M. Betjes

Follicular T helper cells and humoral reactivity in kidney transplant patients

Memory B cells play a pivotal role in alloreactivity in kidney transplantation. Follicular T helper (Tfh) cells play an important role in the differentiation of B cells into immunoglobulin‐producing plasmablasts [through interleukin (IL)‐21]. It is unclear to what extent this T cell subset regulates humoral alloreactivity in kidney transplant patients, therefore we investigated the absolute numbers and function of peripheral Tfh cells (CD4POSCXCR5POS T cells) in patients before and after transplantation. In addition, we studied their relationship with the presence of donor‐specific anti‐human leucocyte antigen (HLA) antibodies (DSA), and the presence of Tfh cells in rejection biopsies. After transplantation peripheral Tfh cell numbers remained stable, while their IL‐21‐producing capacity decreased under immunosuppression. When isolated after transplantation, peripheral Tfh cells still had the capacity to induce B cell differentiation and immunoglobulin production, which could be inhibited by an IL‐21‐receptor‐antagonist. After transplantation the quantity of Tfh cells was the highest in patients with pre‐existent DSA. In kidney biopsies taken during rejection, Tfh cells co‐localized with B cells and immunoglobulins in follicular‐like structures. Our data on Tfh cells in kidney transplantation demonstrate that Tfh cells may mediate humoral alloreactivity, which is also seen in the immunosuppressed milieu.

Human Bone Marrow- and Adipose Tissue-derived Mesenchymal Stromal Cells are Immunosuppressive In vitro and in a Humanized Allograft Rejection Model

Background Recent studies with bone marrow (BM)-derived Mesenchymal Stromal Cells (MSC) in transplant recipients demonstrate that treatment with MSC is safe and clinically feasible. While BM is currently the preferred source of MSC, adipose tissue is emerging as an alternative. To develop efficient therapies, there is a need for preclinical efficacy studies in transplantation. We used a unique humanized transplantation model to study the in vivo immunosuppressive effect of human BM-MSC and adipose tissue-derived MSC (ASC). Methods Gene expression of BM-MSC and ASC and their capacity to inhibit activated PBMC proliferation was evaluated. The in vivo immunosuppressive effect of BM-MSC and ASC was studied in a humanized mouse model. SCID mice were transplanted with human skin grafts and injected with human allogeneic PBMC with or without administration of BM-MSC or ASC. The effect of MSC on skin graft rejection was studied by immunohistochemistry and PCR. Results BM-MSC and ASC expressed TGFβ, CXCL-10 and IDO. IDO expression and acitivity increased significantly in BM-MSC and ASC upon IFN-γ stimulation. IFN-γ stimulated BM-MSC and ASC inhibited the proliferation of activated PBMC in a significant and dose dependent manner. In our humanized mouse model, alloreactivity was marked by pronounced CD45+ T-cell infiltrates consisting of CD4+ and CD8+ T cells and increased IFN-γ expression in the skin grafts which were all significantly inhibited by both BM-MSC and ASC. Conclusion BM-MSC and ASC are immunosuppressive in vitro and suppress alloreactivity in a preclinical humanized transplantation model.

Human Allogeneic Bone Marrow and Adipose Tissue Derived Mesenchymal Stromal Cells Induce CD8+ Cytotoxic T Cell Reactivity

Introduction For clinical applications, Mesenchymal Stromal Cells (MSC) can be isolated from bone marrow and adipose tissue of autologous or allogeneic origin. Allogeneic cell usage has advantages but may harbor the risk of sensitization against foreign HLA. Therefore, we evaluated whether bone marrow and adipose tissue-derived MSC are capable of inducing HLA-specific alloreactivity. Methods MSC were isolated from healthy human Bone Marrow (BM-MSC) and adipose tissue (ASC) donors. Peripheral Blood Mononuclear Cells (PBMC) were co-cultured with HLA-AB mismatched BM-MSC or ASC precultured with or without IFNy. After isolation via FACS sorting, the educated CD8+ T effector populations were exposed for 4 hours to Europium labeled MSC of the same HLA make up as in the co-cultures or with different HLA. Lysis of MSC was determined by spectrophotometric measurement of Europium release. Results CD8+ T cells educated with BM-MSC were capable of HLA specific lysis of BM-MSC. The maximum lysis was 24% in an effector:target (E:T) ratio of 40:1. Exposure to IFNγ increased HLA-I expression on BM-MSC and increased lysis to 48%. Co-culturing of PBMC with IFNγ-stimulated BM-MSC further increased lysis to 76%. Surprisingly, lysis induced by ASC was significantly lower. CD8+ T cells educated with ASC induced a maximum lysis of 13% and CD8+ T cells educated with IFNγ-stimulated ASC of only 31%. Conclusion Allogeneic BM-MSC, and to a lesser extend ASC, are capable of inducing HLA specific reactivity. These results should be taken into consideration when using allogeneic MSC for clinical therapy.

Decreased antigen-specific T-cell proliferation by moDC among hepatitis B vaccine non-responders on haemodialysis

Patients with end-stage kidney disease, whether or not on renal replacement therapy, have an impaired immune system. This is clinically manifested by a large percentage of patients unresponsive to the standard vaccination procedure for hepatitis B virus (HBV). In this study, the immune response to HBV vaccination is related to the in vitro function of monocyte-derived dendritic cells (moDC). We demonstrate that mature moDC from nonresponders to HBV vaccination have a less mature phenotype, compared to responders and healthy volunteers, although this did not affect their allostimulatory capacity. However, proliferation of autologous T cells in the presence of tetanus toxoid and candida antigen was decreased in non-responders. Also, HLA-matched CD4+ hsp65-specific human T-cell clones showed markedly decreased proliferation in the group of non-responders. Our results indicate that impairment of moDC to stimulate antigen-specific T cells provides an explanation for the clinical immunodeficiency of patients with end-stage kidney disease.

CMV Primary Infection Is Associated With Donor-Specific T Cell Hyporesponsiveness and Fewer Late Acute Rejections After Liver Transplantation

Viral infections, including cytomegalovirus (CMV), abrogate transplantation tolerance in animal models. Whether this also occurs in humans remains elusive. We investigated how CMV affects T cells and rejection episodes after liver transplantation (LT). Phenotype and alloreactivity of peripheral and allograft‐infiltrating T cells from LT patients with different CMV status were analyzed by flow cytometry. The association of CMV status with early and late acute rejection was retrospectively analyzed in a cohort of 639 LT patients. CMV‐positivity was associated with expansion of peripheral effector memory T cell subsets after LT. Patients with CMV primary infection showed donor‐specific CD8+ T cell hyporesponsiveness. While terminally differentiated effector memory cells comprised the majority of peripheral donor‐specific CD8+ T cells in CMV primary infection patients, they were rarely present in liver allografts. Retrospective analysis showed that R−D+ serostatus was an independent protective factor for late acute rejection by multivariate Cox regression analysis (hazard ratio [HR]u2009=u20090.18, 95% CIu2009=u20090.04–0.86, pu2009=u20090.015). Additionally, CMV primary infection patients showed the highest Vδ1/Vδ2 γδ T cell ratio, which has been shown to be associated with operational tolerance after LT. In conclusion, our data suggest that CMV primary infection may promote tolerance to liver allografts, and CMV status should be considered when tapering or withdrawing immunosuppression.

Post-transplantation encapsulating peritoneal sclerosis without inflammation or radiological abnormalities

BackgroundPost-transplantation encapsulating peritoneal sclerosis (EPS) causing bowel obstruction has been identified as a serious complication after kidney transplantation in patients previously treated with peritoneal dialysis. Systemic inflammation and abnormalities on an abdominal computed tomography (CT) scan are important hallmarks of EPS. To our knowledge, this is the first report of a case being diagnosed with late-onset post-transplantation EPS without systemic inflammation or abnormalities on a CT scan which could only be diagnosed by laparotomy.Case presentationA 59-year old female presented because of symptoms of bowel obstruction 33xa0months after kidney transplantation. The patient had a 26-month history of peritoneal dialysis before her first kidney transplantation and was treated with peritoneal dialysis for 4xa0years before undergoing a second kidney transplantation. Physical examination was unremarkable and laboratory tests showed no signs of systemic inflammation (C-reactive protein <1xa0mg/L). An abdominal CT scan did not reveal any abnormalities fitting the diagnosis of EPS, except a “feces sign”. Given the severity of the progressive symptoms, a diagnostic laparotomy was performed, visualizing a classical EPS. Total peritonectomy and enterolysis were performed, leading to restoration of peristalsis.ConclusionEPS may occur several years after kidney transplantation in the absence of inflammation and typical radiological abnormalities. Obtaining a diagnosis of post-transplantation EPS is challenging, however, a low threshold for surgical exploration in case of high clinical suspicion and negative findings on the CT scan is mandatory.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

Solid‐phase multiplex‐bead assays are widely used in transplantation to detect anti‐human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex‐bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter‐assay and inter‐machine variability of multiplex‐bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter‐assay and inter‐machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter‐assay MARD was comparable, but showed a higher lot‐to‐lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers recommendations with virtually no differences in HLA antibody assignments.

CD4+CD28null T cells are not alloreactive unless stimulated by interleukin-15

Proinflammatory, cytotoxic CD4+CD28null T cells can be substantially expanded in patients with end‐stage renal disease. These cells have been associated with the risk for rejection, but their alloreactive potential is unknown. CD4+CD28null T cells were stimulated with HLA‐mismatched antigen presenting cells in the absence/presence of exogenous cytokines. Alloreactive potential was evaluated based on proliferation, degranulation, cytotoxicity, and cytokine production. Further, their suppressive capacity was assessed by measuring inhibition of proliferating alloreactive CD28+ T cells. CD4+CD28null T cells contained alloreactive (CD137+) T cells but did not proliferate in response to allogeneic stimulation, unless interleukin (IL)‐15 was added. However, they could proliferate on stimulation with cytomegalovirus antigen without exogenous cytokines. IL‐15 increased the frequency of proliferating alloreactive CD4+CD28null T cells to 30.5% without inducing CD28 expression (P < .05). After allogeneic stimulation together with IL‐15 and IL‐21, frequency of degranulating CD107a+CD4+CD28null T cells increased significantly from 0.6% to 5.8% (P < .001). Granzyme B and perforin positivity remained similar, but production of interferon‐γ and tumor necrosis factor‐α increased by the combination of IL‐15 and IL‐21 (P < .001 and P < .05, respectively). Finally, CD4+CD28null T cells did not show significant suppression. Thus, CD4+CD28null T cells represent a population with absent alloreactivity unless IL‐15 is present.

Tacrolimus intra-patient variability is not associated with chronic active antibody mediated rejection

Background Chronic active antibody mediated rejection (c-aABMR) is a major cause of long-term kidney allograft loss. It is hypothesized that frequent sub-therapeutic exposure to immunosuppressive drugs, in particular tacrolimus (Tac), is a risk factor for the development of c-aABMR. The intra-patient variability (IPV) in Tac exposure may serve as a substitute biomarker for underexposure and/or non-adherence. In this study, the association between Tac IPV and the development of c-aABMR was investigated. Methods We retrospectively included 59 patients diagnosed with c-aABMR and compared them to 189 control patients matched for age, year of transplantation and type of kidney donor. The Tac IPV was calculated from pre-dose tacrolimus concentrations measured over a 3 year period preceding the diagnosis of c-aABMR. The mean Tac predose concentrations (C0), Tac IPV, renal allograft function and graft survival were compared between the groups. Results Tac IPV was 24.4% for the cases versus 23.6% for the controls (p = 0.47). The mean Tac C0 was comparable for the cases (5.8 ng/mL) and control patients (6.1 ng/mL, p = 0.08). Only in the c-aABMR group a significant decline in both mean Tac C0 and allograft function over the timespan of 3 years was observed (p = 0.03 and p<0.001). Additionally, in the group of c-aABMR patients a high IPV was associated with inferior graft survival (p = 0.03). Conclusions A high Tac IPV per se does not predispose to the development of c-aABMR but is associated with inferior graft survival once c-aABMR is diagnosed.

The Influence of Comorbidity On Graft and Patient Survival After Kidney Transplantation.: Abstract# 2207

2184 Kidneys From DCD Donors Are Underutilized in Pediatric Patients. E. King, N. Desai, D. Segev. Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD. Background. In pediatric patients, donation after cardiac death (DCD) organs account for just over 2% of all kidney transplants. It is diffi cult to defi nitively ascertain survival of DCD grafts through single center studies when the frequency of DCD transplantation is so low. A comprehensive study of national data is necessary to better defi ne outcomes and utilization of DCD compared to donation after brain death (DBD) kidneys. Methods. SRTR data (April 1994 to September 2013) of 7,318 pediatric kidneyonly deceased donor transplant recipients (age<18) were analyzed. Death censored graft survival was defi ned as the time from transplantation to graft loss or last date of follow-up with a functional graft. Kaplan Meier survival analysis was used to compare graft loss between DCD and DBD kidneys. Lorenz curves and Gini coeffi cient were used to compare center-level clustering in use of DCD versus DBD kidneys in pediatric recipients across US centers. Results. Graft survival was not statistically signifi cantly different between DCD and DBD organs in pediatric kidney recipients (p=0.88). There was a slight but not statistically signifi cant difference in the occurrence of delayed graft function (2.9% and 0.7%, p=0.06), however the mean creatinine at discharge following transplantation was higher among those receiving DCD kidneys compared to DBD (2.1 mg/dL and 1.5 mg/dL, p < 0.05). Lorenz curves demonstrate that around 25% of centers in the US performed 100% of DCD kidney transplants in pediatric recipients, in a pattern far more center-clustered than DBD transplants. Conclusions. DCD kidneys offer comparable outcomes in pediatric kidney transplantation, but only a small number of pediatric transplant centers currently utilize these organs. Abstract# 2185 Long-Term Data On the Use of Everolimus and Low-Dose Ciclosporin A in Children After Kidney Transplantation (Tx). L. Brunkhorst,1 T. Ahlenstiel,1 F. Lehner,2 L. Pape.1 1Pediatric Nephrology, Hannover Medical School, Hannover, Germany; 2Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany. Background: Actually, only short-term trials using Everolimus in children after Tx have been published. This is the fi rst prospective study to present four-year data using low-dose Ciclosporin A (CsA), Everolimus and steroid elimination. Methods: 35 children (median age: 10.7 ± 5.6 years) received Basiliximab, CsA and Prednisolone after Tx. Everolimus (1.6mg/m2/day) treatment was added two weeks post Tx. In 83% of the patients Prednisolone was withdrawn 8-10 months post Tx. Long-term target trough levels for CsA and Everolimus were 30-50 ng/ ml and 2-4 ng/ml. Clinical outcome and side effects were evaluated prospectively. Results: Patient and graft survival were 100% and median eGFR was 62, 61, 58 and 65 ml/min/1.73m2 1-4 years after Tx. Acute rejection (BANFF ≥ Ia) was diagnosed in indication biopsies in in 2/35 (6%) children in the fi rst year after Tx. In the remaining observation time, acute rejection was only diagnosed in one child in year 2 and 4. De novo Donor specifi c antibodies were found in 4/35 patients in yearly checkups with the histopathological diagnosis of chronic humoral rejection in one patient. In the four years observation time, one child developed EBV associated PTLD that was successfully treated with Rituximab. Two patients developed transient wound healing problems, 17% aphthous stomatitis. No signifi cant albuminuria > 100 mg/ mmol creatinine was recognized. Height SDS was -0.73 / -0.74 two and four years after Tx. Despite an elevation of estradiol in 33% of the girls, all sex hormone levels were within the normal range. Conclusions: After ped. kidney Tx, de novo immunosuppression with low-dose CsA, Everolimus, and a withdrawal of Prednisolone is followed by excellent graft survival and function, good growth and only a small number side effects. DISCLOSURE: Pape, L.: Grant/Research Support, Novartis. 2185 Long-Term Data On the Use of Everolimus and Low-Dose Ciclosporin A in Children After Kidney Transplantation (Tx). L. Brunkhorst,1 T. Ahlenstiel,1 F. Lehner,2 L. Pape.1 1Pediatric Nephrology, Hannover Medical School, Hannover, Germany; 2Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany. Background: Actually, only short-term trials using Everolimus in children after Tx have been published. This is the fi rst prospective study to present four-year data using low-dose Ciclosporin A (CsA), Everolimus and steroid elimination. Methods: 35 children (median age: 10.7 ± 5.6 years) received Basiliximab, CsA and Prednisolone after Tx. Everolimus (1.6mg/m2/day) treatment was added two weeks post Tx. In 83% of the patients Prednisolone was withdrawn 8-10 months post Tx. Long-term target trough levels for CsA and Everolimus were 30-50 ng/ ml and 2-4 ng/ml. Clinical outcome and side effects were evaluated prospectively. Results: Patient and graft survival were 100% and median eGFR was 62, 61, 58 and 65 ml/min/1.73m2 1-4 years after Tx. Acute rejection (BANFF ≥ Ia) was diagnosed in indication biopsies in in 2/35 (6%) children in the fi rst year after Tx. In the remaining observation time, acute rejection was only diagnosed in one child in year 2 and 4. De novo Donor specifi c antibodies were found in 4/35 patients in yearly checkups with the histopathological diagnosis of chronic humoral rejection in one patient. In the four years observation time, one child developed EBV associated PTLD that was successfully treated with Rituximab. Two patients developed transient wound healing problems, 17% aphthous stomatitis. No signifi cant albuminuria > 100 mg/ mmol creatinine was recognized. Height SDS was -0.73 / -0.74 two and four years after Tx. Despite an elevation of estradiol in 33% of the girls, all sex hormone levels were within the normal range. Conclusions: After ped. kidney Tx, de novo immunosuppression with low-dose CsA, Everolimus, and a withdrawal of Prednisolone is followed by excellent graft survival and function, good growth and only a small number side effects. DISCLOSURE: Pape, L.: Grant/Research Support, Novartis. Cardiovascular Complications in Kidney Transplantation II Wednesday, July 30, 2014 4:00 PM 5:30 PM Room 3016/3018 Abstract# 2207 The Infl uence of Comorbidity On Graft and Patient Survival After Kidney Transplantation. M. Laging,1 J. Kal-van Gestel,1 J. van de Wetering,1 J. Ijzermans,2 M. Betjes,1 W. Weimar,1 J. Roodnat.1 1Internal Medicine, Erasmus MC, Rotterdam, Netherlands; 2General Surgery, Erasmus MC, Rotterdam, Netherlands. Background 2207 The Infl uence of Comorbidity On Graft and Patient Survival After Kidney Transplantation. M. Laging,1 J. Kal-van Gestel,1 J. van de Wetering,1 J. Ijzermans,2 M. Betjes,1 W. Weimar,1 J. Roodnat.1 1Internal Medicine, Erasmus MC, Rotterdam, Netherlands; 2General Surgery, Erasmus MC, Rotterdam, Netherlands. Background Nowadays patients with a large variety of comorbidities are referred for transplantation. Acceptance criteria for kidney transplantation are continuously being eased. We questioned to what extent increasing severity and number of comorbidities interfere with graft and patient survival. Methods In our center, 1728 patients were transplanted between January 1, 2000 and December 31, 2012. Four pre-transplant comorbidity categories were defi ned: cardiovascular