Nicole M. van Besouw

Human mesenchymal stem cells are susceptible to lysis by CD8+ T cells and NK cells

There is growing interest in the use of mesenchymal stem cells (MSCs) to improve the outcome of organ transplantation. The immunogenicity of MSCs is, however, unclear and is important for the efficacy of MSC therapy and for potential sensitization against donor antigens. We investigated the susceptibility of autologous and allogeneic MSCs for lysis by CD8+ T-lymphocytes and NK cells in a kidney transplant setting. MSCs were derived from adipose tissue of human kidney donors and were CD90+, CD105+, CD166+, and HLA class I+. They showed differentiation ability and immunosuppressive capacity. Lysis of MSCs by peripheral blood mononuclear cells (PBMCs), FACS-sorted CD8+ T cells, and NK cells was measured by europium release assay. Allogeneic MSCs were susceptible for lysis by cytotoxic CD8+ T cells and NK cells, while autologous MSCs were lysed by NK cells only. NK cell-mediated lysis was inversely correlated with the expression of HLA class I on MSCs. Lysis of autologous MSCs was not dependent on culturing of MSCs in FBS, and MSCs in suspension as well as adherent to plastic were lysed by NK cells. Pretransplant recipient PBMCs did not lyse donor MSCs, but PBMCs isolated 3, 6, and 12 months after transplantation showed increasing lysing ability. After 12 months, CD8+ T-cell-mediated lysis of donor MSCs persisted, indicating there was no evidence for desensitization against donor MSCs. Lysis of MSCs is important to take into account when MSCs are considered for clinical application. Our results suggest that the HLA background of MSCs and timing of MSC administration are important for the efficacy of MSC therapy.

Donor-specific cytokine production by graft-infiltrating lymphocytes induces and maintains graft vascular disease in human cardiac allografts.

BACKGROUND The development of graft vascular disease (GVD) in the allograft is a major impediment for long-term survival of heart transplant recipients. GVD may be mediated by cellular processes, in response to the transplanted heart, and regulated by cytokines. METHODS We studied donor-specific cytokine production patterns in graft-infiltrating lymphocyte cultures propagated from endomyocardial biopsies. The biopsies were derived from patients with and without signs of GVD, as diagnosed by angiography at 1 year after heart transplantation. RESULTS In the first year after transplantation, significantly more T-helper (Th) 1 cytokines (interleukin [IL]-2: P=0.04; interferon-gamma: P=0.01), but not Th2 (IL-4 and IL-6) cytokines, were produced by cultures of patients with GVD compared with patients without GVD. Thereafter, the Th1 cytokine levels in patients with GVD normalized to the levels of patients without GVD. Detectable levels of IL-6 were produced significantly more often (P=0.009) by cultures obtained more than 1 year after transplantation from patients with GVD. CONCLUSIONS The results suggest that high levels of Th1 cytokines produced by graft-infiltrating lymphocytes early after transplantation may be responsible for activation of vascular endothelium, leading to the migration and proliferation of smooth muscle cells that is characteristic of GVD. IL-6, produced later after transplantation, continues this process by promoting smooth muscle cell proliferation.

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 IFNγ-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 IFNγ-Elispot. Proliferative responses and the number of IFNγ-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 frequency of interferon-gproducing cells reflects alloreactivity against minor histocompatibility antigens

Background. In human leukocyte antigen (HLA)-identical living-related renal transplant recipients, no donor-specific alloreactivity can be detected in regular tests (mixed lymphocyte culture, helper T-lymphocyte precursor frequencies, cytotoxic T-lymphocyte precursor frequencies) to identify patients responding to minor histocompatibility antigens (mHag). We questioned whether a more sensitive method like the Elispot-assay could be more appropriate. Methods and Results. Peripheral blood mononuclear cells (PBMC) from 16 HLA-identical living-related kidney transplant patients 3 months (range, 2 weeks to 5 months) after transplantation were tested for the frequency of interferon (IFN)-&ggr; producing cells by the Elispot-assay. PBMC from the recipient were stimulated with irradiated donor PBMC and corrected for backward stimulation. Donor-specific IFN-&ggr; producing cells (pc) in the range of 5 to 115 per million PBMC (median, 30 per million PBMC) were found. To evaluate the frequency of spot forming cells in time, PBMC from six patients who received an HLA-identical renal transplant were stimulated with irradiated donor PBMC before, approximately 3 months after, and 12 months after transplantation. Four patients who received an HLA-mismatched living-related kidney served as positive control. In the HLA-identical group, frequencies in the range of 0 to 10 IFN-&ggr; pc per million PBMC were found before transplantation, 0 to 30 per million PBMC 3 months after transplantation, and 0 to 45 per million PBMC 12 months after transplantation. In the HLA-mismatched group, significantly higher numbers were found: 10 to 480 IFN-&ggr; pc per million PBMC before, 20 to 360 per million PBMC at 3 months, and 30 to 590 per million PBMC 12 months after transplantation. Conclusion. Under immunosuppressive therapy, IFN-&ggr; pc specific for donor mHag can be found after HLA-identical living-related renal transplantation.

Vaccine-induced allo-HLA-reactive memory T cells in a kidney transplantation candidate.

Background. Allo-human leukocyte antigen (HLA) reactivity by naturally acquired viral-specific memory T cells is common. However, the effect of successful vaccination on the alloreactive memory T-cell repertoire is unclear. We hypothesized that vaccination could specifically induce allo-HLA–reactive memory T cells. Methods. A varicella-zoster virus (VZV) immediate early 62 (IE62)-specific CD8 memory T-cell clone was single cell sorted from a VZV seronegative renal transplant candidate after response to live attenuated varicella vaccination. To analyze the allo-HLA reactivity, the VZV IE62-specific T-cell clone was tested against HLA-typed target cells and target cells transfected with HLA molecules, in both cytokine production and cytotoxicity assays. Results. The varicella vaccine–induced VZV IE62-specific T-cell clone specifically produced interferon-&ggr; when stimulated with HLA-B*55:01–expressing Epstein-Barr virus–transformed B cells and HLA-B*55:01–transfected K562 cells (single HLA antigen expressing cell line [SALs]) only. The clone also demonstrated specific cytolytic effector function against HLA-B*55:01 SALs and phytohemagglutinin blasts. Cytotoxicity assays using proximal tubular epithelial cell and human umbilical vein endothelial cell targets confirmed the kidney tissue specificity of the allo-HLA-B*55:01 reactivity, and the relevance of the cross-reactivity to clinical kidney transplantation. The results also suggest that molecular mimicry, and not bystander proliferation, is the mechanism underlying vaccine-induced alloreactivity. Conclusions. Varicella vaccination generated a de novo alloreactive kidney cell–specific cytolytic effector memory T cell in a patient awaiting renal transplantation. Vaccination-induced alloreactivity may have important clinical implications, especially for vaccine timing and recipient monitoring.

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.

Migration of allosensitizing donor myeloid dendritic cells into recipients after liver transplantation

It is thought, but there is no evidence, that myeloid dendritic cells (MDCs) of donor origin migrate into the recipient after clinical organ transplantation and sensitize the recipients immune system by the direct presentation of donor allo‐antigens. Here we show prominent MDC chimerism in the recipients circulation early after clinical liver transplantation (LTx) but not after renal transplantation (RTx). MDCs that detach from human liver grafts produce large amounts of pro‐inflammatory [tumor necrosis factor alpha and interleukin 6 (IL‐6)] and anti‐inflammatory (IL‐10) cytokines upon activation with various stimuli, express higher levels of toll‐like receptor 4 than blood or splenic MDCs, and are sensitive to stimulation with a physiological concentration of lipopolysaccharide (LPS). Upon stimulation with LPS, MDCs detaching from liver grafts prime allogeneic T cell proliferation and production of interferon gamma but not of IL‐10. Soluble factors secreted by liver graft MDCs amplify allogeneic T helper 1 responses. In conclusion, after clinical LTx, but not after RTx, prominent numbers of donor‐derived MDCs migrate into the recipients circulation. MDCs detaching from liver grafts produce pro‐inflammatory and anti‐inflammatory cytokines and are capable of stimulating allogeneic T helper 1 responses, and this suggests that MDC chimerism after clinical LTx may contribute to liver graft rejection rather than acceptance. Liver Transpl 16:12–22, 2010.

Systemic varicella zoster virus reactive effector memory T-cells impaired in the elderly and in kidney transplant recipients

Varicella zoster virus (VZV) infections cause varicella and subsequently herpes zoster upon reactivation. Immune‐compromised individuals and the elderly are at high risk of developing herpes zoster due to waning of VZV‐specific T‐cell immunity. In the present study, a novel functional T‐cell assay was developed to test the correlation between age and VZV‐specific T‐cell responses in peripheral blood from healthy individuals. Secondly, VZV‐specific T‐cell responses from renal transplant recipients were compared with healthy individuals. Monocytes were differentiated into mature monocyte‐derived dendritic cells (moDCs) and were infected with VZV. T‐cells were co‐cultured with autologous moDCs infected with VZV and subjected to flowcytometric analysis to identify the phenotype (i.e., naïve [NA: CCR7+CD45RO−], central [CM: CCR7+CD45RO+] and effector memory [EM: CCR7−CD45RO+] T‐cells) and the frequency of VZV‐reactive T‐cell subsets by intra‐cellular IFN‐γ flowcytometry. In contrast to NA and CM T‐cells, the frequency of VZV‐reactive CD4 and CD8 EM T‐cells was inversely correlated with age (P = 0.0007 and P = 0.01). No difference was found in the percentage of VZV‐reactive CD4 NA, CM and EM T‐cells between transplant recipients and controls. However, the percentage of VZV‐reactive CD8 EM T‐cells was significantly lower in transplant recipients compared to controls (P = 0.02). In conclusion, moDCs infected with VZV are efficient antigen presenting cells applicable to enumerate and characterize the phenotype and differentiation status of the systemic VZV‐specific T‐cell response ex‐vivo. The data suggest that VZV‐reactive EM T‐cells are impaired in the elderly and renal transplant recipients. J. Med. Virol. 84:2018–2025, 2012.

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.

Successful Tapering of Immunosuppression to Low-Dose Monotherapy Steroids After Living-Related Human Leukocyte Antigen-Identical Renal Transplantation

Background. Living-related (LR) human leukocyte antigen (HLA)-identical renal transplant (RTx) recipients often receive standard immunosuppression, despite the absence of mismatched major HLA-antigens and the known complications of long-term use of immunosuppression. No data are available on the need for immunosuppression for these specific patients. We wondered whether their immunosuppressive load could be radically reduced. Method. Between November 1982 and November 2005, 83 LR HLA-identical RTx were performed in our center. Their unadjusted graft survival was 74% at 10 years. In 29 patients (median time after transplantation 5.6 [range 1.0–21.4] years) with stable uncompromised renal function, we tapered their immunosuppression from triple or dual therapy to prednisolone 5 mg/day. Follow up on prednisolone monotherapy was at least 24 months. Results. In 27 of 29 patients reduction of immunosuppression to prednisolone monotherapy was uneventful. One patient, using dual therapy, developed JC-virus nephropathy resulting in graft loss. One refused further discontinuation of his medication. Four (15%) of the 27 patients on monotherapy developed biopsy-proven recurrence of their original disease. Only one of them showed a transient decline in renal function. One additional patient developed minor proteinuria and a rise in serum creatinine level, as a result of chronic urinary tract infections. The remaining 23 of 27 patients (85%) had an uneventful follow up during 24 months prednisolone monotherapy. Conclusion. We conclude that HLA-identical LR RTx recipients who are at least 1 year after transplantation might be treated with low-dose steroid monotherapy. Close surveillance of patients for recurrence of their original disease is recommended to allow for potential early therapeutic intervention.