Pharmacodynamic analysis of tofacitinib and basiliximab in kidney allograft recipients.

BACKGROUND: The common γ-chain (γ(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 γ(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 IC(50) 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: IC(50) 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 γ(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.

FoxP3 T cells and the pathophysiologic effects of brain death and warm ischemia in donor kidneys.

BACKGROUND AND OBJECTIVES: Forkhead box P3 regulatory T cells control inflammatory responses, but it remains unclear whether they inhibit brain death-initiated inflammation and tissue injury in deceased kidney donors. DESIGN, SETTING, PARTICIPANTS, MEASUREMENT: To study the actions of regulatory T cells at various stages of the donation and transplantation procedure, forkhead box P3, regulatory and inflammatory cytokine expression, and tissue injury markers were determined in time 0 kidney biopsies from deceased and living donors. Additionally, the interaction between forkhead box P3+ T cells and kidney injury molecule-1 by activated primary tubular epithelial cells was studied. RESULTS: After cold storage, the deceased donor kidneys expressed the higher mRNA levels of kidney injury molecule-1 and CD3ε. In these samples, the inflammatory cytokines IL-8 and IFN-γ and markers associated with regulation (forkhead box P3, TGF-ß, and IL-10) were highly expressed compared with living donor kidneys. Correlations were found between mRNA expression levels of forkhead box P3 and kidney injury molecule-1 and forkhead box P3 and IFN-γ. Immunohistochemical analysis confirmed the presence of forkhead box P3+ T cells in donor kidneys. Renal function (analyzed by serum creatinine levels) at the first week posttransplantation correlated with kidney injury molecule-1 and forkhead box P3 mRNA levels. In vitro studies showed that kidney injury molecule-1 expression by primary tubular epithelial cells was 63% (mean) lower when cocultured with regulatory T cells compared with control T cells. CONCLUSIONS: These results show that donor forkhead box P3+ T cells infiltrate the deceased donor kidney, where they may control inflammatory and injury responses.

Generation of donor-specific regulatory T-cell function in kidney transplant patients.

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.

End-stage renal failure and regulatory activities of CD4+CD25bright+FoxP3+ T-cells.

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(+)CD25(bright+)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 ml/min/ 1.73 m(2). RESULTS: The basal IL-2 mRNA level was high in patient-PBMC (P = 0.0002 versus healthy controls). The absolute number of CD4(+)CD25(bright+) 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(+)CD25(bright+) T-cells was determined in the setting of direct allorecognition. First, the effect of depletion of CD25(bright+) cells from patient-PBMC on proliferation was low. Second, co-culture of CD25(bright+) cells with CD25(neg/dim) cells (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(+)CD25(bright+)FoxP3(+) T-cells is also impaired.

Functional CD25(bright+) alloresponsive T cells in fully immunosuppressed renal allograft recipients.

BACKGROUND: Evidence from animal studies indicate a crucial role for CD25(bright+) regulatory T cells in transplantation tolerance. METHODS: To assess whether peripheral CD25(bright+) 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 CD25(bright+) 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 CD25(bright+) 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(-/dim) T cells plus CD25(bright+) T cells. At a 1:2 ratio, the CD25(bright+) T cells suppressed the proliferation of CD25(-/dim) donor- and third party-stimulated responder T cells. CONCLUSIONS: CD25(bright+) 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 CD25(bright+) T cells indeed play a role in graft acceptance after organ transplantation in patients remains to be elucidated.

FTY720 treatment of kidney transplant patients: a differential effect on B cells, naïve T cells, memory T cells and NK cells.

FTY720 alters lymphocyte recirculation and homing by interfering with S1P receptors on lymphocytes, possibly in combination with chemokine receptors, and induces a decrease in PBL counts. In fresh, whole blood samples of 14 kidney transplant patients, we analyzed by flow cytometry the effect of FTY on the number of NK cells, monocytes, naïve (CCR7+) T cells, memory (CCR5+) T cells and B cells. Patients treated with 0.5, 2.5 or 5mg FTY/day showed a strong decrease in T and B cell numbers. NK cells and monocytes were not affected. FTY reduced primarily naïve T cells. From the memory T cells (CCR5+), predominantly CD8 cells, 40-60% remained in the circulation. The majority of the CCR7+ cells disappeared from the circulation within 3-6h, while a further reduction was achieved later. The more slowly decrease in naïve CCR7+ T cell numbers was also observed in the group treated with 0.25mg FTY/day. Elispot assays revealed no IL-4 producing cells and a low frequency of IFN-gamma producing cells. We suggest that both CCR7 dependent and independent mechanisms are involved in the depletion of T cells from peripheral blood.

Differential effect of calcineurin inhibitors, anti-CD25 antibodies and rapamycin on the induction of FOXP3 in human T cells.

BACKGROUND: The transcription factor FOXP3 has been identified as the molecule associated with the regulatory function of CD25+ T cells. METHODS: To understand the biology of FOXP3+ T cells in allogeneic reactions, we measured FOXP3 mRNA expression levels in allostimulated CD25 cells and CD25 cells and in peripheral blood mononuclear cells (PBMC). The effect of immunosuppressive drugs on FOXP3 expression was studied in mixed lymphocyte reactions (MLR) in the presence and absence of calcineurin inhibitors (CNI), alphaCD25 mAb, and Rapamycin (Rapa), and analyzed in biopsies from cardiac allograft recipients during acute rejection by quantitative (Q)-PCR. RESULTS: FOXP3 mRNA expression was restricted to the CD25 population that inhibited the proliferation of allostimulated CD25 cells. In the MLR FOXP3 was readily induced after allostimulation. Kinetic examination of the MLR showed a 10-20-fold higher FOXP3 mRNA expression level after 5 days of culture. The CNI Cyclosporin and Tacrolimus, and alphaCD25 mAb inhibited in vitro induced FOXP3 gene transcription (range 70%-90%), whereas Rapa did not inhibit the induction. After clinical heart transplantation the highest FOXP3 mRNA expression levels were measured in biopsies during acute rejection (P=0.03). CONCLUSIONS: The high FOXP3 mRNA levels during allogeneic responses in vivo and in vitro suggests that regulatory activities of CD25 T cells or the generation of these cells is an intrinsic part of activation. CNI and alphaCD25 mAb in contrast to Rapa, did interfere with this immunosuppressive counter-mechanism and as a result might have an inhibitory effect to tolerance induction after transplantation.

The beneficial effects of recipient-derived vascular endothelial growth factor on graft survival after kidney transplantation.

BACKGROUND: Vascular endothelial growth factor (VEGF) is crucial for preservation of microvasculature and contributes to cytoprotection of the graft after kidney transplantation. METHODS: The authors investigated the influence of VEGF single-nucleotide polymorphism (SNP) on graft survival after renal transplantation. The SNP at positions -2578, -1154, and, -7 were analyzed in 306 donors and 387 recipients of renal transplants. RESULTS: The authors observed no effect of those recipient or donor SNP on acute rejection. However, graft survival was associated with recipient SNP at position -2578 C/A. Recipients with a genetic basis for high production of VEGF had significantly better graft survival compared with recipients with low production of VEGF. Homozygotes for the A allele (low producers of VEGF) had worse graft survival compared with high producers, the heterozygotes and homozygotes for C allele (P=0.03). Multivariate analysis in which the effects of donor age, recipient race, cold ischemia time, donor origin, and number of human leukocyte antigen mismatches were included showed that the status of noncarriers of -2578 C allele of recipients was an independent factor for graft failure (odds ratio, 1.8; 95% confidence interval, 1.0-3.0; P=0.03). CONCLUSIONS: The authors conclude that homozygote recipients for the -2578 A allele, the low producers of VEGF, are more vulnerable to tissue injury, resulting in worse graft survival.

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

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.

Differential intragraft cytokine messenger RNA profiles during rejection and repair of clinical heart transplants. A longitudinal study.

After clinical heart transplantation, ischemia, acute rejection, and repair mechanisms can trigger the up-regulation of cytokines. To investigate the cytokine profile early after transplantation, we monitored messenger RNA (mRNA) expression levels of tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta (TGF-beta), platelet-derived growth factor-A (PDGF-A), and basic fibroblast growth factor (bFGF) by reverse transcriptase-polymerase chain reaction (RT-PCR) in serial endomyocardial biopsies ( n=123) from 16 cardiac allograft recipients during the first 3 post-operative months. In the first month, mRNA expression levels of MCP-1, TNF-alpha, TGF-beta, and bFGF were significantly higher than in the period thereafter (acute rejection episodes excluded). Acute rejection (International Society for Heart and Lung Transplantation (ISHLT) rejection grade >2) was strongly associated with the level of TNF-alpha mRNA. After acute rejection episodes, rising mRNA expression levels of PDGF-A and bFGF were found. The association between TNF-alpha mRNA and acute rejection reflects the importance of this cytokine in allogeneic responses. Elevated growth factor expression levels indicate repair responses after tissue damage due to either the transplantation procedure (surgery, ischemia, reperfusion) or acute allograft rejection.

Sequential monitoring of intragraft cytokine mRNA expression in relation to diastolic left ventricular wall thickness and function early after heart transplantation.

Because production of immune regulatory proteins may play a role in early graft dysfunction after heart transplantation, we analyzed whether intragraft cytokine messenger RNA (mRNA) expression levels are associated with diastolic left ventricular function in cardiac allografts. We intensively monitored 16 cardiac allograft recipients during the first 3 months after transplantation. The mRNA expression levels of tumor necrosis factor (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1), transforming growth factor (TGF-beta), platelet derived growth factor (PDGF-A), and basic fibroblast growth factor (bFGF) were measured in endomyocardial biopsies (n = 123) by quantitative RT-PCR. To determine diastolic allograft function, concurrent M-mode and two-dimensional Doppler echocardiograms were analyzed for the following parameters: left ventricular total wall thickness, maximal early and atrial mitral flow velocity, deceleration time of maximal early mitral flow velocity, and isovolumetric relaxation period. During the first 3 months post-transplant an overall decrease in mRNA expression levels of almost all measured cytokines was observed, which paralleled an improvement in diastolic left ventricular wall thickness and function. However, no straightforward relationship could be found between a specific cytokine mRNA expression pattern and the studied echocardiographic parameters. Our data suggest that the improvement in diastolic left ventricular function is associated with a general reduction of inflammation within the allograft, rather than related to a specific cytokine expression pattern.