Discontinuation of calcineurin inhibitors treatment allows the development of FOXP3+ regulatory T-cells in patients after kidney transplantation.

This study investigated specific gene expression profiles in patients with donor-specific cytotoxic-hyporesponsiveness, reflected by cytotoxic T-lymphocyte precursor frequency (CTLpf). The effect of calcineurin inhibitor (CNI) withdrawal was studied on markers for cytotoxicity (perforin, granzyme B), apoptosis (Fas,FasL), Th1 and Th2 cytokines (IL-2, IL-10), Th1 and Th2 transcription factors (T-bet, GATA 3), Th17 transcription factor and cytokine (RORγt, IL-17), and for immune regulation/activation (CD25, FOXP3). Peripheral blood samples from renal allograft recipients (n = 18) more than two yr after transplantation with stable renal function were analyzed before and four months after CNI withdrawal. Additionally, systolic and diastolic blood pressure, cholesterol, serum creatinine and proteinuria were evaluated, and no significant differences were measured before and after CNI withdrawal. However, CNIs' discontinuation influenced peripheral gene expression profiles. After CNI withdrawal, the mRNA expression of Granzyme B, Perforin, Fas, FasL, T-bet, GATA3 and CD25 were significantly lower than during CNI treatment. After CNI discontinuation, donor-specific CTLpf decreased, while FOXP3 expression discriminated between detectable and non-detectable donor-specific cytolysis reactivity; FOXP3 transcript values were highest in absence of donor-specific cytotoxicity (p < 0.01). Our study shows CNI withdrawal in stable kidney transplant recipients two yr after transplantation is safe. Moreover, discontinuation of CNIs' treatment allows FOXP3+ regulatory T-cells development, resulting in a significant decrease of anti-donor immune reactivity.

Preeclampsia is associated with increased cytotoxic T-cell capacity to paternal antigens.

OBJECTIVE: During an uncomplicated pregnancy the conceptus is a semiallogeneic entity in which rejection is prevented by suppression of the maternal immune system. We hypothesized that this suppression is disturbed in patients with preeclampsia and that a maternal immune response to fetal (foreign/paternal) antigens in the fetal-maternal interface may be responsible for local inflammation, with subsequent endothelial dysfunction and systemic disease. STUDY DESIGN: Blood samples were obtained from 14 women with preeclampsia (cases), 14 gestational-age and parity-matched women with uncomplicated pregnancies (controls), and their partners. We determined the partner-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) and the CTLpf directed to unrelated partners with uncomplicated pregnancies. We measured the CTLpf in peripheral blood mononuclear cells (PBMCs) from cases and controls using limited-dilution assays. In addition, proliferation was tested in a mixed-lymphocyte culture (MLR). RESULTS: The partner-specific CTLpf was significantly higher in cases compared with controls (median, 183 [15-338] vs 67 [9-232] per million PBMCs, P = .02). In contrast, in women with uncomplicated pregnancies, the partner-specific CTLpf was down-regulated compared with the CTLpf directed to an unrelated partner who fathered uncomplicated pregnancies (P = .02). No difference was found in partner-specific MLR response between cases and controls. CONCLUSION: These results suggest that women with preeclampsia have a higher cytotoxic T-cell response to paternal antigens compared with pregnant controls. This insufficiently suppressed immune response may eventually lead to the development of preeclampsia.

Calcineurin inhibitor withdrawal in stable kidney transplant patients decreases the donor-specific cytotoxic T lymphocyte precursor frequency.

BACKGROUND: In a prospective study, calcineurin inhibitors (CNI) were withdrawn in patients two years after kidney transplantation. We questioned whether stopping CNI had an effect on the donor-specific reactivity, as CNI might hinder immune responses leading to graft acceptance. METHODS: We measured the donor-specific cytotoxic T lymphocyte (CTL) precursor frequency (CTLpf) in 54 patients before and after withdrawal of CNI. In addition, the T-cell reactivity of PBMC to donor and third-party antigens was tested in MLR, and in IFNgamma-Elispot. Reactivity to tetanus toxoid (TET) was studied as well. RESULTS: Donor-specific CTLpf significantly decreased after CNI withdrawal (P=0.0001). In contrast, no difference was observed in third-party reactive CTLpf, donor and third-party reactive MLR and IFNgamma-Elispot. Proliferative responses and the number of IFNgamma-producing cells to TET also decreased after CNI withdrawal. The decrease in CTLpf correlated with the time between the two blood samples (before and after stopping CNI, P=0.05). This decrease was caused by stopping CNI, because there was no correlation between CTLpf and the duration of the CNI treatment after transplantation. Moreover, the percentage of regulatory T cells in the peripheral blood increased after CNI withdrawal. CONCLUSIONS: We report here that after withdrawal of CNI the donor-specific CTLpf decreases. We hypothesize that CNI suppress regulatory mechanisms that have the potential to down-regulate donor-specific CTL responses and reactivity to TET.

The granzyme B and interferon-gamma enzyme-linked immunospot assay as alternatives for cytotoxic T-lymphocyte precursor frequency after renal transplantation.

BACKGROUND: The interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay has gained increased popularity as a surrogate marker of cytotoxic T-lymphocyte (CTL) activity. However, the functional activity of CTL might be a more relevant surrogate marker of CTL. Therefore, the authors wondered whether the granzyme B (GrB) ELISPOT assay is a better marker for determining the number of CTL than the IFN-gamma ELISPOT assay. METHOD.: Peripheral blood mononuclear cells (PBMC) from 19 kidney transplant patients were stimulated with donor cells or third-party cells. The authors determined the CTL precursor frequency (CTLpf) and simultaneously measured the number of IFN-gamma- and GrB-producing cells (pc) by ELISPOT assay. RESULTS: In all three different assays, the reactivity to donor cells was significant lower than the reactivity to third-party cells: CTLpf, median: 9 versus 60/10(6) PBMC (P=0.0002); number of IFN-gamma pc: 10 versus 90/10(6) PBMC (P=0.0001); number of GrB pc: 60 versus 205/10(6) PBMC (P=0.05). When the authors compared the CTLpf after third-party stimulation with the corresponding ELISPOT results, they found a positive correlation between the CTLpf and the number of IFN-gamma pc (r(s)=0.47, P=0.05). No correlation was found between the CTLpf and the number of GrB pc (r(s)=0.23, P=0.36). However, when they compared the donor-specific CTLpf with the corresponding ELISPOT results, no correlation with the ELISPOT for IFN-gamma (r(s)=0.10, P=0.69) or GrB (r(s)=-0.24, P=0.34) was found. CONCLUSIONS: The authors feel that the CTLpf, as a measure of the actual endpoint of cytolytic activity and independent of the pathway of killing, remains the "gold standard" for determining donor-specific cytolytic activity after clinical organ transplantation.

Tapering immunosuppression in recipients of living donor kidney transplants.

We have previously suggested that the in vitro donor-specific cytotoxic T-lymphocyte precursor (CTLp) assay can guide us to identify patients in which the immunosuppressive load can be tapered. In a clinical trial we had observed that a low (<10/10(6) PBMC) frequency of these CTLp was predictive for an uneventful rejection-free clinical course in patients that were converted from calcineurin inhibitors to mycophenolate mofetil or azathiopine. In the present prospective study in 81 stable kidney transplant recipients, already converted from calcineurin inhibitors, we measured CTLp frequencies and reduced the immunosuppressive load on a routine basis when CTLp were <10/10(6) PBMC. Donor-specific cytotoxicity could not be measured in 50/81 patients, while their reactivity against third-party lymphocytes was not impaired. These 50 patients were tapered in their immunosuppression. Only in one patient, who had stopped all his medication, was a rejection episode diagnosed. We conclude that in patients with a low donor-specific CTLp frequency it is safe to reduce the immunosuppression.

Formation of donor-specific human leukocyte antigen antibodies after kidney transplantation: correlation with acute rejection and tapering of immunosuppression.

BACKGROUND: Before kidney transplantation, a serological crossmatch is routinely performed between donor and recipient to prevent hyperacute rejection by donor-specific anti-human leukocyte antigen (HLA) antibodies. After transplantation, the presence of these antibodies is not routinely monitored. We wanted to know whether donor-specific anti-HLA antibodies are detectable during acute rejection (AR), before or after reduction of immunosuppression in kidney transplant recipients who were converted from cyclosporine A (CsA) to the less nephrotoxic azathioprine (AZA) or mycophenolate mofetil (MMF) at 1 year after transplantation. METHODS: Plasma samples were collected before transplantation, at several time points after transplantation, and during AR. Antibodies were measured in 29 patients: 5 patients with AR during the first year after transplantation (before conversion), 14 patients with AR after conversion or dose-reduction of AZA or MMF, and a control group of 10 patients without AR during a follow-up of 2 years (1 year before and 1 year after conversion of immunosuppression). Antibodies were measured by complement-dependent cytotoxicity assay, enzyme-linked immunosorbent assay (ELISA), and flow-cytometry in a crossmatch with donor spleen cells. RESULTS: Donor-specific antibodies were not detectable after transplantation in the control group without AR, nor in patients with AR shortly after transplantation during CsA therapy. After conversion from CsA to AZA or MMF, antibodies appeared only in one patient after graft failure followed by transplantectomy and in patients during AR on AZA but not on MMF therapy. CONCLUSION: In this patient group, we could not detect donor-specific antibodies during CsA treatment, not even at the time of AR using three different techniques. Donor-specific antibodies were primarily present during AR in patients converted from CsA to AZA and were not found in the sera from patients converted to MMF.