their induction and function, i.e. calcineurin inhibitors. CD25neg T cells were stimulated with rATG or control rabbit immunoglobulin G (rIgG) in the absence and presence of tacrolimus for 24 h. Flow cytometry was performed for CD4, CD25, FoxP3 and CD127 and the function of CD25+ T cells was examined in suppression assays. MRNA expression profiles were composed to study the underlying mechanisms. After stimulation, the percentage CD4+CD25+FoxP3+CD127−/low increased (from 2% to 30%, mean, P < 0·01) and was higher in the rATG samples than in control rIgG samples (2%, P < 0·01). Interestingly, FoxP3+T cells were also induced when tacrolimus was present in the rATG cultures. Blockade of the interleukin (IL)-2 pathway did not affect the frequency of rATG-induced FoxP3+ T cells. The rATG tacrolimus-induced CD25+ T cells inhibited proliferative responses of alloantigen-stimulated effector T cells as vigorously as rATG-induced and natural CD4+CD25+FoxP3+CD127−/low T cells (67% ± 18% versus 69% ± 16% versus 45% ± 20%, mean ± standard error of the mean, respectively). At the mRNA-expression level, rATG-induced CD25+ T cells abundantly expressed IL-10, IL-27, interferon (IFN)-γ, perforin and granzyme B in contrast to natural CD25+ T cells (all P = 0·03), while FoxP3 was expressed at a lower level ( P = 0·03). These mRNA data were confirmed in regulatory T cells from kidney transplant patients. Our findings demonstrate that tacrolimus does not negatively affect the induction, phenotype and function of CD4+CD25+ T cells, suggesting that rATG may induce regulatory T cells in patients who receive tacrolimus maintenance therapy. [ABSTRACT FROM AUTHOR]

Introduction: Rabbit Anti-Thymocyte Globulin (r-ATG) is a polyclonal antibody preparation, used to prevent and treat acute rejection episodes after organ transplantation. However, despite more than 40 years of clinical use, the optimal dose of r-ATG is still not defined. To find a better balance between efficacy and infectious complications, we embarked on a controlled study and monitored the effect of low and ultra-low dosages Thymoglobulin (Genzyme) on peripheral T, B, and NK cells. Patients and methods: Kidney transplant recipients received either 0.5 mg/kg, 1.0 mg/kg or 2.0 mg/kg on the first 3 consecutive days post-transplantation. Thus, total doses were 1.5 mg/kg, 3.0 mg/kg and 6.0 mg/kg. A total of 40 patients were enrolled, including 11 controls. All patients were treated with Prednisolon, Advagraf (Astellas) and Mycophenolate Mofetil (Roche). T (CD3 +), B (CD19 +) and NK (CD3-CDt6 + 56 +) cells were analyzed by flow cytometry. Baseline cell counts were compared to forty age and sex matched healthy persons. Post-transplantation cell counts of the 3 Thymoglobulin groups were compared to the 11 control patients, who received no induction therapy. Results: Absolute numbers of T, B, and NK cells were comparable in all patients pre-transplantation, but T and B cells were lower than in healthy persons (p = 0.007 and p = 0.0003, Mann Whitney test). In the first week, T cells and NK cells were significantly lower in all Thymoglobulin groups compared to controls. B cells were not affected. One month after Thymoglobulin NK cells had returned to control numbers in all groups, while T cells had already recovered to control counts in the 1.5 mg/kg group. During follow-up, T cells in the 3.0 mg/ kg group also returned to control values, but at one year the patients in the 6.0 mg/kg group still had significantly lower T cells (p = 0.03). Patient and graft survival, rejection and infection incidence and renal function did not differ between groups. Conclusion: Patients with end stage renal disease have significantly lower peripheral T and B cell counts than healthy persons. (Ultra-) low Thymoglobulin schedules deplete peripheral lymphocytes in a dose dependent way. Knowledge of the duration of this depletion contributes to finding the optimal immunosuppressive strategy for kidney transplant recipients. [ABSTRACT FROM AUTHOR]