Mol, W.M.

Pharmacodynamic analysis of tofacitinib and basiliximab in kidney allograft recipients (Article)

2012-09-15T00:00:00Z

BACKGROUND: The common γ-chain (γc) 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 γccytokines 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 γccytokine-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. Copyright

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

2010-08-01T00:00:00Z

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)-, 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.

Monitoring of the immunomodulatory effect of CP-690,550 by analysis of the JAK/STAT pathway in kidney transplant patients (Article)

2009-10-01T00:00:00Z

BACKGROUND.: The small molecule drug CP-690,550 inhibits Janus kinase 3 at nanomolar concentrations and has recently been shown to prevent allograft rejection in rodents and nonhuman primates. METHODS.: As part of a phase 1 clinical trial, we investigated the effect of CP-690,550 after 29 days of 30 mg twice daily treatment at the cellular level in eight kidney transplant patients by studying ex vivo phosphorylation of STAT5 (P-STAT5), the key substrate of JAK3. RESULTS.: As determined by quantitative fluorescent western blotting, interleukin-2-induced P-STAT5 in YT cells was reduced by a median of 73% (P<0.01) in the presence of serum collected on day 29 compared with pretreatment baseline. When evaluated by phosphospecific flow cytometry, CP-690,550 also reduced interleukin-2-induced P-STAT5 in CD3 (median 20%; P<0.05), CD3CD4 (median 37%; P<0.05), and CD3CD8 (median 34%; P<0.01) populations in patient-derived peripheral blood mononuclear cells. At the functional level, the inhibitory effect of CP-690,550 was confirmed by determining the expression of several STAT5 targets genes. CONCLUSION.: Analysis of P-STAT5 may, therefore, be used to determine the immunomodulatory effect of CP-690,550 at the cellular level in transplant patients.

End-stage renal failure and regulatory activities of CD4 +CD25bright+FoxP3+ T-cells (Article)

2009-06-01T00:00:00Z

Background. The defensive immune system in patients with end-stage renal failure is impaired at multiple levels. This state of immune incompetence is associated with continuous activation of the immune system. An additional explanation for this state of activation may be the disturbed function of CD4+CD25bright+FoxP3+regulatory T-cells.Methods. The phenotype and function of peripheral regulatory T-cells from patients with end-stage renal failure (N = 80) and healthy controls (N = 17) was studied by flow cytometry, RT-PCR and mixed lymphocyte reaction. Patients were on haemodialysis (N = 40), peritoneal dialysis (N = 26) or not treated with dialysis yet (N = 14). The latter group had a glomerular filtration rate of <20 mlmin 1.73 m2.Results. The basal IL-2 mRNA level was high in patient-PBMC (P = 0.0002 versus healthy controls). The absolute number of CD4+CD25bright+T-cells was low in patients (P < 0.05 versus healthy controls). Furthermore, proliferation of patient-PBMC upon allogeneic stimulation was impaired (P < 0.0001 versus healthy controls). The regulatory function of CD4+CD25bright+T-cells was determined in the setting of direct allorecognition. First, the effect of depletion of CD25bright+cells from patient-PBMC on proliferation was low. Second, co-culture of CD25bright+cells with CD25negdimcells (1:10 ratio) showed impaired regulatory function (P < 0.001 versus healthy controls), which was especially pronounced in patients on dialysis. The FOXP3 mRNA level was also low upon stimulation (P = 0.0002 versus healthy controls).Conclusions. In line with previous studies, we observed an overactivated but functionally compromised immune system in patients with end-stage renal failure. It now appears that in this setting, regulation by CD4+CD25bright+FoxP3+T-cells is also impaired.

Generation of donor-specific regulatory t-cell function in kidney transplant patients (Article)

2009-02-15T00:00:00Z

Background.: In the search for mechanisms that can induce and maintain transplant tolerance, donor-specific CD4CD25FoxP3 regulatory T cells have been frequently mentioned. However, it remains to be demonstrated, whether these cells are generated after clinical transplantation. Methods.: We prospectively analyzed the phenotype and function of peripheral regulatory CD4CD25 T cells of 79 patients before, 3, 6, and 12 months after kidney transplantation. The immune regulatory capacities of CD4CD25 T cells were assessed by their depletion from peripheral blood mononuclear cells and in co-culture with CD25 responder T-cells in the mixed lymphocyte reactions. Results.: In the first year after transplantation, the number and proportion of CD4CD25 T cells significantly decreased (P<0.05 and P<0.001, respectively). In the mixed lymphocyte reactions, we observed donor-specific hyporesponsiveness in the presence of significantly increased proliferation to third and fourth Party-Ag, (P<0.001 and P<0.05, respectively). Furthermore, functional analysis of CD25 cells showed that the effect of depletion of these cells from peripheral blood mononuclear cells, and their suppressive capacities in co-culture with donor-Ag stimulated CD25 responder T-cells (1:10 ratio) significantly improved (P<0.01 and P<0.001, respectively). Moreover, the difference between the stimulation with donor-Ag and third Party-Ag became apparent at 6 months after transplantation. Conclusions.: These findings demonstrate that donor-specific CD4CD25 regulatory T-cell function is generated in fully immunosuppressed renal recipients in the first year after transplantation.

Functional CD25bright+ alloresponsive T cells in fully immunosuppressed renal allograft recipients (Article)

2007-01-01T00:00:00Z

Background: Evidence from animal studies indicate a crucial role for CD25bright+regulatory T cells in transplantation tolerance. Methods: To assess whether peripheral CD25bright+T cells control immune responses in immunosuppressed kidney transplant patients, we analyzed the suppressive capacities of these cells using mixed lymphocytes reactions. Results: Allogeneic stimulation of patients peripheral blood mononuclear cells was associated with IL-2 production and T-cell proliferation. Depletion of CD25bright+T cells resulted in a 35% (median) higher IL-2 production and a 38% higher proliferative response against third party cells, showing that functional regulatory CD25bright+T cells were present (p = 0.03 and 0.02 respectively). In eight out of 11 patients, we also demonstrated regulation activity against donor-activated T cells (p = 0.03). These data were confirmed in coculture experiments with isolated CD25-/dimT cells plus CD25bright+T cells. At a 1:2 ratio, the CD25bright+T cells suppressed the proliferation of CD25-/dimdonor- and third party-stimulated responder T cells. Conclusions: CD25bright+T cells with immune regulatory activities against anti-donor-responsive T cells are readily detectable in renal allograft recipients during treatment with full dosage immunosuppression. Whether CD25bright+T cells indeed play a role in graft acceptance after organ transplantation in patients remains to be elucidated.

Differential expression of heme oxygenase-1 and vascular endothelial growth factor in cadaveric and living donor kidneys after ischemia-reperfusion (Article)

2003-01-01T00:00:00Z

The extent of graft damage after ischemia-reperfusion reflects the balance between deleterious events and protective factors. Heme oxygenase-1 (HO-1) and vascular endothelial growth factor (VEGF) may contribute to cytoprotection by their anti-inflammatory and antiapoptotic properties. For investigating whether HO-1 and VEGF play a role in the adaptive response to ischemia-reperfusion injury after renal transplantation, kidney biopsies were analyzed from living (n = 45) and cadaveric (n = 16) donors, obtained at three time points: at the end of cold storage T(-1), after warm ischemia T(0), and after reperfusion T(+1). The mRNA expression levels of HO-1, VEGF(165), Bcl-2, Bax, and hypoxia inducible factor-1alpha were quantified by real-time reverse transcriptase-PCR, and the HO-1 and VEGF proteins were analyzed by immunohistochemistry. Cadaveric donor kidneys presented higher mRNA expression levels of hypoxia inducible factor-1alpha. In contrast, mRNA expression levels of HO-1, VEGF(165), and Bcl-2 were significantly lower in kidneys from cadaveric donors. Overall, a significant correlation was observed between mRNA expression of Bcl-2 and VEGF(165), between Bcl-2 and HO-1, and between HO-1 and VEGF(165). Moreover, protein expression of HO-1 and VEGF was detected in the same anatomical kidney compartments (glomerulus, arteries, and distal tubules). Renal function at the first week posttransplantation (analyzed by serum creatinine levels) showed a significant correlation with both HO-1 and VEGF mRNA expression, reinforcing the protective role of both genes in the early events of transplantation. It is concluded that the lower expression of HO-1, VEGF(165), and Bcl-2 in cadaveric donor kidneys can reflect a defective adaptation against ischemia-reperfusion injury that may affect their function in the short term.

Intragraft interleukin 2 mRNA expression during acute cellular rejection and left ventricular total wall thickness after heart transplantation (Article)

2002-01-01T00:00:00Z

OBJECTIVE: To assess whether diastolic graft function is influenced by intragraft interleukin 2 (IL-2) messenger RNA (mRNA) expression in rejecting cardiac allografts. DESIGN: 16 recipients of cardiac allografts were monitored during the first three months after transplantation. The presence of IL-2 mRNA in endomyocardial biopsies (n = 123) was measured by reverse transcriptase polymerase chain reaction. To determine heart function, concurrent M mode and two dimensional Doppler echocardiograms were analysed. RESULTS: Histological signs of acute rejection (International Society for Heart and Lung Transplantation (ISHLT) rejection grade > 2) were strongly associated with IL-2 mRNA expression (IL-2 mRNA was present in 12 of 20 endomyocardial biopsies (60%) with acute rejection and in 24 of 103 endomyocardial biopsies (23%) without acute rejection, p = 0.002). No significant relation was found between either histology or IL-2 mRNA expression alone and the studied echocardiographic parameters. However, stratification of the echocardiographic data into those of patients with and those without acute rejection showed that during acute rejection IL-2 mRNA expression was significantly associated with increased left ventricular total wall thickness (mean change in total wall thickness was +0.22 cm in patients with IL-2 mRNA expression versus -0.18 cm in patients without IL-2 mRNA expression, p = 0.048). CONCLUSIONS: An increase in left ventricular total wall thickness precedes IL-2 positive acute rejection after heart transplantation. Thus, cardiac allograft rejection accompanied by intragraft IL-2 mRNA expression may be indicative of more severe rejection episodes.