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Donor-specific hyporesponsiveness as occurs after allogeneic kidney transplantation may be mediated by repression of effector cells by a specific subset of T-cells: the CD4+ CD25bright+ FoxP3+ regulatory T-cells (Tregs). Here, we examined the suppressive capacity of Tregs isolated from the leukafereses product of 6 kidney transplant recipients, by reconstituting Tregs to responder T-cells at several time-points after initiation of proliferation. We show that Tregs derived from kidney transplant patients potently restrain proliferation to donor-antigens and 3rd party-antigens in classic reconstitution assays (i.e. addition of Tregs at the start of the co-incubation). However, when Tregs were added 5 days after initiation of proliferation, they were still capable of suppressing proliferation to donor-antigens (by 38%) but no longer to 3rd party-antigens. Thus, we conclude that the potency of Tregs to suppress reactivity to specific antigens should be determined by reconstitution to ongoing reactions. [ABSTRACT FROM AUTHOR]

Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare but hazardous complication caused by transfusion of leucocyte-containing blood. It is not clear why some patients are at risk for TA-GVHD, but others are not. We studied TA-GVHD in immunocompetent individuals by looking at donor-anti-host reactivity after transfusion of leucocyte-containing blood. We tested reactivity in 62 donor-recipient combinations in vitro before and at different times after blood transfusion with mixed lymphocyte culture. One patient was studied in more detail. Reactivity of blood donors against hosts gradually decreased after blood transfusion. This decrease significantly correlated with time (p < 0.001). Studies in a single patient showed that absence of donor-host reactivity was due to host T cells that inhibited the response. These data indicate that an active mechanism exists in immunocompetent individuals which inhibits the graft-versus-host reaction of the donor against the host. This mechanism might be exploited in organ transplantation by pre-transplant blood transfer. [ABSTRACT FROM AUTHOR]

The two regions of the maternal decidua, decidua basalis and decidua parietalis, differ in the extent of trophoblast invasion and consequently in cytokines and other biological mediators, extracellular matrix and cellular components. Our aim was to compare the phenotypic features of macrophages from the two decidual regions across a broad gestational age range. We isolated macrophages by enzymatic digestion from healthy decidua samples obtained after elective abortions, at 9-18-week and at 19-23-weeks, or after term deliveries (caesarean sections at term and spontaneous term vaginal deliveries). Macrophages were analysed by flow cytometry applying the same instrument settings to all the samples to allow semi-quantitative comparison of the expression of a particular marker between different samples. We found higher expressions of CD80, CD86 and HLA-DR, suggestive of a more activated phenotype of decidual macrophages, at early/mid pregnancy than at term. Marginal differences were found between term decidual macrophages obtained after spontaneous vaginal deliveries or caesarean sections which imply that the parturient process is not associated with decidual macrophage activation. The expressions of CD105, DC-SIGN and MMR were the strongest in decidua basalis of mid pregnancy and indicate the importance of decidual macrophages in tissue homeostasis at the uteroplacental interface.

Donor-specific hyporesponsiveness as occurs after allogeneic kidney transplantation may be mediated by repression of effector cells by a specific subset of T-cells: the CD4(+) CD25(bright+) FoxP3(+) regulatory T-cells (Tregs). Here, we examined the suppressive capacity of Tregs isolated from the leukafereses product of 6 kidney transplant recipients, by reconstituting Tregs to responder T-cells at several time-points after initiation of proliferation. We show that Tregs derived from kidney transplant patients potently restrain proliferation to donor-antigens and 3rd party-antigens in classic reconstitution assays (i.e. addition of Tregs at the start of the co-incubation). However, when Tregs were added 5 days after initiation of proliferation, they were still capable of suppressing proliferation to donor-antigens (by 38%) but no longer to 3rd party-antigens. Thus, we conclude that the potency of Tregs to suppress reactivity to specific antigens should be determined by reconstitution to ongoing reactions.

During pregnancy several maternal and fetal mechanisms are established to prevent a destructive immune response against the allogeneic fetus. Despite these mechanisms, fetus specific T-cells persist throughout gestation but little is known about the regulation of these T-cells. Recently, CD4(+)CD25(+) regulatory T-cells have been identified in human decidua. Human decidua forms the maternal part of the fetal-maternal interface and is subdivided in two distinct regions: the decidua (d.) basalis and the decidua (d.) parietalis. The aim of this study was to determine the distribution of specific T-cell subsets in d. basalis and d. parietalis in early and term pregnancy, with a special emphasis on the presence of CD4(+)CD25(bright) (regulatory) T-cells and CD8(+)CD28(-) (suppressor) T-cells. In addition, we compared phenotypic characteristics of decidua derived T-cell subsets with maternal peripheral blood (mPBL) T-cells and T-cells from non-pregnant controls. We identified significantly higher percentages of CD4(+)CD25(bright) and CD8(+)CD28(-) T-cells in decidua compared to peripheral blood suggesting an important role for these T-cell subsets locally at the fetal-maternal interface. The major differences in T-cell subset distribution and the presence of additional phenotypic differences between T-cells in d. basalis, d. parietalis and mPBL may reflect specific immunomodulatory functions of these T-cell subsets at these different sites during pregnancy.

Background: Tapering of immunosuppressive medication is indicated to prevent long-term side effects. Recently, we have shown that renal transplant recipients can safely be converted from calcineurin inhibitors to MMF or AZA when their donor-specific cytotoxic T-lymphocyte precursor frequencies (CTLpf) are below 10/10(6) PBMC. We wondered whether a low CTLpf also had predictive value when immunosuppressive medication was reduced in patients only on MMF or AZA and steroid medication., Methods: Renal transplant recipients with stable renal function at least 2 years after transplantation and with low (<10/10(6) PBMC) CTLpf were included. Their MMF or AZA dose was reduced to 75% and to 50% of the original dose at 4 months and 8 months after inclusion. Endpoint of the study was 12 months after inclusion or developing acute rejection., Results: Forty-five patients have reached the 1-year follow up endpoint. Their median time after transplantation was 4.2 years (range 2.0-15.5 years). Acute rejection was seen in one patient only (who had discontinued all his medication)., Conclusion: In patients with low CTLpf long after kidney transplantation, a 50% reduction of immunosuppression is safe and further decreasing their immunosuppressive load is the obvious next step.

Background: Pre-transplant blood transfusion (BT) results in better graft survival in organ transplant recipients, especially when BT donor and allograft recipient share an HLA-DR antigen. Although the immunologic mechanisms involved are still poorly understood, we wanted to know whether down regulation of donor-reactive T cells play a role., Methods: In a retrospective study, we analyzed the clinical effects of BT for 45 heart transplant (HTx) patients who had each received 1 BT that shared an HLA-DR-antigen with the recipient, and 55 who had a DR-mismatched BT before heart transplantation. From 30 patients, 15 with DR-shared BT and 15 with DR-mismatched BT, peripheral blood lymphocytes (PBL) were available. From each patient, we analyzed PBL samples taken at the day of transplantation (pre-transplant), and 1 to 2 months, 5 to 7 months, 9 to 14 months, 2 years, and 6 to 7 years after transplantation. Cytotoxic T-lymphocyte precursors (CTLp) and helper T-lymphocyte precursors (HTLp) were measured in a combined limiting dilution assay., Results: Analysis of survival during the first 10 years revealed a significantly (p = 0.016) better survival rate in the group of patients who had received HLA-DR-shared BT compared with the group who had received HLA-DR-mismatched BT. Patients of the DR-shared group experienced significantly (p = 0.042) less acute rejections compared with the patients who received DR-mismatched BT. We found no differences in the development of graft vascular disease. Frequencies of CTLp specific for the organ donor did not change with time after transplantation in the individual patients, nor did we detect any differences between the two BT groups. We found the same for organ donor-specific HTLp frequencies., Conclusions: These data suggest again that transfusion effect depends on HLA-DR compatibility between the heart transplant recipient and the pre-transplant BT donor. The mechanism that caused better survival rate was not down regulation of the donor-reactive T-cell frequency.

Background: The introduction of cyclosporine (CsA) in kidney transplantation has improved early graft survival. However, its long-term use is associated with impairment of renal function and increased cardiovascular risk factors. To avoid CsA-related long-term adverse effects, patients were converted to either azathioprine (AZA) or mycophenolate mofetil (MMF) 1 year after transplantation., Methods: Between September 1995 and January 1997, 64 stable renal transplant recipients on CsA and prednisone treatment were included in a prospective, randomized study. Patients were randomized for conversion of CsA to 2 mg/kg AZA (n=30) or 1 g of MMF twice daily (n=34). All patients remained on low-dose steroids. To decrease the total immunosuppressive load, a dose reduction in MMF and AZA was performed at 4 and again at 8 months after conversion. Mycophenolic acid trough levels were measured at regular intervals., Results: After conversion, a decrease in serum creatinine was found for both groups: for MMF, 132 to 109 micromol/L (P=0.016); and for AZA, 123 to 112 micromol/L (P<0.0001). After conversion, more acute rejections occurred in the AZA group (11/30) compared to the MMF group (4/34) (P=0.04). Dose reduction of MMF to 500 mg twice daily and of AZA to 1.0 mg/kg elicited three rejections in both groups. The incidence of side effects and infections were similar., Conclusion: Discontinuation of CsA spared renal function. In patients converted to MMF significantly less rejections occurred compared to patients converted to AZA. Furthermore, dose reduction of both AZA and MMF is possible in the majority (72%) of the patients.

Background: To reduce the side effects of long-term immunosuppressive therapy, stable renal transplant patients were routinely converted from cyclosporine to either azathioprine or mycophenolate mofetil. Thereafter, the azathioprine and mycophenolate mofetil dose was reduced to 75% at 4 months and to 50% at 8 months after conversion. We questioned whether the T-cell reactivity before conversion was able to predict which patients could be safely converted and tapered in their immunosuppressive load, while remaining free from acute rejection., Methods: Before conversion, the T-cell reactivity of peripheral blood mononuclear cells against donor and third-party spleen cells were tested in mixed lymphocyte cultures. We measured the frequency of donor and third-party reactive helper T-lymphocyte (HTLpf) and cytotoxic T-lymphocyte (CTLpf) precursors and their avidity for HLA class I antigens using limiting dilution analysis. Peripheral blood mononuclear cells were also stimulated with tetanus toxoid to test the general immune response., Results: The tetanus toxoid response, reactivity to donor and third-party cells as measured in mixed lymphocyte cultures and HTLpf, and the avidity of cytotoxic T-lymphocyte precursors were not predictive for the development of acute rejection. However, significant differences were found in donor-specific CTLpf before conversion, between patients with and without acute rejection after conversion in immunosuppression. The donor-specific CTLpf was significantly lower in patients without compared to those with acute rejection (P=0.01). Additionally, when no CTLpf was detectable before conversion, acute rejection did not occur after conversion. Acute rejection was only diagnosed in patients with detectable CTLpf before conversion., Conclusion: The number of donor-specific cytotoxic T-lymphocytes identifies patients in whom the immunosuppressive load can be safely reduced.

Cyclosporine (CsA) is thought to enhance transforming growth factor (TGF)-beta1 production in vitro and in vivo and this may have a negative effect on long-term graft survival. Therefore, we studied TGF-beta1, plasma levels in 30 patients before kidney transplantation, after transplantation during CsA treatment and after conversion from CsA to azathioprine (AZA) or mycophenolate mofetil (MMF). We questioned whether TGF-beta1 plasma levels would decrease after the discontinuation of CsA and whether the TGF-beta1 plasma levels did correlate with CsA trough levels and kidney function, measured by serum creatinine levels. TGF-beta1 plasma levels measured 1 yr after transplantation were lower compared to levels measured before transplantation, however not significantly (p = 0.08). After conversion from CsA to MMF or AZA, a slight increase was observed in some patients, but in the total group TGF-beta1 levels remained unaffected. No correlation was found between the TGF-beta1 levels and CsA trough levels nor with creatinine levels. In conclusion, we did not observe higher TGF-beta1 plasma levels in plasma levels of patients receiving CsA treatment compared to blood from the same patients while on AZA or MMF.

The ultimate goal in organ transplantation is the induction of donor-specific transplantation tolerance. The fact that in some patients it is possible to withdraw immunosuppressive therapy completely, suggests that immunological adaptation or donor-specific nonresponsiveness can occur following transplantation. In earlier studies we have shown that after blood transfusion, the mixed lymphocyte reactivity of the donor against patient peripheral blood mononuclear lymphocytes taken after blood transfusion gradually decreased with time. This may reflect the induction of an immunoregulatory mechanism, which protects the recipient against an immune reaction of the donor, enhancing a state of mixed chimerism. A similar phenomenon might also play a role in the immunological mechanism leading to transplantation tolerance. Therefore, we studied responses in patients with a well-functioning liver and heart transplant using a primed lymphocyte test (PLT) and a mixed lymphocyte culture (MLC). Two years after liver transplantation the PLT and MLC responses of patient against donor were decreased significantly compared to the situation before transplantation. The response of donor against patient was also lower two years after transplantation. The decreased responses were donor-specific since responses to third-party cells generally remained unchanged. In heart transplant recipients we could not detect a donor-specific downregulation. The reversed response, of donor against patient, was not different from responses of third-party against patient cells. Therefore, we conclude that donor-specific nonresponsiveness is not induced in patients with well-functioning heart transplants. In contrast, after a successful liver transplantation the response of patient against donor is decreased, as is the reversed response. It may be valuable to test whether in liver transplant patients withdrawing or reducing of maintenance immunosuppression is permitted for patients who appear to have developed two-way donor-specific hyporeactivity.

Pretransplant blood transfusion can have a favorable effect on renal and cardiac graft survival. In a previous study, we found that HLA-DR-shared blood transfusions led to better graft survival after kidney transplantation. In the present study, we tried to confirm and extend these findings by analyzing the effects of blood transfusion on the clinical course following heart transplantation. According to a predefined protocol, cardiac allograft recipients received random blood transfusion before surgery. A beneficial effect of HLA-DR-shared blood transfusion could be observed on survival and on the number of rejection episodes. Patients who had received an HLA-mismatched transfusion had significantly more infections, and in this group, more patients died due to malignancies. To study a possible role of immunomodulating T cells, patients who had or had not received prophylactic anti-T cell therapy were analyzed separately. In both groups, a beneficial effect of HLA-DR-shared blood transfusion could be demonstrated. These results indicate that the administration of HLA-DR-matched blood transfusion before transplantation is beneficial in cardiac allograft recipients, and that regulatory T cells are either not directly involved or (partly) resistant to modulation by anti-T cell antibody therapy.

Recent data suggest that the favorable effect of pretransplant blood transfusion (BT) on transplant outcome depends on the HLA match. HLA-DR or haplotype shared transfusions lead to transplantation tolerance, and HLA-mismatched BT leads to immunization. The immunological mechanism involved is still unknown. To investigate the effect of HLA compatibility between blood donor and recipient on the T cell compartment, we determined the frequency of cytotoxic and helper T cell precursors specific for blood donor cells (n=20) and the T cell receptor Vbeta (TCRBV) repertoire of the CD4- and CD8-positive peripheral blood mononuclear cells before, at 2 weeks after, and at more than 10 weeks after BT (n=10). Patients had received one transfusion of a nonstored (<24 hr after withdrawal) erythrocyte concentrate without buffy coat containing on average 6x10(8) leukocytes. Eight patients shared an HLA-B and -DR antigen, nine patients shared one HLA-DR antigen, and three patients shared no HLA class II antigens with the blood donor. All patients showed a significant increase in both cytotoxic and helper T cell precursor frequencies against the blood donor 2 weeks after BT. In most patients, the frequencies reached pretransfusion levels again long after BT. In 5 of 10 patients, an expansion of one or more TCRBV families was observed in either the CD4 or CD8 compartment. This study demonstrates that BT, irrespective of the degree of HLA matching, induces activation of the T cell compartment. The degree of sharing of HLA antigens was not correlated with quantitative changes in cytotoxic T lymphocyte precursor or helper T lymphocyte precursor frequencies, or changes induced in the TCRBV repertoire. Cytotoxic and helper T lymphocyte precursor frequencies and TCRBV repertoire determined after BT do not give an indication for a state of tolerance prior to transplantation.