Conversion from cyclosporin A to tacrolimus is safe and decreases blood pressure, cholesterol levels and TGF-beta 1 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)-beta 1 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-beta 1 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-beta 1 type I receptor expression.

Quantitative flow cytometry shows activation of the TNF-alpha system but not of the IL-2 system at the single cell level in renal replacement therapy.

BACKGROUND: Immunological dysfunction in patients on haemodialysis may be related to imbalanced cytokine systems, such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-2. Despite activation of these systems, haemodialysis patients show high susceptibility for infections and malignancies, and have a poor immunological reaction to T-cell-dependent antigens, like hepatitis B vaccination. In this study we have determined the activation status of the two different cytokine systems, at the single cell level, using quantitative flow cytometry. METHODS: Using fluorescein isothiocyanate- or phycoerythrin-conjugated antibodies directed against TNF-R2 (CD120b), IL-2Ralpha (CD25) and IL-2Rbeta (CD122), we measured the expression of these receptors at the single cell level in order to determine the level of activation of monocytes and T-lymphocytes. RESULTS: Significantly higher expression of the TNF-alpha receptor, TNF-R2, was present on both monocytes and T-lymphocytes in patients on renal replacement therapy (RRT) compared with pre-dialysis chronic renal failure (CRF) patients and controls, indicating activation of the TNF-alpha system. In contrast, IL-2R expression was comparable in all groups studied, which may reflect a non-activated state of the IL-2 system. CONCLUSIONS: The present study illustrates an activated state of the TNF-alpha system in patients on RRT, at the single cell level, while the IL-2 system seems to be unaffected. These findings support the hypothesis that the interaction between the TNF-alpha and IL-2 cytokine systems is disturbed.

T cells activate the tumor necrosis factor-alpha system during hemodialysis, resulting in tachyphylaxis.

BACKGROUND: The immunosuppressive state of hemodialysis (HD) patients is accompanied by activation of antigen-presenting cell-derived cytokines, for example, tumor necrosis factor-alpha (TNF-alpha), which are required for T-cell activation. To test whether an activated TNF-alpha system results in impaired T-cell response in these patients, we analyzed parameters of their antigen-presenting cell (APC) function (for example, TNF-alpha system) and T-cell function [for example, interleukin-2 (IL-2) system]. METHODS: By quantitative flow cytometry, the expression of the TNF-receptor 2 (TNF-R2 = CD120b) and the alpha and beta chain of the IL-2 receptor (IL-2R; CD25, CD122) was measured. Using reverse transcriptase-polymerase chain reaction, the mRNA for TNF-alpha, IL-2, and IL-2R were determined. Phyto-hemagglutinin (PHA)- and IL-2-stimulated proliferation and cytokine production were measured. Biological activity of soluble receptors was measured by adding recombinant cytokines to the patient's plasma. RESULTS: CD120b expression was significantly increased in HD patients, whereas CD25 and CD122 was comparable to controls. In contrast to mRNA for IL-2 and IL-2R, mRNA for TNF-alpha was increased in HD. This resulted in significantly increased TNF-alpha levels in HD patients. In peripheral blood of HD patients, high levels of soluble TNF-R (R1 and R2) and IL-2R were found. These receptors were capable of binding 40% of added TNF-alpha and 55% of added IL-2. PHA-induced TNF-alpha production by T cells from HD patients was significantly lower, while their PHA-stimulated IL-2 production and proliferation capacity by T cells were comparable to controls. CONCLUSIONS: We conclude that although the TNF-alpha system is activated during HD, the TNF-alpha production of T cells is impaired, suggesting that tachyphylaxis of T cells occurs for TNF-alpha, as their proliferative capacity and IL-2 production capacity do not imply an intrinsic T-cell defect.

Quantitative flow cytometry to measure the TNF-alpha and IL-2 system after heart transplantation.

After heart transplantation a high incidence of infections and malignancies is found. Not only immunosuppression, but also intrinsic cytokine systems with some unbalance, e. g. TNF-alpha and IL-2, can result in impaired immune competence and may have a role in these complications. The aim of this study was to assess the activity of the TNF-alpha and IL-2 systems after heart transplantation. In peripheral blood we measured expression of activation markers of TNF-alpha (TNF-R2) and IL-2 (IL-2R alpha, IL-2R beta-chain) on monocytes and lymphocytes using quantitative flow-cytometric analysis. TNF-R2 expression was significantly enhanced on monocytes and lymphocytes in patients after heart transplantation, while the expression of IL-2R alpha and IL-2R beta was not elevated. Increased TNF-R2 expression in peripheral blood after heart transplantation reflects an activated TNF-alpha system, leading to high levels of active sTNF-R, which impairs TNF-alpha bioavailibility and consequently leads to immune incompetence.

Circulating donor-specific cytotoxic T lymphocytes with high avidity for donor human leukocyte antigens in pediatric and adult cardiac allograft valved conduit recipients.

OBJECTIVE: Specific immunological responses may be involved in the process of cryopreserved allograft valved conduit (AVC) degeneration, which is more frequently seen in young recipients. Rejection of heart and corneal allografts is preceded by an increase in the fraction of cytotoxic T lymphocytes (CTL) with high avidity for donor human leukocyte antigens (HLA) circulating in both peripheral blood and the affected graft. These donor-specific high-avidity CTLs are regarded as the destructive cells capable of causing graft damage. To monitor the precursors of these cells (CTLp) in young and adult AVC recipients, in vitro quantitative tests were performed on sequentially taken blood samples to quantitate CTLp frequencies and their avidity for donor antigens. METHOD: Six children and nine adults who received a cryopreserved AVC in the period between 1994 and 1997 were included in the study. From these patients, two to six blood samples were obtained up to 3 years after valve implantation. The number of circulating CTLp present within the peripheral blood mononuclear cell (PBMC) population was determined by limiting dilution analysis (LDA). The fraction of CTLp with high avidity for donor HLA class I was determined by addition of CD8 monoclonal antibodies (mAb) during the cytotoxic phase of the assay. Third-party stimulator cells were used to verify the donor-specificity of the response. RESULTS: The number of donor-specific CTLp increased significantly in the period 6-12 months after AVC implantation, while third-party-specific CTLp frequencies were not affected. Additionally, we found a significant increase of the high-avidity fraction of CTLp directed against donor antigens as early as during the first 6 months after AVC implantation. The fraction of high-avidity CTLp remained significantly higher post- compared with pre-implantation, even after 12 months. We observed no significant difference in the kinetics of CTLp frequencies between pediatric and adult AVC recipients. CONCLUSION: Implantation of cryopreserved human AVC induces an increase in the total number of circulating CTLp directed against donor HLA class I in both adults and children. The shift towards more destructive high-avidity CTLp in the peripheral blood indicates their potential damaging effect towards the heart valve allograft.

Functional responses of T cells blocked by anti-CD25 antibody therapy during cardiac rejection.

BACKGROUND: Despite anti-CD25 (interleukin [IL]-2 receptor alpha chain) monoclonal antibody (mAb) therapy, rejection can still occur. T-cell activation through the IL-2 receptor beta and gamma chains by IL-2 or other growth factors may contribute to this rejection. Recently, we have demonstrated that the T-cell growth factor IL-15 was abundantly present in rejecting cardiac grafts during anti-CD25 mAb treatment. METHODS: To test whether IL-2- and IL-15-responsive T cells play an active role in rejection during anti-CD25 mAb therapy, we measured the frequency of IL-2- and IL-15-proliferative T cells in peripheral blood from treated patients during rejection (n=12). Measurements were made by limiting dilution analysis in the absence and presence of extra in vitro-added mouse anti-human CD25 mAb. RESULTS: In the absence of anti-CD25 mAb, the frequencies of peripheral T cells responding to recombinant human (rh)IL-2 and rhIL-15 from patients were lower than those measured in samples of healthy controls (n=7): median of IL-2-responding T cells 78 per 10(6) (range 31-210 per 10(6)) vs. 154 per 10(6) (122-484 per 10(6), P=0.008) and median of IL-15-responding T cells 62 per 10(6) (range 19-207 per 10(6)) vs. 129 per 10(6) (range 79-192 per 10(6), P=0.02), respectively. In the presence of extra in vitro-added anti-CD25 mAb, frequencies of IL-2-responding T cells from patients significantly decreased, although a considerable number of T cells still proliferated on rhIL-2 (median 85%, range 46-100%). In contrast, the frequencies of IL-15 T cells still responding remained stable (median 2%, range 0-50%, P<0.001). CONCLUSIONS: Treatment with anti-CD25 mAbs cannot provide complete suppression of T-cell function because significant numbers of IL-2- and IL-15-responsive T cells remain present in the peripheral blood of allograft recipients during anti-CD25 mAb treatment.

Anti-CD25 therapy reveals the redundancy of the intragraft cytokine network after clinical heart transplantation.

BACKGROUND: Despite blockade of the interleukin-2/interleukin 2 receptor (IL-2/IL-2R) pathway by the murine anti-CD25 (i.e., IL-2R alpha chain) monoclonal antibody BT563, cardiac rejection can still occur. In these cases, growth factors other than IL-2 may contribute to allograft rejection. We studied the expression of IL-15, a macrophage-derived cytokine associated with T-cell activation, which interacts with the beta and gamma chains of the IL-2R during rejection episodes under anti-CD25 therapy. METHODS: We measured intragraft IL-15 mRNA expression and the number of IL-15- and CD68-positive cells in posttransplantation endomyocardial biopsies (EMBs; n=45) and in nontransplanted, donor-heart specimens (n=11) by competitive template reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS: IL-15 mRNA expression was present in the majority of posttransplantation EMB specimens (91%, 41/45) and in nontransplanted donor-heart specimens (91%, 10/11). Relative IL-15 mRNA levels were neither associated with transplantation nor with rejection status. After transplantation, the number of IL-15- and CD68-positive cells significantly increased (P<0.001), but IL-15-positive cell counts did not reflect the histological rejection grade. Anti-CD25 treatment, in contrast to its effects on the IL-2/IL-2R complex, had no influence on intragraft IL-15 mRNA and protein production. In rejection EMB specimens, during (n=5) and after (n=8) anti-CD25 therapy, no differences in relative IL-15 mRNA levels, or in IL-15- and CD68-positive cell counts, were measured. CONCLUSIONS: After heart transplantation, high numbers of IL-15- and CD68-positive cells infiltrate the graft. This phenomenon is independent of the rejection status. IL-15 remains present during blockade of the IL-2/IL-2R pathway by anti-CD25 monoclonal antibodies, and it may participate in T cell-dependent donor-directed immune responses, thereby explaining the occurrence of rejection in the absence of IL-2.

Kinetics of circulating cytotoxic T lymphocyte precursors that have a high avidity for donor antigens: correlation with the rejection status of the human cardiac allograft.

Studies on graft infiltrating cells demonstrated that accumulation of cytotoxic T lymphocytes (CTL) with high avidity for donor antigens (Ag) coincided with acute cardiac rejection. In the present study, we analyse whether such high-avidity CTL are present within the peripheral blood of cardiac transplant recipients and whether their kinetics correspond with the rejection status of the allograft. Using limiting dilution analysis (LDA), donor-specific CTL were enumerated in serial blood samples of seven patients. From each patient, 7-11 samples were obtained during the first year after transplantation and up to three samples were obtained at a later date. Enumerated donor-specific CTL were divided into CTL with high or low avidity for donor Ag, depending on their sensitivity to CD8-blocking. In contrast to the situation in the graft, the donor-specific CTL present within the peripheral blood were CTL precursors (pCTL) and not fully mature CTL (cCTL). The number of donor-specific pCTL among peripheral blood cells fluctuated irrespective of the rejection grade of the allograft, indicating that the frequency of circulating donor-specific CTL does not reflect the immunological status of the allograft. During acute cardiac rejection, 66% (median) of the circulating donor-specific pCTL had a high avidity for donor Ag. This percentage significantly exceeded pre- and postrejection values obtained during the first year post-transplantation (median, 39% and 37%, respectively). The disparity in avidity increased even further more than 1 year after transplantation, when stable engraftment was achieved. Among donor-specific pCTL in peripheral blood, those with a high avidity were absent (median, 0%). Hence the avidity of circulating donor-specific CTL might inform us about the immune status of the cardiac allograft.

C1.7 monoclonal antibody designates high-avidity CD4+ cytotoxic T lymphocytes involved in clinical heart rejection.

BACKGROUND: It is assumed that not all donor-specific cytotoxic T lymphocytes (CTLs), but only those with a high avidity for donor antigens, can function as terminal effector cells in transplant rejection. METHODS: In the present study, we searched for markers that would exclusively designate these high-avidity CTL. RESULTS: FACS analysis of donor-specific CTL clones obtained from heart transplant patients revealed that high- and low-avidity CTL varied in their expression of p38, a surface molecule involved in signal transduction, which is stained by the antibody C1.7. High- and low-avidity CD8+ CTL and high-avidity CD4+ CTL expressed p38, whereas low-avidity CD4+ CTL did not. Noncytotoxic and naive CD4+ lymphocytes also lacked p38 surface expression. CONCLUSION: Therefore, we conclude that p38 is a marker for CD4+ lymphocytes with the potency to damage the transplanted heart. Accordingly, p38 might be used to analyze the contribution of CD4+ CTL in immune responses, such as transplant rejection.