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Summary: Background: Currently, there is no licensed treatment for chronic norovirus infections, but the use of intra-duodenally-delivered immunoglobulins is promising; nevertheless, varying results have limited their wide use. Little is known about the relationship between norovirus genetic diversity and treatment efficacy. Methods: We analyzed the norovirus within-host diversity and evolution in a cohort of 20 immunocompromised individuals using next-generation sequencing (NGS) and clone-based sequencing of the capsid (VP1) gene. Representative VP1s were expressed and their glycan receptor binding affinity and antigenicity were evaluated. Findings: The P2 domain, within the VP1, accumulated up to 30-fold more non-synonymous mutations than other genomic regions. Intra-host virus populations in these patients tended to evolve into divergent lineages that were often antigenically distinct. Several of these viruses were widely resistant to binding-blocking antibodies in immunoglobulin preparations. Notably, for one patient, a single amino-acid substitution in the P2 domain resulted in an immune-escape phenotype, and it was likely the main contributor to treatment failure. Furthermore, we found evidence for transmission of late-stage viruses between two immunocompromised individuals. Interpretation: The findings demonstrated that within-host noroviruses in chronic infections tend to evolve into antigenically distinct subpopulations. This antigenic evolution was likely caused by the remaining low immunity levels exerted by immunocompromised individuals, possibly undermining antiviral treatment. Our observations provide insights into norovirus (within-host) evolution and treatment. Funding: Erasmus MC grant mRACE, the European Union's Horizon 2020 research and innovation program under grant agreement No. 874735 (VEO), and the NWO STEVIN award (Koopmans).

Delayed graft function (DGF) after kidney transplantation heralds a worse prognosis. In patients with hyperoxaluria, the incidence of DGF is high. Oxalic acid is a waste product that accumulates when kidney function decreases. We hypothesize that residual diuresis and accumulated waste products influence the DGF incidence. Patients transplanted between 2018–2022 participated in the prospective cohort study. Pre-transplant concentrations of oxalic acid and its precursors were determined. Data on residual diuresis and other recipient, donor or transplant related variables were collected. 496 patients were included, 154 were not on dialysis. Oxalic acid, and glyoxylic acid, were above upper normal concentrations in 98.8%, and 100% of patients. Residual diuresis was ≤150 mL/min in 24% of patients. DGF occurred in 157 patients. Multivariable binary logistic regression analysis demonstrated a significant influence of dialysis type, recipient BMI, donor type, age, and serum creatinine on the DGF risk. Residual diuresis and glycolic acid concentration were inversely proportionally related to this risk, glyoxylic acid directly proportionally. Results in the dialysis population showed the same results, but glyoxylic acid lacked significance. In conclusion, low residual diuresis is associated with increased DGF incidence. Possibly accumulated waste products also play a role. Pre-emptive transplantation may decrease the incidence of DGF.

Background. The aim of this open-label, multicenter, randomized controlled study was to investigate whether the life cycle pharma (LCP)-tacrolimus compared with the extended-release (ER)-tacrolimus formulation results in a difference in the prevalence of posttransplant diabetes, hypertension and chronic kidney disease (CKD) at 12 mo after liver transplantation. Methods. Patients were 1:1 randomized to either of the 2 tacrolimus formulations. The primary endpoint was defined as a composite endpoint of any of 3 events: sustained (>3 mo postrandomization) posttransplant diabetes, new-onset hypertension, and/or CKD, defined as estimated glomerular filtration rate 3 m during the follow-up. Results. In total, 105 patients were included. In the intention-to-treat analysis, a statistically significant lower proportion of liver transplant recipients in the LCP-tacrolimus group reached the composite primary endpoint at 12 mo compared with the ER-tacrolimus group (50.9% [27/53], 95% confidence interval [CI], 37.9%-63.9% versus 71.2% [37/52], 95% CI, 57.7%-81.7%; risk difference: 0.202; 95% CI, 0.002-0.382; P = 0.046). No significant difference was found in the per protocol analysis. In the intention-to-treat and per protocol population, fewer liver transplant recipients in the LCP-tacrolimus group developed CKD and new-onset hypertension compared with the ER-tacrolimus group. No differences in rejection rate, graft and patient survival were found. Conclusions. A statistically significant and clinically relevant reduction in the prevalence of the composite primary endpoint was found in the LCP-tacrolimus group compared with the ER-tacrolimus group in the first year after liver transplantation with comparable efficacy.

Tissue-resident lymphocytes (TRLs) are critical for local protection against viral pathogens in peripheral tissue. However, it is unclear if TRLs perform a similar role in transplanted organs under chronic immunosuppressed conditions. In this study, we aimed to characterize the TRL compartment in human kidney transplant nephrectomies and examine its potential role in antiviral immunity. The TRL compartment of kidney transplants contained diverse innate, innate-like, and adaptive TRL populations expressing the canonical residency markers CD69, CD103, and CD49a. Chimerism of donor and recipient cells was present in 43% of kidney transplants and occurred in all TRL subpopulations. Paired single-cell transcriptome and T cell receptor (TCR) sequencing showed that donor and recipient tissue–resident memory T (TRM) cells exhibit striking similarities in their transcriptomic profiles and share numerous TCR clonotypes predicted to target viral pathogens. Virus dextramer staining further confirmed that CD8 TRM cells of both donor and recipient origin express TCRs with specificities against common viruses, including CMV, EBV, BK polyomavirus, and influenza A. Overall, the study results demonstrate that a diverse population of TRLs resides in kidney transplants and offer compelling evidence that TRM cells of both donor and recipient origin reside within this TRL population and may contribute to local protection against viral pathogens.

Alemtuzumab is used as lymphocyte-depleting therapy for severe or glucocorticoid-resistant kidney transplant rejection. However, the long-term efficacy and toxicity of alemtuzumab therapy are unclear. Therefore, all cases of alemtuzumab anti-rejection therapy between 2012 and 2022 in our institution were investigated. Graft survival, graft function, lymphocyte depletion, serious infections, malignancies, and patient survival were analyzed and compared with a reference cohort of transplanted patients who did not require alemtuzumab anti-rejection therapy. A total of 225 patients treated with alemtuzumab were identified and compared with a reference cohort of 1,668 patients. Over 60% of grafts was salvaged with alemtuzumab therapy, but graft survival was significantly poorer compared to the reference cohort. The median time of profound T- and B lymphocyte depletion was 272 and 344 days, respectively. Serious infection rate after alemtuzumab therapy was 54.1/100 person-years. The risk of death (hazard ratio 1.75, 95%-CI 1.28–2.39) and infection-related death (hazard ratio 2.36, 95%-CI 1.35–4.11) were higher in the alemtuzumab-treated cohort. In conclusion, alemtuzumab is an effective treatment for severe kidney transplant rejection, but causes long-lasting lymphocyte depletion and is associated with frequent infections and worse patient survival outcomes.

Abstract Extracellular vesicles (EVs) are tissue-specific particles containing valuable diagnostic information. However, single EV analysis in blood is challenging due to their physical properties, the molecular complexity of plasma, and a lack of robust data interpretation methods. We assess the applicability of our recently-developed calibrated Imaging Flow Cytometry (IFCM)-based methodology to detect/characterize circulating tissue-specific EV subsets in the clinical setting of kidney transplantation. Platelet-poor plasma was generated from 36 HLA-A3 mismatched donor (HLA-A3 +) and kidney transplant recipients (KTRs; HLA-A3-). Samples taken before transplantation, 3 days, 7 days, and 6 months after transplantation as well as before ‘for-cause’ kidney transplant biopsies were stained with anti-CD9 (plasma EV-marker) and anti-HLA-A3. Before transplantation, no significant differences in total CD9 + EV concentrations were detected between donor and KTR samples. Tissue-specific EVs were identified as CD9 + HLA-A3 + . Serial dilution experiments of HLA-A3 + in HLA-A3- PPP showed that single CD9 + HLA-A3 + EVs were detectable down to ~ 1% above the recipient ‘self-signal’. After transplantation, CD9 + HLA-A3 + EVs were detected above pre-transplantation concentrations in individuals with stable allograft function, but not in individuals with allograft dysfunction. These results demonstrate the applicability of our calibrated IFCM-based methodology in the direct detection of tissue-specific EV subsets in clinical samples. We believe that this EV methodology is applicable in a variety of clinical contexts.

Abstract A high intrapatient variability (IPV) in tacrolimus exposure is a risk factor for poor long‐term outcomes after kidney transplantation. The main objective of this trial was to investigate whether tacrolimus IPV decreases after switching patients from immediate‐release (IR)‐tacrolimus to either extended‐release (ER)‐tacrolimus or LifeCyclePharma (LCP)‐tacrolimus. In this randomized, prospective, open‐label, cross‐over trial, adult kidney transplant recipients on a stable immunosuppressive regimen, including IR‐tacrolimus, were randomized for conversion to ER‐tacrolimus or LCP‐tacrolimus, and for the order in which IR‐tacrolimus and the once‐daily formulations were taken. Patients were followed 6 months for each formulation, with monthly tacrolimus predose concentration assessments to calculate the IPV. The IPV was defined as the coefficient of variation (%) of dose corrected predose concentrations. Ninety‐two patients were included for analysis of the primary outcome. No significant differences between the IPV of IR‐tacrolimus (16.6%) and the combined once‐daily formulations (18.3%) were observed (% difference +1.7%, 95% confidence interval [CI] −1.1% to ‒4.5%, p = 0.24). The IPV of LCP‐tacrolimus (20.1%) was not significantly different from the IPV of ER‐tacrolimus (16.5%, % difference +3.6%, 95% CI −0.1% to 7.3%, p = 0.06). In conclusion, the IPV did not decrease after switching from IR‐tacrolimus to either ER‐tacrolimus or LCP‐tacrolimus. These results provide no arguments to switch kidney transplant recipients from twice‐daily (IR) tacrolimus formulations to once‐daily (modified‐release) tacrolimus formulations when the aim is to lower the IPV.

Background. Memory T cells are important mediators of transplant rejection but are not routinely measured before or after kidney transplantation. The aims of this study were as follows: (1) validate whether pretransplant donor-reactive memory T cells are reliable predictors of acute rejection (AR) (2) determine whether donor-reactive memory T cells can distinguish AR from other causes of transplant dysfunction. Methods. Samples from 103 consecutive kidney transplant recipients (2018–2019) were obtained pretransplantation and at time of for-cause biopsy sampling within 6 mo of transplantation. The number of donor-reactive interferon gamma (IFN-γ) and interleukin (IL)-21-producing memory T cells was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay. Results. Of the 63 patients who underwent a biopsy, 25 had a biopsy-proven acute rejection (BPAR; 22 aTCMR and 3 aAMR), 19 had a presumed rejection, and 19 had no rejection. Receiver operating characteristic analysis showed that the pretransplant IFN-γ ELISPOT assay distinguished between patients who later developed BPAR and patients who remained rejection-free (area under the curve [AUC] 0.73; sensitivity 96% and specificity 41%). Both the IFN-γ and IL-21 assays were able to discriminate BPAR from other causes of transplant dysfunction (AUC 0.81; sensitivity 87% and specificity 76% and AUC 0.81; sensitivity 93% and specificity 68%, respectively). Conclusions. This study validates that a high number of donor-reactive memory T cells before transplantation is associated with the development of AR after transplantation. Furthermore, it demonstrates that the IFN-γ and IL-21 ELISPOT assays are able to discriminate between patients with AR and patients without AR at the time of biopsy sampling.

Background. Mast cells are potential contributors to chronic changes in kidney transplants (KTx). Here, the role of mast cells (MCs) in KTx is investigated in patients with minimal inflammatory lesions. Methods. Fourty-seven KTx biopsies (2009–2018) with borderline pathological evidence for T cell-mediated rejection according to the Banff’17 Update were retrospectively included and corresponding clinical data was collected. Immunohistochemistry for tryptase was performed on formalin-fixed paraffin-embedded sections. Cortical MCs were counted and corrected for area (MC/mm²). Interstitial fibrosis was assessed by Sirius Red staining and quantified using digital image analysis (QuPath). Results. Increased MC number was correlated to donor age (spearman’s r = 0.35, P = 0.022), deceased donor kidneys (mean difference = 0.74, t [32.5] = 2.21, P = 0.035), and delayed graft function (MD = 0.78, t [33.9] = 2.43, P = 0.020). Increased MC number was also correlated to the amount of interstitial fibrosis (r = 0.42, P = 0.003) but did not correlate with transplant function over time (r = −0.14, P = 0.36). Additionally, transplant survival 2 y post-biopsy was not correlated to MC number (mean difference = −0.02, t [15.36] = −0.06, P = 0.96). Conclusions. MC number in suspicious (borderline) for acute T cell-mediated rejection is correlated to interstitial fibrosis and time post-transplantation, suggesting MCs to be a marker for cumulative burden of tissue injury. There was no association between MCs and transplant function over time or transplant survival 2 y post-biopsy. It remains unclear whether MCs are just a bystander or have pro-inflammatory or anti-inflammatory effects in the KTx with minimal lesions.

Abstract Pre-transplant screening focuses on the detection of anti-HLA alloantibodies. Previous studies have shown that IFN-γ and IL-21 producing T cells are associated with the development of acute rejection (AR). The aim of this study, was to assess whether pre-transplant donor-reactive T cells and/or B cells are associated with increased rejection risk. Samples from 114 kidney transplant recipients (transplanted between 2010 and 2013) were obtained pre-transplantation. The number of donor-reactive IFN-γ and IL-21 producing cells was analyzed by ELISPOT assay. The presence of donor specific antibodies (DSA) was also determined before transplantation. Numbers of donor-reactive IFN-γ producing cells were similar in patients with or without AR whereas those of IL-21 producing cells were higher in patients with AR (p = 0.03). Significantly more patients with AR [6/30(20%)] had detectable DSA compared to patients without AR [5/84(5.9%), p = 0.03]. Multivariate logistic regression showed that donor age (OR 1.06), pre-transplant DSA (OR 5.61) and positive IL-21 ELISPOT assay (OR 2.77) were independent predictors of an increased risk for the development of AR. Aside from an advanced donor-age and pre-transplant DSA, also pre-transplant donor-reactive IL-21 producing cells are associated with the development of AR after transplantation.

BackgroundSolid organ transplant recipients are at high risk to develop (complicated) herpes zoster (HZ). Booster vaccination could prevent HZ. However, end-stage renal disease (ESRD) patients show poor immunological responses to vaccinations. We studied the effect of a live attenuated VZV booster vaccine on VZV-specific B and T cell memory responses in ESRD patients and healthy controls. NL28557.000.09, www.toetsingonline.nlMethodsVZV-seropositive patients, aged ≥50 years, awaiting kidney transplantation, were vaccinated with Zostavax®. Gender and age-matched VZV-seropositive potential living kidney donors were included as controls. VZV-specific IgG titers were measured before, at 1, 3 and 12 months post-vaccination. VZV-specific B and T cell responses before, at 3 months and 1 year after vaccination were analysed by flow-cytometry and Elispot, respectively. Occurrence of HZ was assessed at 5 years post-vaccination.Results26 patients and 27 donors were included. Median VZV-specific IgG titers were significantly higher at all time-points post-vaccination in patients (mo 1: 3104 IU/ml [1967-3825], p

Duration of infectious SARS-CoV-2 shedding is an important measure for improved disease control. Here, the authors use virus cultures of respiratory tract samples from COVID-19 patients and observe a median shedding duration of 8 days and a drop below 5% after 15,2 days post onset of symptoms.

Abstract Aims Many heart transplant recipients will develop end‐stage renal disease in the post‐operative course. The aim of this study was to identify the long‐term incidence of end‐stage renal disease, determine its risk factors, and investigate what subsequent therapy was associated with the best survival. Methods and results A retrospective, single‐centre study was performed in all adult heart transplant patients from 1984 to 2016. Risk factors for end‐stage renal disease were analysed by means of multivariable regression analysis and survival by means of Kaplan–Meier. Of 685 heart transplant recipients, 71 were excluded: 64 were under 18 years of age and seven were re‐transplantations. During a median follow‐up of 8.6 years, 121 (19.7%) patients developed end‐stage renal disease: 22 received conservative therapy, 80 were treated with dialysis (46 haemodialysis and 34 peritoneal dialysis), and 19 received a kidney transplant. Development of end‐stage renal disease (examined as a time‐dependent variable) inferred a hazard ratio of 6.45 (95% confidence interval 4.87–8.54, P < 0.001) for mortality. Tacrolimus‐based therapy decreased, and acute kidney injury requiring renal replacement therapy increased the risk for end‐stage renal disease development (hazard ratio 0.40, 95% confidence interval 0.26–0.62, P < 0.001, and hazard ratio 4.18, 95% confidence interval 2.30–7.59, P < 0.001, respectively). Kidney transplantation was associated with the best median survival compared with dialysis or conservative therapy: 6.4 vs. 2.2 vs. 0.3 years (P < 0.0001), respectively, after end‐stage renal disease development. Conclusions End‐stage renal disease is a frequent complication after heart transplant and is associated with poor survival. Kidney transplantation resulted in the longest survival of patients with end‐stage renal disease.

Abstract Background In solid organ transplant (SOT) recipients, transplant rejection during immune checkpoint inhibitor (ICI) treatment for cancer is a clinical problem. Donor-derived cell-free DNA (dd-cfDNA) can be detected in blood and is a sensitive biomarker for diagnosis of acute rejection in SOT recipients. To our best knowledge, this is the first case report of a kidney transplant recipient with advanced cancer treated with ICI who was monitored with dd-cfDNA. Case presentation A 72-year old female with a long-standing renal transplant was diagnosed with advanced melanoma in 2018 and was treated with the anti-PD1 antibody nivolumab. Within 12 days after the first administration of nivolumab, dd-cfDNA ratio increased to 23%, suggesting allograft rejection. Her kidney transplant function deteriorated and acute rejection was confirmed by renal transplant biopsy. As the rejection could not be controlled despite immunosuppressive treatment, a transplant nephrectomy was necessary and haemodialysis was started. Immunological analysis of the renal explant showed infiltration of alloreactive, nivolumab-saturated, PD1+ cytotoxic T cells. After transplant nephrectomy, she experienced nivolumab-related toxicity and rapid disease progression. Conclusion Clinicians prescribing ICIs should be aware that SOT recipients are at risk of transplant rejection as a result of T cell activation. Dd-cfDNA is a sensitive biomarker and should be further studied for early detection of transplant rejection. Immunological analysis of the kidney explant showed marked graft infiltration with alloreactive PD-1+ cytotoxic T cells that were saturated with nivolumab.

Background. Cholesterol embolization syndrome (CES) is an uncommon but well-known cause of renal failure in native kidneys, but little is known about CES in kidney transplant recipients. The aim of this study was to determine the incidence, clinical characteristics, histopathology, and prognosis of CES after kidney transplantation. Methods. CES cases in both transplanted and native kidneys (control group) were identified by searching the databases of the divisions of Nephrology and Pathology of our institution. Clinical data were retrospectively collected. Biopsies were classified according to the latest Banff 2019 Update. Second, a systematic literature search was performed (December 01, 2020) of Ovid MEDLINE, EMBASE, the Cochrane Central Register of controlled trials, Google Scholar, and Web of Science. Results. CES was observed in for-cause biopsies of 11 out of 2350 (0.47%) kidney transplant recipients transplanted between January 1, 2006, and December 31, 2018 (0.0009 cases per person-year). All patients had ≥1 cardiovascular risk factor, and 9 donors were expanded criteria donors. Graft loss occurred in 27.3% of the patients diagnosed with CES. Eight transplant biopsies with CES were also classified as biopsy-proven acute rejection. Transplant biopsies showed signs of inflammation (arteritis, n = 7; interstitial inflammation, n = 5; tubulitis, n = 7). One patient with CES in a native kidney was identified. The biopsy of the native kidney only showed arteritis and classified as an isolated “v” lesion. The literature search resulted in 188 unique articles of which 20 were included. A total of 47 cases of CES after kidney transplantation was reported. Cholesterol emboli were found in

Cytochrome P450 3A4 (CYP3A4) is the most important drug metabolizing enzyme in the liver, responsible for the oxidative metabolism of ∼50% of clinically prescribed drugs. Therefore, genetic variation in CYP3A4 could potentially affect the pharmacokinetics, toxicity and clinical outcome of drug treatment. Thus far, pharmacogenetics for CYP3A4 has not received much attention. However, the recent discovery of the intron 6 single-nucleotide polymorphism (SNP) rs35599367C > T, encoding the CYP3A4∗22 allele, led to several studies into the pharmacogenetic effect of CYP3A4∗22 on different drugs. This allele has a relatively minor allele frequency of 3-5% and an effect on CYP3A4 enzymatic activity. Thus far, no review summarizing the data published on several drugs is available yet. This article therefore addresses the current knowledge on CYP3A4∗22. This information may help in deciding if, and for which drugs, CYP3A4∗22 genotype-based dosing could be helpful in improving drug therapy. CYP3A4∗22 was shown to significantly influence the pharmacokinetics of several drugs, with currently being most thoroughly investigated tacrolimus, cyclosporine, and statins. Additional studies, focusing on toxicity and clinical outcome, are warranted to demonstrate clinical utility of CYP3A4∗22 genotype-based dosing.

The endothelium plays a key role in acute and chronic rejection of solid organ transplants. During both processes the endothelium is damaged often with major consequences for organ function. Also, endothelial cells (EC) have antigen-presenting properties and can in this manner initiate and enhance alloreactive immune responses. For decades, knowledge about these roles of EC have been obtained by studying both in vitro and in vivo models. These experimental models poorly imitate the immune response in patients and might explain why the discovery and development of agents that control EC responses is hampered. In recent years, various innovative human 3D in vitro models mimicking in vivo organ structure and function have been developed. These models will extend the knowledge about the diverse roles of EC in allograft rejection and will hopefully lead to discoveries of new targets that are involved in the interactions between the donor organ EC and the recipient's immune system. Moreover, these models can be used to gain a better insight in the mode of action of the currently prescribed immunosuppression and will enhance the development of novel therapeutics aiming to reduce allograft rejection and prolong graft survival.

Background. The relationship between circulating effector memory T and B cells long after transplantation and their susceptibility to immunosuppression are unknown. To investigate the impact of antirejection therapy on T cell-B cell coordinated immune responses, we assessed IFN-γ-producing memory cells and natural antibodies (nAbs) that potentially bind to autoantigens on the graft. Methods. Plasma levels of IgG nAbs to malondialdehyde (MDA) were measured in 145 kidney transplant recipients at 5–7 years after transplantation. In 54 of these patients, the number of donor-reactive IFN-γ-producing cells was determined. 35/145 patients experienced rejection, 18 of which occurred within 1 year after transplantation. Results. The number of donor-reactive IFN-γ-producing cells and the levels of nAbs were comparable between rejectors and nonrejectors. The nAbs levels were positively correlated with the number of donor-reactive IFN-γ-producing cells (rs = 0.39, p=0.004). The positive correlation was only observed in rejectors (rs = 0.53, p=0.003; nonrejectors: rs = 0.24, p=0.23). Moreover, we observed that intravenous immune globulin treatment affected the level of nAbs and this effect was found in patients who experienced a late ca-ABMR compared to nonrejectors (p=0.008). Conclusion. The positive correlation found between alloreactive T cells and nAbs in rejectors suggests an intricate role for both components of the immune response in the rejection process. Treatment with intravenous immune globulin impacted nAbs.

Background: FoxP3+ follicular regulatory T cells (Tfr) have been identified as the cell population controlling T follicular helper (Tfh) cells and B cells which, are both involved in effector immune responses against transplanted tissue.Methods: To understand the biology of Tfr cells in kidney transplant patients treated with tacrolimus and mycophenolate mofetil (MMF) combination immunosuppression, we measured circulating (c)Tfh and cTfr cells in peripheral blood by flow cytometry in n = 211 kidney transplant recipients. At the time of measurement patients were 5–7 years after transplantation. Of this cohort of patients, 23.2% (49/211) had been previously treated for rejection. Median time after anti-rejection therapy was 4.9 years (range 0.4–7 years). Age and gender matched healthy individuals served as controls.Results: While the absolute numbers of cTfh cells were comparable between kidney transplant recipients and healthy controls, the numbers of cTfr cells were 46% lower in immunosuppressed recipients (p < 0.001). More importantly, in transplanted patients, the ratio of cTfr to cTfh was decreased (median; 0.10 vs. 0.06), indicating a disruption of the balance between cTfr and cTfh cells. This shifted balance was observed for both non-rejectors and rejectors. Previous pulse methylprednisolone or combined pulse methylprednisolone + intravenous immunoglobulin anti-rejection therapy led to a non-significant 30.6% (median) and 51.2% (median) drop in cTfr cells, respectively when compared to cTfr cell numbers in transplant patients who did not receive anti-rejection therapy. A history of alemtuzumab therapy did lead to a significant decrease in cTfr cells of 85.8% (median) compared with patients not treated with anti-rejection therapy (p < 0.0001). No association with tacrolimus or MMF pre-dose concentrations was found.Conclusion: This cross-sectional study reveals that anti-rejection therapy with alemtuzumab significantly lowers the number of cTfr cells in kidney transplant recipients. The observed profound effects by these agents might dysregulate cTfr functions.

Rabbit anti-thymocyte globulin (rATG) is currently the treatment of choice for glucocorticoid-resistant, recurrent, or severe acute allograft rejection (AR). However, rATG is associated with severe infusion-related side effects. Alemtuzumab is incidentally given to kidney transplant recipients as treatment for AR. In the current study, the outcomes of patients treated with alemtuzumab for AR were compared with that of patients treated with rATG for AR. The patient-, allograft-, and infection-free survival and adverse events of 116 alemtuzumab-treated patients were compared with those of 108 patients treated with rATG for AR. Propensity scores were used to control for differences between the two groups. Patient- and allograft survival of patients treated with either alemtuzumab or rATG were not different [hazard ratio (HR) 1.14, 95%-confidence interval (CI) 0.48–2.69, p = 0.77, and HR 0.82, 95%-CI 0.45–1.5, p = 0.52, respectively). Infection-free survival after alemtuzumab treatment was superior compared with that of rATG-treated patients (HR 0.41, 95%-CI 0.25–0.68, p < 0.002). Infusion-related adverse events occurred less frequently after alemtuzumab treatment. Alemtuzumab therapy may therefore be an alternative therapy for glucocorticoid-resistant, recurrent, or severe acute kidney transplant rejection.

Interleukin (IL)-21 supports induction and expansion of CD8+ T cells, and can also regulate the differentiation of B cells into antibody-producing plasma cells. We questioned whether the number of circulating donor-specific IL-21 producing cells (pc) can predict kidney transplant rejection, and evaluated this in two different patient cohorts. The first analysis was done on pre-transplantation samples of 35 kidney transplant recipients of whom 15 patients developed an early acute rejection. The second study concerned peripheral blood mononuclear cell (PBMC) samples from 46 patients obtained at 6 months after kidney transplantation of whom 13 developed late rejection. Significantly higher frequencies of donor-specific IL-21 pc were found by Elispot assay in both patients who developed early and late rejection compared to those without rejection. In addition, low frequencies of donor-specific IL-21 pc were associated with higher rejection-free survival. Moreover, low pre-transplant donor-specific IL-21 pc numbers were associated with the absence of anti-HLA antibodies. Donor-reactive IL-21 was mainly produced by CD4+ T cells, including CD4+ follicular T helper cells. In conclusion, the number of donor-specific IL-21 pc is associated with an increased risk of both early and late rejection, giving it the potential to be a new biomarker in kidney transplantation.

Background. Belatacept-based therapy in kidney transplant recipient has been shown to increase long-term renal allograft and patient survival compared with calcineurin inhibitor–based therapy, however, with an increased risk of acute T cell-mediated rejection (aTCMR). An improved understanding of costimulation blockade-resistant rejections could lead to a more personalized approach to belatacept therapy. Here, immunomic profiles of aTCMR biopsies of patients treated with either tacrolimus or belatacept were compared. Methods. Formalin-fixed paraffin-embedded renal transplant biopsies were used for immunohistochemistry and gene expression analysis using the innovative NanoString technique. To validate NanoString, transcriptomic profiles of patients with and without biopsy-proven aTCMR were compared. Biopsies from 31 patients were studied: 14 tacrolimus-treated patients with aTCMR, 11 belatacept-treated patients with aTCMR, and 6 controls without rejection. Results. A distinct pattern was seen in biopsies with aTCMR compared to negative controls: 78 genes had a higher expression in the aTCMR group (false discovery rate P value

BackgroundCalcium oxalate (CaOx) deposition in the kidney may lead to loss of native renal function but little is known about the prevalence and role of CaOx deposition in transplanted kidneys.MethodsIn patients transplanted in 2014 and 2015, all for-cause renal allograft biopsies obtained within 3 months post-transplantation were retrospectively investigated for CaOx deposition. Additionally, all preimplantation renal biopsies obtained in 2000 and 2001 were studied.ResultsIn 2014 and 2015, 388 patients were transplanted, of whom 149 had at least one for-cause renal biopsy. Twenty-six (17%) patients had CaOx deposition. In the population with CaOx deposition: Patients had significantly more often been treated with dialysis before transplantation (89 vs. 64%; p = 0.011); delayed graft function occurred more frequently (42 vs. 23%; p = 0.038); and the eGFR at the time of first biopsy was significantly worse (21 vs. 29 ml/min/1.73m2; p = 0.037). In a multivariate logistic regression analysis, eGFR at the time of first biopsy (OR 0.958, 95%-Cl: 0.924-0.993, p = 0.019), dialysis before transplantation (OR 4.868, 95%-Cl: 1.128-21.003, p = 0.034) and the time of first biopsy after transplantation (OR 1.037, 95%-Cl: 1.013-1.062, p = 0.002) were independently associated with CaOx deposition. Graft survival censored for death was significantly worse in patients with CaOx deposition (p = 0.018). In only 1 of 106 preimplantation biopsies CaOx deposition was found (0.94%).ConclusionCaOx deposition appears to be primarily recipient-derived and is frequently observed in for-cause renal allograft biopsies obtained within 3 months post-transplantation. It is associated with inferior renal function at the time of biopsy and worse graft survival.

Abstract. The introduction of immunosuppressant belatacept, an inhibitor of the CD28-80/86 pathway, has improved 1-year outcomes in kidney transplant recipients with preexistent diabetes mellitus and has also reduced the risk of posttransplant diabetes mellitus. So far, no studies have compared a tacrolimus-based with a belatacept-based immunosuppressive regimen with regard to improving glucose tolerance after kidney transplantation. Here, we present the case of a 54-year-old man with type 2 diabetes mellitus who was converted from belatacept to tacrolimus 1 year after a successful kidney transplantation. Thereafter, he quickly developed severe hyperglycemia, and administration of insulin was needed to improve metabolic control. Six months after this episode, he was converted back to belatacept because of nausea, diarrhea, and hyperglycemia. After switching back to belatacept and within 4 days after stopping tacrolimus glucose tolerance improved and insulin therapy could be discontinued. Although belatacept is considered less diabetogenic than tacrolimus, the rapid improvement of glucose tolerance after switching to belatacept is remarkable. In this article, the potential mechanisms of this observation are discussed.

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.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.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

BACKGROUND:Therapeutic drug monitoring (TDM) of tacrolimus, based on blood concentrations, shows an imperfect correlation with the occurrence of rejection. Here, we tested whether measuring NFATc1 amplification, a member of the calcineurin pathway, is suitable for TDM of tacrolimus. MATERIALS AND METHODS:NFATc1 amplification was monitored in T cells of kidney transplant recipients who received either tacrolimus- (n = 11) or belatacept-based (n = 10) therapy. Individual drug effects on NFATc1 amplification were studied in vitro, after spiking blood samples of healthy volunteers with either tacrolimus, belatacept or mycophenolate mofetil. RESULTS:At day 30 after transplantation, in tacrolimus-treated patients, NFATc1 amplification was inhibited in CD4+ T cells expressing the co-stimulation receptor CD28 (mean inhibition 37%; p = 0.01) and in CD8+CD28+ T cells (29% inhibition; p = 0.02), while this was not observed in CD8+CD28- T cells or belatacept-treated patients. Tacrolimus pre-dose concentrations of these patients correlated inversely with NFATc1 amplification in CD28+ T cells (rs = -0.46; p < 0.01). In vitro experiments revealed that 50 ng/ml tacrolimus affected NFATc1 amplification by 58% (mean; p = 0.02). CONCLUSION:In conclusion, measuring NFATc1 amplification is a direct tool for monitoring biological effects of tacrolimus on T cells in whole blood samples of kidney transplant recipients. This technique has potential that requires further development before it can be applied in daily practice.

Tacrolimus (Tac) is a profoundly effective immunosuppressant that reduces the risk of rejection after solid organ transplantation. However, its use is hampered by its narrow therapeutic window along with its highly variable pharmacological (pharmacokinetic [PK] and pharmacodynamic [PD]) profile. Part of this variability is explained by genetic polymorphisms affecting the metabolic pathway. The integration of CYP3A4 and CY3A5 genotype in tacrolimus population-based PK (PopPK) modeling approaches has been proven to accurately predict the dose requirement to reach the therapeutic window. The objective of the present study was to develop an accurate PopPK model in a cohort of 59 kidney transplant patients to deliver this information to clinicians in a clear and actionable manner. We conducted a non-parametric non-linear effects PopPK modeling analysis in Pmetrics®. Patients were genotyped for the CYP3A4∗22 and CYP3A5∗3 alleles and were classified into 3 different categories [poor-metabolizers (PM), Intermediate-metabolizers (IM) or extensive-metabolizers (EM)]. A one-compartment model with double gamma absorption route described very accurately the tacrolimus PK. In covariate analysis, only CYP3A genotype was retained in the final model (Δ-2LL = -73). Our model estimated that tacrolimus concentrations were 33% IC95%[20–26%], 41% IC95%[36–45%] lower in CYP3A IM and EM when compared to PM, respectively. Virtually, we proved that defining different starting doses for PM, IM and EM would be beneficial by ensuring better probability of target concentrations attainment allowing us to define new dosage recommendations according to patient CYP3A genetic profile.

Humoral alloreactivity has been recognized as a common cause of kidney transplant dysfunction. B-cell activation, differentiation, and antibody production are dependent on IL-21+CXCR5+follicular T-helper (Tfh) cells. Here, we studied whether belatacept, an inhibitor of the costimulatory CD28-CD80/86-pathway, interrupts the crosstalk between Tfh- and B-cells more efficiently than the calcineurin inhibitor tacrolimus. The suppressive effects of belatacept and tacrolimus on donor antigen-driven Tfh–B-cell interaction were functionally studied in peripheral blood mononuclear cells from 40 kidney transplant patients randomized to a belatacept- or tacrolimus-based immunosuppressive regimen. No significant differences in uncultured cells or donor antigen-stimulated cells were found between belatacept- and tacrolimus-treated patients in the CXCR5+Tfh cell generation and activation (upregulation of PD-1). Belatacept and tacrolimus in vitro minimally inhibited Tfh-cell generation (by ~6–7%) and partially prevented Tfh-cell activation (by ~30–50%). The proportion of IL-21+-activated Tfh-cells was partially decreased by in vitro addition of belatacept or tacrolimus (by ~60%). Baseline expressions and proportions of activated CD86+ B-cells, plasmablasts, and transitional B-cells after donor antigen stimulation did not differ between belatacept- and tacrolimus-treated patients. Donor antigen-driven CD86 upregulation on memory B-cells was not fully prevented by adding belatacept in vitro (~35%), even in supratherapeutic doses. In contrast to tacrolimus, belatacept failed to inhibit donor antigen-driven plasmablast formation (~50% inhibition vs. no inhibition, respectively, p

Monocytes and macrophages play key roles in many disease states, including cellular and humoral rejection after solid organ transplantation (SOT). To suppress alloimmunity after SOT, immunosuppressive drug therapy is necessary. However, little is known about the effects of the immunosuppressive drugs tacrolimus and mycophenolic acid (MPA) on monocyte activation and function. Here, the effect of these immunosuppressants on monocytes was investigated by measuring phosphorylation of three intracellular signaling proteins which all have a major role in monocyte function: p38MAPK, ERK and Akt. In addition, biological functions downstream of these signaling pathways were studied, including cytokine production, phagocytosis and differentiation into macrophages. To this end, blood samples from healthy volunteers were spiked with diverse concentrations of tacrolimus and MPA. Tacrolimus (200 ng/ml) inhibited phosphorylation of p38MAPK by 30% (mean) in CD14+ monocytes which was significantly less than in activated CD3+ T cells (max 60%; p < 0.05). This immunosuppressive agent also partly inhibited p-AKT (14%). MPA, at a therapeutic concentration showed the strongest effect on p-AKT (27% inhibition). p-ERK was inhibited with a maximum of 15% after spiking with either tacrolimus or MPA. The production of IL-1β and phagocytosis by monocytes were not affected by tacrolimus concentrations, whereas MPA did inhibit IL-1β production by 50%. Monocyte/macrophage polarization was shifted to an M2-like phenotype in the presence of tacrolimus, while MPA increased the expression of M2 surface markers, including CD163 and CD200R, on M1 macrophages. These results show that tacrolimus and MPA do not strongly affect monocyte function, apart from a change in macrophage polarization, to a clinically relevant degree.