No abstract

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.

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 (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.

De novo donor-specific HLA antibodies (dnDSA) are recognized as a risk factor for premature allograft failure. Determinants of DSA specificity are generated via the indirect allorecognition pathway. Here, we present supportive data for the relevance of predicted indirectly recognizable HLA epitopes (PIRCHE) to predict dnDSA following kidney transplantation. A total of 2787 consecutive kidney transplants performed between 1995 and 2015 without preformed DSA have been analyzed. De novo DSA were detected by single antigen bead assay. HLA epitope mismatches were determined by the HLAMatchmaker and PIRCHE approach and correlated in uni- and multivariate analyses with 10-year allograft survival and incidence of dnDSA. The PIRCHE-II score moderately predicted allograft survival. However, the predictive value of elevated PIRCHE-II scores >9 for the incidence of dnDSA was statistically significant (p < 0.001). In a multivariate Cox regression analysis adjusted for antigen mismatch and HLAMatchmaker epitopes, the PIRCHE-II score could be identified as an independent risk factor for dnDSA. The PIRCHE-II score independently from the antigen mismatch and HLAMatchmaker epitopes could be revealed as being a strong predictor for dnDSA. PIRCHE may help to identify acceptable mismatches with decreased risk of dnDSA and thus improve long-term renal allograft survival.

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.

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.

Cell-mediated immunity plays an essential role in the control of infection with the human cytomegalovirus (HCMV). However, only a few CD8+-T-cell epitopes are known, with the majority being contained in the pp65 phosphoprotein, which is believed to dominate the CD8+-T-cell response to HCMV. Here, we have readdressed the issue of CD8+ T cells specific for the 72-kDa major immediate-early protein (IE-1), which is nonstructural but is found very early and throughout the replicative cycle. Using a novel flow-cytometric assay, we were able to identify CD8+-T-cell epitopes (by IE-1 peptide-specific induction of cytokine synthesis) and simultaneously measure the frequency of cells directed against them. For this purpose, 81 pentadecamer peptides covering the complete 491-amino-acid sequence of IE-1 were tested on peripheral blood mononuclear cells of anti-HCMV immunoglobulin G-seropositive donors. At least 10 new epitopes were identified, and the fine specificity and presenting HLA molecule of the first of them was determined. The frequencies of CD8+ T cells directed against IE-1 were similar to those directed against pp65 in donors tested with known pp65-derived peptides. Importantly, additional testing of a corresponding set of peptides covering the complete sequence of pp65 on 10 of these donors identified individuals whose CD8+ T cells recognized IE-1 but not pp65 and vice versa, clearly illustrating that either protein may be a major target. In summary, our results suggest that IE-1 is far more important as a CD8+-T-cell target than current opinion suggests.