Wendy M. Mol

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 bright+ cells and CD25 intermediate( int)/- 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), &agr;CD25 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 bright+ population that inhibited the proliferation of allostimulated CD25 int/- 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 &agr;CD25 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 bright+ T cells or the generation of these cells is an intrinsic part of activation. CNI and &agr;CD25 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.

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.

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.

CD4+ CD25bright+ Regulatory T Cells Can Mediate Donor Nonreactivity in Long‐Term Immunosuppressed Kidney Allograft Patients

CD4+ CD25bright+ FoxP3+ T cells are potent regulators of T‐cell reactivity, but their possible involvement in donor‐specific nonresponsiveness after clinical kidney transplantation remains to be elucidated. We assessed the proliferative donor‐reactivity in 33 kidney allograft recipients who were maintained on a combination of proliferation inhibitors (mycophenolate mofetil (MMF) or Azathioprine (Aza)) and prednisone, long (>5 years) after transplantation. Of the 33 patients, 8 still exhibited donor‐reactivity, whereas 25 were classified as donor nonreactive patients. Within these 25 donor nonreactive patients, we assessed the involvement of CD4+ CD25bright+ regulatory T cells both by depleting them from the responder population as well as by reconstituting them to the CD25−/dim effector population. The absence of proliferation in these 25 patients, was abolished in 7 (28%) recipients upon depletion of the CD4+ CD25bright+ T cells. Reconstitution of these cells suppressed the donor‐reactivity in a dose‐dependent manner. Adding‐back CD4+ CD25bright+ T cells inhibited the anti‐third party response in all recipients, indicating that functional CD4+ CD25bright+ T cells circulate despite more then 5 years of immunosuppressive treatment.

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

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), CD3+CD4+ (median 37%; P<0.05), and CD3+CD8+ (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.

Conversion from cyclosporin A to tacrolimus is safe and decreases blood pressure, cholesterol levels and TGF-β1 type I receptor expression

To determine whether conversion from cyclosporin A (CsA) to tacrolimus (TAC)‐based immunosuppressive therapy is safe and might lead to improvement in the clinical side effect profile we studied 55 cardiac allograft recipients. Ten stable patients were electively converted (0.2–1.5 yr after transplantation; group I) and 45 patients were converted on indication (0.5–14 yr after transplantation; group II). 
We studied blood pressure, cholesterol level and renal function in all patients. To unravel the mechanisms by which CsA may exert its toxic effects and to evaluate whether conversion is associated with immune activation, we analyzed the transforming growth factor (TGF)‐β1 system and intragraft interleukin (IL)‐2 and IL‐15 mRNA expression by real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) and quantitative flow cytometry in the selectively converted patients (group I). 
Conversion did not result in immune activation as no clinical, histological or molecular signs of immune activation (increased intragraft IL‐2 and IL‐15 messenger RNA (mRNA) expression) leading to rejection were found. It did not improve renal function neither in patient group I nor in patient group II. However, after conversion the blood pressure decreased (group I: systolic 154±16 vs 143±21 mmHg, p=0.03, diastolic: 99±11 vs 90±11, p=0.02 and group II: systolic 155±17 vs 142±14, p<0.001, diastolic: 99±11 vs 91±8 mmHg, p<0.001). Likewise, the cholesterol levels improved (group I: 6.6±0.5 vs 5.7±0.3 mmol/L, p=0.001 and group II: 7.1±1.7 vs 6.1±1.7 mmol/L, p=0.001). When patients were treated with TAC the ongoing rejections (n=4) resolved and gum hyperplasia disappeared (n=5). Conversion was associated with a two‐fold lower TGF‐β1 type I receptor expression on peripheral lymphocytes and monocytes (p=0.02 and p=0.002, respectively). 
Conversion from CsA to TAC results in improvement of blood pressure and cholesterol levels and does not induce immune activation. These beneficial effects were accompanied with lower TGF‐β1 type I receptor expression.

Living kidney donors and hypoxia-inducible factor-1alpha

Background. Hypoxia inducible factor (HIF)-1, a heterodimeric transcription factor composed of &agr; and &bgr; subunits, is induced in the adaptive response to hypoxia and is critical for initiating the transcriptional activation of growth factors. We speculate that prolonged ischemia and hypoxia time leads to the production of HIF-1&agr;, which in turn induces the production of fibrogenic cytokines in the graft. Methods. To investigate our hypothesis, we measured the expression of HIF-1&agr; in time-zero biopsy specimens from living-donor kidneys (≤2.5 hr of ischemia) and cadaveric donor kidneys (12–32 hr of ischemia). Results. By real time reverse-transcriptase polymerase chain reaction analysis, the mRNA expression level of HIF-1&agr; was fivefold lower in time-zero biopsy specimens from living-donor kidneys than in specimens from cadaveric donor kidneys. In these time-zero biopsy specimens, the mRNA expression level of the fibrogenic cytokine transforming growth factor-&bgr; was also significantly lower (twofold). Conclusions. Low HIF-1&agr; mRNA expression levels correlate with short ischemia times and prevent the transcription of fibrogenic cytokines that initiate the irreversible process of graft fibrosis.

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 CD4+CD25bright+FoxP3+ 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 CD4+CD25bright+ T cells of 79 patients before, 3, 6, and 12 months after kidney transplantation. The immune regulatory capacities of CD4+CD25bright+ T cells were assessed by their depletion from peripheral blood mononuclear cells and in co-culture with CD25neg/dim responder T-cells in the mixed lymphocyte reactions. Results. In the first year after transplantation, the number and proportion of CD4+CD25bright+ 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 CD25brigth+ 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 CD25neg/dim 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 CD4+CD25bright+ 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

Abstract:  Background:  Evidence from animal studies indicate a crucial role for CD25bright+ regulatory T cells in transplantation tolerance.

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

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.