Constanze Schönemann

Anti-human leukocyte antigen and donor-specific antibodies detected by luminex posttransplant serve as biomarkers for chronic rejection of renal allografts.

Background. Although the incidence of early acute rejection could have been diminished in the past, the long-term renal allograft survival could not benefit from the introduction of more effective immunosuppressive regimens mainly aiming at cellular rejection mechanisms. The cause of chronic rejection is still discussed controversially. Here, we demonstrate to what extent human leukocyte antigen (HLA) antibodies (HLAab) posttransplant contribute to late graft outcome. Methods. A total of 1014 deceased kidney transplant recipients transplanted at the Charité hospital were monitored in a cross-sectional manner for the development of HLAab using Luminex Single Antigen beads. Patients with stable kidney function at a median of 5-years posttransplant were tested once for HLAab and monitored for 5.5 years after testing. Results. Thirty percent of recipients showed HLAab. Donor-specific antibodies (DSA) were found in 31% of antibody positive patients. The presence of DSA was associated with a significantly lower graft survival of 49% vs. 83% in the HLAab negative group (P≤0.0001). Non-DSAs also had an adverse effect on graft survival (70% vs. 83%; P=0.0001). In a prospective analysis of 195 patients with repeatedly no detectable HLAab, the survival probability was 94% as opposed to 79% survival among patients who developed HLAab de novo after the first testing (P=0.05). Conclusions. We confirmed that HLAab produced even late after transplantation are detrimental to graft outcome. DSA were proven to have a strong adverse impact on graft survival. The results indicate that a posttransplant HLAab monitoring routine could be appropriate to improve long-term results.

Analysis of CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides

The frequencies of human cytomegalovirus (HCMV) protein‐specific CD8 T cells, identified by the presence of intracellular IFN‐γ, were measured by flow cytometry following stimulation of freshly isolated peripheral blood mononuclear cells (PBMC) with comprehensive peptide pools. These pools spanned the entire amino acid sequences of the HCMV pp65 and major immediate early (IE‐1) proteins and consisted of 15‐amino acid peptides with at least nine overlaps between neighboring peptides. As a result all potential CD8 T cell epitopes contained in these proteins were provided by the complete pools and, therefore, unlike with single epitopes, testing was independent of donor HLA type. Individual stimulating peptides from the same pools were identified in parallel experiments. Thus we found that our results with the complete pools using PBMC from 26 healthy HCMV‐seropositive donors were 100 % sensitive and specific with respect to predicting the presence of recognized epitopes in the respective proteins. In addition, cells from 15 renal transplant patients were tested with complete pools alone. While our results confirmed our previous contention that HCMV IE‐1 is an important CD8 T cell target, the technical improvement we made in order to address this question has clearly wider implications. Similar pools may be applied to examine the role of proteins from other pathogens, in autoimmune disease or following vaccination.

Donor‐Specific HLA Antibodies in a Cohort Comparing Everolimus With Cyclosporine After Kidney Transplantation

Donor‐specific HLA antibodies (DSA) have a negative impact on kidney graft survival. Therefore, we analyzed the occurrence of DSA and antibody‐mediated rejection (AMR) in patients from two prospective randomized trials in our center. At 3–4.5 months posttransplant 127 patients were randomized to continue cyclosporine or converted to everolimus therapy. The presence of DSA was prospectively assessed using Luminex assays. AMR was defined according to the Banff 2009 classification. Antibody screening was available in 126 patients with a median follow‐up of 1059 days. Seven out of 65 (10.8%) patients on cyclosporine developed DSA after a median of 991 days. In comparison, 14/61 patients (23.0%) randomized to everolimus developed DSA after 551 days (log‐rank: p = 0.048). Eight patients on everolimus compared to two patients on cyclosporine developed AMR (log‐rank: p = 0.036). Four of 10 patients with AMR—all in the everolimus group—lost their graft. A multivariate regression model revealed everolimus, >3 mismatches and living donor as significant risk factors for DSA. Acute rejection within the first year, >3 mismatches, everolimus and living donor were independent risk factors for AMR. This single center analysis demonstrates for the first time that everolimus‐based immunosuppression is associated with an increased risk for the development of DSA and AMR.

Enzyme-linked immunosorbent spot assay for donor-reactive interferon-gamma-producing cells identifies T-cell presensitization and correlates with graft function at 6 and 12 months in renal-transplant recipients.

Background. A major goal in clinical transplantation is the individualization of immunosuppression. This requires a definition of markers that identify patients at heightened risk of acute rejection and immune-mediated chronic allograft nephropathy. Methods. Frequencies of interferon–&ggr;-producing donor-reactive cells were serially determined in unselected renal-transplant patients in an enzyme-linked immunosorbent spot assay (ELISPOT) before transplantation (n=42) and up to 10 (mean 5.0) times during the first 6 months posttransplantation (n=48) to determine detailed kinetics and analyze for correlation with acute rejection and graft function at 6 and 12 months posttransplantation. Results. Pretransplant ELISPOT frequencies were significantly higher in patients with acute rejection (16/42) versus nonrejecters (26/42). Highly elevated pretransplant frequencies (>200 spots/300,000 peripheral blood mononuclear cells [PBMCs], n=5/42) were associated with a risk of severe acute rejection episodes but were independent of risk factors such as high panel reactive antibodies. Early graft failure exclusively occurred in this group. Importantly, mean ELISPOT frequencies at weeks 2 and 3 but not at month 6 posttransplant correlated inversely with 6 and 12 months glomerular filtration rate. The correlation between ELISPOT frequencies and renal function showed the highest significance in patients without acute rejection. Conclusions. The pretransplant ELISPOT assay might be useful to identify T-cell presensitized patients, who are at heightened risk for severe early acute rejection. An analysis of ELISPOT donor-reactive cells during the early posttransplant period might allow an identification of patients at risk for immune-mediated graft deterioration.

Soluble CD30 as a predictor of kidney graft outcome.

Background. In the present study, we investigated whether the soluble form of CD30 (sCD30), a marker for T helper 2‐type cytokine‐producing T cells, is increased in sera of potential kidney graft recipients. We also investigated whether the pretransplantation serum sCD30 content is related to kidney graft survival. Methods. Pretransplantation sera of 844 cadaver kidney recipients from three transplant centers in Germany were tested for serum sCD30 content using a commercially available ELISA kit. Results. Kidney graft recipients showed a significantly higher serum sCD30 content than healthy controls (P<0.0001). High sCD30 serum content was associated with graft rejection. The 2‐year graft survival rate in recipients with a high pretransplantation serum sCD30 was 68±6%, significantly lower than the 86±1% rate in recipients with a low sCD30 (P<0.0001). Importantly, high sCD30 was indicative of an increased risk of graft loss even in recipients without lymphocytotoxic alloantibodies. Conclusion. These data show that an elevated pretransplantation serum sCD30 reflects an immune state that is detrimental for kidney graft survival.

Comparison between bortezomib and rituximab in the treatment of antibody-mediated renal allograft rejection

BACKGROUND Antibody-mediated rejection (ABMR) following kidney transplantation is associated with poor allograft survival. Conventional treatment based on plasmapheresis (PPH) and the administration of intravenous immunoglobulins (IVIG) is not satisfactory. Two compounds, more specifically targeting B cells and plasma cells, may help to improve the prognosis: rituximab, a B-cell-depleting monoclonal antibody, and bortezomib, a proteasome inhibitor causing apoptosis of plasma cells. METHODS Starting in February 2009, we treated 10 consecutive patients with ABMR according to current Banff criteria with one cycle of bortezomib [1.3 mg/m(2) intravenously (i.v.), Day 1, 4, 8, 11]. This group was compared to a historical control group of patients (n = 9) treated with a fixed single dose of rituximab (500 mg i.v.). All patients received PPH (6×) and IVIG (30 g). Patients with acute ABMR additionally received methylprednisolone (3 × 500 mg i.v.). RESULTS Patient survival in both groups was 100%. At 18 months after treatment, graft survival was 6/10 in the bortezomib group as compared to 1/9 functioning grafts in the rituximab group (P = 0.071). Renal function was superior in patients treated with bortezomib as compared to rituximab-treated patients (serum creatinine at 9 months: 2.5 ± 0.6 versus 5.1 ± 2.1 mg/dL, P = 0.008). Proteinuria was not different between both groups (9 months: 1.3 ± 1.0 versus 1.6 ± 1.6 g/day, P = n.s.). CONCLUSIONS Treatment of ABMR with bortezomib in addition to standard therapy was partially effective, whereas treatment with a fixed dose of rituximab in addition to standard therapy with PPH and IVIG did not result in sufficient long-term graft survival. In the future, new strategies including the combination of both substances and the application of higher doses must be discussed.

Intact HLA Not β2m-free Heavy Chain-Specific HLA Class I Antibodies Are Predictive of Graft Failure

Background. We investigated the effects of intact and &bgr;2m-free heavy chain (HC)-specific human leukocyte antigen (HLA) class I antibodies on long-term graft survival. Methods. HLA class I mixed antigen beads were used to detect intact and &bgr;2m-free HC-specific antibodies, whereas elution buffer-treated beads were used to detect antibodies against &bgr;2m-free HC. Donor-specific antibodies (DSAs) were identified using single-antigen beads. Complement-dependent cytotoxicity assays were performed to determine the cytotoxicity of DSA. Results. Three hundred seventy-nine of 994 of patients (38%) had antibodies against intact HLA and &bgr;2m-free HC. There was no survival rate difference between antibody-positive and -negative groups. When the 379 antibody-positive patients were further tested with &bgr;2m-free HC-coated beads, 179 of them with antibodies only against intact form of antigens had a 4-year graft survival rate of 76%, which is significantly lower than that of 200 patients with antibodies against &bgr;2m-free HC of HLA antigens (88%, P=0.0056). Patients with intact antigen specific DSAs had a significantly lower graft survival rate as compared with those with no DSAs (70% vs. 89%, P=0.0073). More patients with strong donor-specific cytotoxic antibodies lost allografts than those with weak-cytotoxic or noncytotoxic antibodies. However, cytotoxic activity of DSA was not correlated to antibody level. Conclusions. We concluded that intact antigen-specific antibodies, especially DSAs, are predictive of graft failure. DSAs were not always cytotoxic. Strong cytotoxic activity of DSA was associated with a higher rate of graft loss but not correlated to the antibody level. Antibodies against &bgr;2m-free HC negatively interfere with the predictive value of intact antigen-specific antibodies.

Identification of Dialysis Patients with Panel-Reactive Memory T Cells before Kidney Transplantation Using an Allogeneic Cell Bank

Donor-reactive cellular sensitization does not routinely suggest humoral sensitization and vice versa, but both predict poor kidney transplant outcome. Irrespective of donor reactivity, panel-reactive antibody (PRA) screening identifies patients who are at enhanced risk. Therefore, it was hypothesized that panel-reactive memory T cell reactivity (PRT) might be an additional risk assessment factor of dialysis patients who are on the transplant waiting list. IFN-gamma-enzyme-linked immunosorbent spot memory T cell frequencies were determined in 10 healthy volunteers and 41 hemodialysis patients using for stimulation an allogeneic cell bank (ACB) from 17 healthy individuals who represented the most frequent white HLA antigens. Positive responses to ACB were analogous to PRA defined as percentage of positive assays of the ACB sets. Hemodialysis patients expressed higher PRT levels compared with healthy volunteers. Five of 10 PRT++ patients were PRA negative, and only four of 10 PRA++ patients exhibited PRT reactivity, suggesting independence of humoral and cellular sensitization. Pretransplantation PRT testing of recipients might improve individual risk assessment to make individualized therapy decisions.

High levels of CMV-IE-1-specific memory T cells are associated with less alloimmunity and improved renal allograft function.

BACKGROUND Cytomegalovirus (CMV) infection has been associated with allograft rejection in solid organ transplantation. However, the immunologic mechanisms behind this observation have not been elucidated. One proposed mechanism is direct cross-reactivity of antiviral T-cells with allogeneic MHC/peptide complexes, a process termed heterologous immunity. Another model favours indirect stimulation of alloimmunity by CMV-induced proinflammatory cytokines and upregulation of MHC class II and adhesion molecules. Recently, we found that protection from CMV disease was correlated with high levels of CMV-immediate early-1 (IE-1) specific IFN-gamma-producing T-cell responses in heart and lung transplant recipients. The aim of this study was to define the relation of CMV-specific T-cell responses to acute rejection, donor-reactive memory T cells, and allograft function after kidney transplantation. METHODS To address this issue, IFN-gamma-producing T-cell responses following ex-vivo stimulation with pools of overlapping peptides representing the CMV pp65 and IE-1 proteins, as well as donor-reactive IFN-gamma-producing T-cells were determined at multiple time points before (pre-Tx) and during the first 6 months posttransplant (post-Tx) in 36 kidney transplant recipients using an enzyme linked immunoabsorbent spot assay (ELISPOT). RESULTS CMV-specific T cells were not exclusively detectable in CMV seropositive patients, as 3/12 seronegative patients had significant pre- and post-Tx pp65/IE-1-specific T-cell responses. In patients with detectable anti-CMV antibody or T-cell responses, no difference in CMV-specific T-cell frequencies was found between patients with versus without acute rejection. However, early (week 1, r=0.457, p=0.037) and average IE-1-specific T-cell responses (r=-0.415, p=0.032) during 6 months post-Tx showed a significant inverse correlation with average post-Tx donor-reactive T-cell responses. Furthermore, average post-Tx IE-1-specific T-cell responses correlated significantly with 6 and 12 months glomerular filtration rate (GFR). In contrast, pp65-specific T-cell responses did not correlate with donor-reactive T cells or graft function. Only 2/36 patients developed CMV disease, both showing very weak IE-1-specific T-cell responses during the whole monitoring period. CONCLUSION No evidence for heterologous immunity could be found in patients with high levels of CMV-specific T cells. On the contrary, less alloreactivity and improved graft function were found in patients with strong IE-1-specific T-cell responses. These results emphasize the importance of immediate early antigens (IE) as targets for T-cell immunity to CMV. We hypothesize that IE-1-specific T cells might effectively suppress IE-1-induced indirect effects such as inflammation and upregulation of MHC class II and adhesion molecules.

Heightened Expression of the Cytotoxicity Receptor NKG2D Correlates with Acute and Chronic Nephropathy After Kidney Transplantation

The activating cytotoxicity receptor NKG2D binds to stress‐regulated molecules encoded by the major histocompatibility complex class I chain‐related (MIC) and UL‐16‐binding protein (ULBP)/retinoic acid early transcript (RAET) gene family. To assess whether acute allograft rejection leads to an induction of these inducible ligands and their receptor NKG2D, we examined the mRNA profiles in kidney transplant biopsies. Expression levels were correlated with the incidence of acute rejection (aRx) episodes and chronic allograft nephropathy (CAN) proven by histology. Whereas MICA, ULBP1/3 and RAET1‐E did not display heightened gene expression, elevated levels of NKG2D mRNA could be associated with aRx (p < 0.001). Immunohistology of kidney biopsies diagnosed with aRx revealed NKG2D+ cells in tubulointerstitial areas positive for CD8+ cells. Most importantly, elevated levels of NKG2D mRNA were associated with restricted long‐term graft function assessed by the glomerular filtration rate at 6, 12 and 18 months posttransplantation. Induced NKG2D mRNA expression was still observable in biopsies diagnosed with CAN (p < 0.001), demonstrating a higher sensitivity and specificity compared to CD3, granzyme B and granulysin mRNA measurement. Significant elevated levels of NKG2D mRNA could be further detected in urine sediment prior to aRx, suggesting this receptor as a new candidate marker for the diagnosis of acute and chronic allograft rejection.