Marcia M.L. Kho

The effect of rabbit anti-thymocyte globulin induction therapy on regulatory T cells in kidney transplant patients

BACKGROUND Prevention of alloreactivity by rabbit anti-thymocyte globulins (rATG) may not only result from immunodepletion but also from the induction of T cells that control allogeneic immune responses. In the present prospective and controlled study, we investigated the effect of rATG on the frequency, function and phenotype of peripheral immunoregulatory CD4+ T cells in kidney transplant (KTx) patients. METHODS After transplantation, 16 patients received ATG-induction therapy and triple therapy consisting of tacrolimus, MMF and steroids. The control group (n = 18) received triple therapy only. By flow cytometry, T cells were analysed for CD25, FoxP3, CD127, CD45RO and CCR7. To study their suppressive capacities, CD25bright T cells were co-cultured with CD25(-/dim) effector T cells (Teff) in mixed lymphocyte reactions (MLR), stimulated with donor and third party (3P) antigens. RESULTS Pre-transplant levels of FoxP3+CD127(-/low) T cells were 6% of CD4+ T cells. One week post-ATG treatment, no measurable numbers of regulatory T cells were present (P < 0.01). After 4 weeks, the cell numbers of CD4+FoxP3+CD127(-/low) T cells slowly reappeared and thereafter remained low (P < 0.01). At 14 weeks, a significant shift towards the CD45RO+CCR7+ (central memory) phenotype within CD4+FoxP3+ T cells was observed (P < 0.01). At 26 weeks, the proliferative alloresponses of the PBMC and CD25(-/dim) Teff profoundly decreased compared to pre-transplant (P = 0.01 and P = 0.02 respectively), while the regulatory capacity of the CD25bright T cells, of which 90% consisted of FoxP3+CD127(-/low) T cells, remained unaffected. The CD25bright T cells suppressed the anti-donor (94%) and 3P responses (93%). CONCLUSION Our findings show that rATG therapy does not spare peripheral immunoregulatory T cells in vivo, but after regeneration preserves their suppressive activity.

The effect of low and ultra-low dosages Thymoglobulin on peripheral T, B and NK cells in kidney transplant recipients

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-CD16+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 3.0mg/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.

The impact of induction therapy on the homeostasis and function of regulatory T cells in kidney transplant patients

BACKGROUND To evaluate the influence of induction therapy on Tregs we investigated their origin, kinetics and function in kidney transplant patients after treatment with T-cell depleting rabbit antithymocyte globulin (rATG) or IL-2 receptor antagonist basiliximab. METHODS Flow cytometry was used to study thymopoiesis by CD31+ naïve Tregs, homeostatic proliferation by Ki-67+ Tregs and Treg origin by the expression of Helios (nTreg-marker). FACSsorted Tregs were analysed for the demethylation status of the Treg-specific demethylated region (TSDR) of the FoxP3 gene, and Treg-suppressive function. RESULTS Differential effects of rATG and basiliximab induction therapies were measured on the repopulation kinetics of Tregs. While decreased absolute numbers of Tregs were found in both study arms, increased percentages of Tregs were found in rATG treated patients and decreased percentages in basiliximab treated patients. In both groups, Treg repopulation was the result of homeostatic proliferation and not of thymopoiesis. At 1 month after rATG and 6 months after basiliximab therapy, high percentages of Ki-67+ Treg were measured, which in the rATG group, was accompanied by low percentages of Ki-67+Helios+ Treg, and by cells with a demethylated TSDR in the FoxP3 gene. After both rATG and basiliximab therapy, repopulated Tregs inhibited proliferation of allo-antigen activated T effector cells (Teff). CONCLUSIONS In kidney transplant patients, repopulation of Treg after rATG and basiliximab therapy is the result of homeostatic proliferation and not of thymopoiesis. These repopulated Treg were functional after both induction strategies; however only after rATG therapy, were increased proportions of Helios(-) methylated FoxP3 Treg found.

Accumulation of unfavorable clinical and socioeconomic factors precludes living donor kidney transplantation

Background. In the past 30 years, the number of living donor kidney transplantations has increased considerably and nowadays outnumbers the deceased donor transplantations in our center. We investigated which socioeconomic and clinical factors influence who undergoes living or deceased donor kidney transplantation. Methods. This retrospective study included all 1338 patients who received a kidney transplant between 2000 and 2011 in the Erasmus MC Rotterdam. Clinical and socioeconomic variables were combined in our study. Clinical variables were recipient age, gender, ethnicity, original disease, retransplants, ABO blood type, panel-reactive antibody, previous treatment, and transplantation year. Each recipients postcode was linked to a postcode area information data base, to extract demographic information on urbanization level, percentage non-Europeans in the area, income, and housing value. Chi-square, analysis of variance, and univariate and multivariate logistic regression analyses were performed. Results. There were significant differences between the recipients of a living versus deceased donor kidney transplantation. In multivariate logistic regression analyses, 10 variables had a significant influence on the chance of receiving living donor kidney transplantation. Clinical and socioeconomic factors had an independent influence on this chance. Patients with ABO blood type O and B have smaller chances. Highly sensitized and elderly patients have smaller chances especially when combined with a collection of other unfavorable factors. Accumulation of unfavorable factors in non-Europeans prevents their participation in living donation programs. Conclusion. Both clinical and socioeconomic factors are associated with participation in living or deceased donor kidney transplantation. This study highlights the populations that would benefit from educational intervention regarding living donor transplantation.

Kinetics of homeostatic proliferation and thymopoiesis after rATG induction therapy in kidney transplant patients.

Background Lymphocyte-depleting therapy is associated with long-lasting effects on repopulated T cells and subsequent increased rates of infections and malignancies. The mechanisms of T-cell repopulation and their posttransplantation kinetics are not fully understood. Methods We studied thymopoiesis by CD31+ naïve T cells (recent thymic emigrants) and homeostatic proliferation by Ki-67+ T cells in rabbit antithymocyte globulin (rATG)–treated patients the first 6 months after transplantation. Patients receiving basiliximab or no induction therapy served as controls. Results At 6 months after transplantation, T-cell numbers were lower than before transplantation in rATG-treated patients, whereas T-cell numbers remained stable in both control groups. In this time period, thymopoiesis was similar between the three treatment groups; CD8+ T cells showed the highest percentage of recent thymic emigrants. At month 1, percentages of Ki-67+ naïve and memory CD4+ and CD8+ T cells were the highest in rATG-treated patients, but these percentages declined in the months thereafter. When CD31 was used to distinguish between cytokine- and antigen-driven proliferation in naïve T cells, we found evidence for cytokine-dependent proliferation. Cytokine-dependent proliferation was also shown by in vivo increased percentages of phosphorylated STAT5 and high expression levels of the interleukin-7 receptor-&agr; and interleukin-15 receptor-&agr; by T cells. Conclusion These findings demonstrate that, in the first month after rATG therapy, cytokine-induced homeostatic proliferation is involved in T-cell repopulation of both naïve and memory T cells. At later time points, the contribution of homeostatic proliferation diminished, which explains the observed incomplete T-cell recovery.

Current immunosuppressive treatment after kidney transplantation

Introduction: Since 1995, several immunosuppressive drugs have entered the field of organ transplantation: tacrolimus, mycophenolate and the mTOR-inhibitors. Now treating physicians have a choice. Areas covered: The authors review the important studies on immunosuppressive drugs used at present after kidney transplantation, published in the last three decades. This review also discusses the available evidence for selecting one of the calcineurin inhibitors, antiproliferative agents and induction therapy. Interesting new drugs are discussed briefly. Expert opinion: Calcineurin inhibitors (CNIs) are considered, especially in de novo transplantation, to be the most effective maintenance drugs to prevent acute rejection. Combining CNI with mycophenolate or an mTOR-inhibitor has made it possible to reduce CNI dose and diminish nephrotoxicity. Uniform treatment regimes according to guidelines are useful but should leave room for adjustment to the needs of individual patients. Longer follow-up studies are needed to decide on the optimal maintenance treatment.

Characterization of rabbit antithymocyte globulins-induced CD25+ regulatory T cells from cells of patients with end-stage renal disease.

Background. Rabbit antithymocyte globulins (rATGs) are known to convert CD4+CD25−FoxP3− T cells from healthy individuals to CD4+CD25+FoxP3+ T cells. In this study, we investigated the effect of rATG on the induction of regulatory T cells (Tregs) from blood cells of patients with end-stage renal disease who are candidates for transplantation and rATG-induction therapy. The induced Tregs were analyzed and compared with naturally occurring CD4+CD25+FoxP3+T cells. Methods. The CD25− T cells of pretransplant patients (n=7) and healthy controls (n=4) were stimulated with rATG or control rabbit immunoglobulins for 24 hr. The phenotype of induced Tregs was examined by flow cytometry, and their function was studied in the conventional suppression assay. Further characterization was performed by mRNA analyses. Results. After 24 hr, the percentage of CD4+CD25+FoxP3+CD127−/low T cells and CD8+CD25+FoxP3−CD127+ T cells became higher in the rATG-treated samples compared with the rabbit immunoglobulin-treated samples (P<0.01). The rATG-induced CD25+T cells, whether CD4+ or CD8+ inhibited the allogeneic responses of CD25−/dim effector T cells as vigorously as natural CD25+T cells. However, the proportion of FoxP3+ within the top 2% rATG-induced CD4+CD25+T-cells was lower than within the natural CD4+CD25+T-cells (11%±2% vs. 95%±5%, P<0.01). The mRNA-expression levels of interleukin-27, interleukin-10, interferon-γ, perforin, and granzyme B were markedly higher compared with natural CD25+T-cells (all P=0.03), whereas CTLA4 (P=0.03), transforming growth factor-β (P=0.02), and RORγt (P=0.04) were lower. Conclusion. rATG allows the induction of Tregs from patient peripheral blood mononuclear cell in vitro. In comparison with natural Tregs, the rATG-induced Tregs are phenotypically distinct but have similar regulatory activities. rATG may beneficially contribute to the mechanisms that control alloreactivity.

The calcineurin inhibitor tacrolimus allows the induction of functional CD4+CD25+ regulatory T cells by rabbit anti-thymocyte globulins

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.

Conversion from twice-daily to once-daily tacrolimus does not reduce intrapatient variability in tacrolimus exposure

Background: Intrapatient variability (IPV) in tacrolimus exposure is associated with renal allograft failure. The aim of this study was to investigate whether conversion from the twice-daily tacrolimus formulation (Tac-TD) to a once-daily formulation (Tac-OD) leads to a lower IPV in tacrolimus exposure. Methods: Two hundred forty-seven stable renal transplant recipients were converted from Tac-TD to Tac-OD (Advagraf) on a 1:1-mg total daily dose basis. After conversion, patients were followed for 12 months and tacrolimus predose whole-blood concentrations (C0), serum creatinine, estimated glomerular filtration rate, and proteinuria were measured. These parameters were compared with those collected at all outpatient visits in the 12-month period (±3 months) before conversion (Tac-TD period). The IPV was calculated based on the dose-adjusted tacrolimus C0. Results: The Tac-OD formulation provided an excellent graft survival (100%), a low acute rejection rate (0.8%), and good tolerability. Renal function remained stable: estimated glomerular filtration rate 48 (16–90) versus 46 (12–90) mL/min (P = 0.15) before and after conversion, respectively. After conversion to Tac-OD, mean C0 was significantly lower, decreasing from 5.7 ± 1.5 to 5.0 ± 1.5 ng/mL, corresponding to a 12% reduction (P < 0.01). Both drugs had similar IPVs (Tac-TD: 17.3% ± 1.6% versus Tac-OD: 16.4% ± 1.6%, P = 0.31). Conclusions: Although conversion from Tac-TD to Tac-OD significantly reduces tacrolimus exposure as measured by C0 and seems safe, it does not reduce IPV in tacrolimus exposure.

Pharmacodynamic analysis of tofacitinib and basiliximab in kidney allograft recipients

Background The common &ggr;-chain (&ggr;c) cytokines signal through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and play pivotal roles in lymphocyte activation. We investigated the effect of immunosuppressive drugs targeting this pathway, the JAK inhibitor tofacitinib (CP-690,550) and the anti–interleukin (IL)-2R antibody basiliximab, as part of a phase 2 study. Methods After whole-blood activation with the &ggr;c cytokines IL-2, IL-7, and IL-15, STAT5 phosphorylation was determined in T cells of de novo kidney transplantation patients treated with tofacitinib/basiliximab (n=5), calcineurin inhibitor (CNI) (cyclosporine A)/basiliximab (n=4) or CNI (tacrolimus)-based immunosuppression (n=6). The IC50 for phosphorylated STAT (P-STAT) 5 inhibition by tofacitinib was determined in cytokine-activated CD4+ and CD8+ T cells from healthy individuals (n=4). Results IC50 was 26, 72, and 37 ng/mL for IL-2, IL-7, and IL-15 activation, in CD4+ T cells, respectively; and 35, 61, and 76 ng/mL for IL-2, IL-7, and IL-15 activation, in CD8+ T cells, respectively. In kidney transplantation patients, 7 days after starting tofacitinib/basiliximab treatment, cytokine-induced P-STAT5 was inhibited in CD4+ T cells (92% for IL-2 activation, 60% for IL-7, and 75% for IL-15), which persisted for the 2-month study period. In contrast, CNI/basiliximab treatment did not affect IL-7–activated or IL-15–activated P-STAT5; only IL-2–activated P-STAT5 was reduced by 77% on day 7 and recovered to pretreatment levels within 2 months. CD8+ T cells showed a comparable profile to CD4+ T cells. P-STAT5 was not inhibited in CNI-treated control patients. Conclusions Tofacitinib therapy strongly inhibits &ggr;c cytokine-induced JAK/STAT5 activation, whereas basiliximab suppresses IL-2–stimulated activation only. Pharmacodynamic monitoring offers a unique tool to evaluate the biologic effects of immunosuppressive drugs.