Despite advancements in donor selection, conditioning regimens, and immunosuppressive therapies, graft-versus-host disease (GvHD) remains a frequent complication of allogeneic hematopoietic stem cell transplantation (HSCT). Regulatory T cells (Tregs), key mediators of immune tolerance, have been investigated for use in GvHD, with promising preclinical and early clinical data. Here we report two pediatric HSCT recipients who underwent successful adoptive Treg therapy for severe, treatment-refractory acute GvHD.In both patients, conventional GvHD therapy in the form of immunosuppression, including calcineurin inhibitors, ruxolitinib, and extracorporeal photopheresis, did not achieve disease control. Both patients experienced life-threatening disease progression refractory to all conventional therapy. Tregs were isolated from original stem cell donors and administered following lymphodepletion. Patient 1 achieved partial remission of intestinal GvHD after initial Treg infusion, with sustained response following multiple doses, allowing for long-term immunosuppressant tapering. Patient 2 showed substantial improvement in cutaneous GvHD after repeated Treg infusions. Complete remission was achieved and immunosuppression successfully discontinued.These findings support the feasibility, safety, and efficacy of adoptive Treg therapy in refractory GvHD. Long-term GvHD control without significant infusion-related complications highlight the potential of Tregs as a potent GvHD-directed therapy.

The composition of the gut microbiota (GM) is altered in solid organ transplantation (SOT) recipients, where the degree of dysbiosis is associated with long-term survival and is believed to be influenced by immunosuppression therapy. At the interface stands secretory (S)IgA, however, little is known about its role in governing dysbiosis in the context of SOT. We performed quantitative metagenomic analyses of the GM accompanied by SIgA sequencing in 48 pediatric SOT recipients (age = 10.6 ± 4.7 y) receiving either heart (n = 11), kidney (n = 10) or liver transplantation (n = 27), and compared the results to age-matched healthy controls (HC, n = 16). We confirmed compositional and functional dysbiosis in SOT recipients, with the degree of dysbiosis being associated with tacrolimus (TAC) levels. Overall, SOT recipients exhibited higher SIgA levels than HC, along with an increased percentage of bacteria targeted and altered target spectra. Furthermore, altered SIgA responses were associated with the degree of dysbiosis. A mechanistic model connecting immunosuppression, GM composition and SIgA-targeting is proposed, suggesting that GM dysbiosis in SOT recipients is mediated by the immune system through the SIgA response; direct drug-mediated effects on fecal communities were not observed in in vitro experiments. Our study provides new insights into factors that contribute to persisting dysbiosis in SOT recipients.

Background Composition of gut microbiota (GM) is altered in solid organ transplantation (SOT) patients, where the degree of dysbiosis is associated with long-term survival. Factors governing dysbiosis are poorly understood with immunosuppression therapy believed to be involved in altering GM composition in those patients, either directly or via the immune system. At the interface stands secretory (S)IgA, however, little is known on its role governing dysbiosis in the context of SOT. In this study, we performed quantitative metagenomic analyses of GM accompanied by SIgA sequencing in 48 pediatric SOT recipients (age = 10.6 ± 4.7 years) receiving either a heart (HTR, n=10), a kidney (KTR, n=11) or a liver (LTR, n=27) and compared results to healthy controls (HC, n=16). Results We confirmed compositional and functional dysbiosis in SOT patients that broadly clustered into two groups, where cluster 1 (n=23) comprised most LTR patients and was closer to HC compared with cluster 2 (n=24) that exhibited stronger dysbiosis and included most HTR and KTR patients. The degree of dysbiosis was associated with Tacrolimus (TAC) levels. Overall, patients exhibited higher SIgA levels than HC, along with an increased percentage of bacteria targeted and altered target spectra. Furthermore, altered SIgA responses were associated with the degree of dysbiosis and were especially increased in cluster 2, in particular in HTR patients. A mechanistic model connecting immunosuppression, GM composition and SIgA targeting is proposed. Conclusion Our study suggests that altered SIgA responses play an important role for GM alterations observed in SOT patients. It opens new therapeutic angles to combat GM dysbiosis and associated long-erm complications. ### Competing Interest Statement The authors have declared no competing interest.

In children, little is known about gut microbiota (GM) in end-stage liver disease and its association with graft function after pediatric liver transplantation (pLT). We analyzed GM composition and function in children before pLT, longitudinally post-pLT and in long-term survivors (LT-pLT) in order to assess the impact of disease severity, treatment, and pLT on GM and delineate associations with graft and patient health. Fecal samples (FS) of 29 children [17f (female), age 2.6 (0.2-15.7) years] awaiting pLT were included with longitudinal follow-ups until 12 months post-transplant in 18, and compared with 38 LT-pLT [21f, age 11 (2.7-17.7) years, 7.8 (1.0-17.0) years post-pLT] and 94 healthy controls (HCs). Samples were analyzed using quantitative 16S rRNA gene analyses combined with shotgun metagenomics (subset of samples). Pre-pLT patients showed reduced alpha-diversities and altered GM composition compared with LT-pLT and HC, associated with disease severity and anti-pruritic treatment with rifampicin. Dysbiosis increased after pLT and started to recover after 3M (months). Although bacterial concentrations, alpha diversity, and gene richness increased post-pLT, levels remained below those of HC. Abundances of key functions, for example, the capacity to synthesize butyrate, also remained reduced. Quantitative analyses revealed the true extent of differences between patients and HC that were underestimated using relative abundance data. GM diversity and functional capacities correlated negatively with transaminase levels mid-term and long-term after pLT. Random Forest analyses based on GM were able to predict hepatocellular damage at high accuracy (AUC: 0.89). We provide comprehensive, quantitative insights into GM composition and function before and after pLT. A link between GM alterations and (long-term) graft health was uncovered, providing possible targets to modulate GM function in order to increase graft and patient health.

Steroid-free immunosuppression protocols gained popularity in pediatric liver transplantation (pLT) after the introduction of IL-2–receptor blockade for induction therapy. We analyzed the clinical and immunologic outcome data of the multicenter prospective observational ChilSFree study to compare the impact of steroid-free versus steroid-containing immunosuppressive therapy following pLT in a real-life scenario. Two hundred forty-six children [55.3% male, age at pLT median: 2.4 (range: 0.2–17.9) y] transplanted for biliary atresia (43%), metabolic liver disease (9%), acute liver failure (4%), hepatoblastoma (9%), and other chronic end-stage liver diseases (39%) underwent immune monitoring and clinical data documentation over the first year after pLT. Patient and graft survival at 1 year was 98.0% and 92.7%, respectively. Primary immunosuppression was basiliximab induction followed by tacrolimus (Tac) monotherapy (55%), Tac plus steroid tapering over 3 months (29%), or cyclosporine and steroid tapering (7%). One center used intraoperative steroids instead of basiliximab followed by Tac plus mycophenolate mofetil (7% of patients). N = 124 biopsy-proven T-cell–mediated rejections were documented in n = 82 (33.3%) patients. T-cell–mediated rejection occurred early (median: 41 d, range: 3–366 d) after pLT. Patients initially treated with Tac plus steroids experienced significantly fewer episodes of rejection than patients treated with Tac alone (chi-square p <0.01). The use of steroids was associated with earlier downregulation of proinflammatory cytokines interferon (IFN)-γ, Interleukin (IL)-6, CX motif chemokin ligand (CXCL)8, IL-7, and IL-12p70. Both primary immunosuppression with Tac plus steroids and living donor liver transplantation were independent predictors of rejection-free survival 1 year after pLT on logistic regression analysis. Adjunctive steroid therapy after pLT leads to earlier suppression of the post-pLT proinflammatory response and significantly reduced rejection rates during the first year after pLT (15.9%). Fifty-one percent of patients initially treated without steroids remain steroid-free over the first 12 months without rejection.

Background & Aims After pediatric liver transplantation (pLT), children undergo life-long immunosuppression since reliable biomarkers for the assessment of rejection probability are scarce. In the multicentre (n=7) prospective clinical cohort “ChilSFree” study, we aimed to characterize longitudinal dynamics of soluble and cellular immune mediators during the first year after pLT and identify early biomarkers associated with outcome. Methods Using paired Luminex-based multiplex technique and flow cytometry, we characterized longitudinal dynamics of soluble immune mediators (SIM, n=50) and immune cells in the blood of 244 patients at 8 visits over one year: before, 7/14/21/28 days, 3/6/12 months after pLT. Results The unsupervised clustering of patients based on SIM profiles revealed 6 unique SIM signatures associated with clinical outcome. From 3 signatures linked to improved outcome, one was associated with one-year-long rejection-free survival and stable graft function and was characterized by low levels of pro-inflammatory (CXCL8/9/10/12, CCL7, SCGF-β, sICAM-1), high levels of regenerative (SCF, TNF-β), and pro-apoptotic (TRAIL) SIM (all, p<0.001, fold change >100). Of note, this SIM signature appeared two weeks after pLT and remained stable over the entire year, pointing towards its potential as a novel early biomarker for minimizing or weaning immunosuppression. In the blood of these patients, a higher frequency of CD56bright NK cells (p<0.01), a known hallmark also associated with operationally tolerant pLT patients, was detected. The concordance of the model for prediction of rejection based on identified SIM signatures was 0.715, and 0.795, in combination with living-related transplantation as co-variate, respectively. Conclusions SIM blood signatures may enable the non-invasive and early assessment of rejection risks in the first year after pLT, paving the way to improved therapeutic options.

Background: The current gold standard to diagnose T-cell-mediated acute rejection (TCMR) requires liver histology. Using data from the ChilSFree study on immune response after paediatric liver transplantation (pLT), we aimed to assess whether soluble cytokines can serve as an alternative diagnostic tool in children suspected to have TCMR. Methods: A total of n = 53 blood samples obtained on the day of or up to 3 days before liver biopsy performed for suspected TCMR at median 18 days (range 7–427) after pLT in n = 50 children (38% female, age at pLT 1.8 (0.5–17.5) years) were analysed for circulating cytokine levels using Luminex-based Multiplex technology. Diagnostic accuracy of cytokine concentrations was assessed using a multivariable model based on elastic net regression and gradient boosting machine analysis. Results: TCMR was present in 68% of biopsies. There was strong evidence that patients with TCMR had increased levels of soluble CXCL8, CXCL9, CXCL10, IL-16, IL-18, HGF, CCL4, MIF, SCGF-β, and HGF before biopsy. There was some evidence for increased levels of sCD25, ICAM-1, IL-6, IL-3, and CCL11. Diagnostic value of both single cytokine levels and a combination of cytokines and clinical markers was poor, with AUROCs not exceeding 0.7. Conclusion: Patients with TCMR showed raised levels of cytokines and chemokines reflective of T-cell activation and chemotaxis. Despite giving insight into the mechanisms of TCMR, the diagnostic value of soluble cytokines for the confirmation of TCMR in a clinical scenario of suspected TCMR is poor.

Epidemiological evidence suggests that thrombophilic factors, including male sex, non-O blood type, MTHFRnt677TT mutation, factor V Leiden G1691A mutation, and prothrombin G20210A polymorphism, may contribute to the progression of fibrosis and occurrence of portal vein thrombosis in liver disease. We retrospectively investigated the effect of potentially thrombophilic factors on native liver survival as a patient-relevant endpoint of disease progression in a cohort of 142 children being followed up for biliary atresia at Hannover Medical School from April 2017 to October 2019. No significant association could be determined. There was no evidence for relevant differences in native liver survival for the Factor V Leiden G1691A mutation (hazard ratio [HR] = 0.86, 95% confidence interval [CI] 0.38–1.98, p = 0.73), prothrombin G20210A polymorphism (HR = 0.96, 95%CI 0.24–3.65, p = 0.96), non-O blood type (HR = 0.79, 95%CI 0.51–1.21, p = 0.28) or MTHFRnt677TT mutation (HR = 1.24, 95%CI 0.60–2.56, p = 0.56). A certain, albeit not strong, evidence of reduced native liver survival in male patients after Kasai hepatoportoenterostomy, particularly during the first 2000 days (42%; HR = 1.41, 95%CI 0.92–2.18, p = 0.11) was found. All children with pre-transplant portal vein thrombosis (n = 7) had non-O blood types. Larger multi-centre studies are necessary to show if the male sex or other thrombophilic factors could be potentially associated with reduced native liver survival.

Background: Bile salt export pump (ABCB11) deficiency [Progressive familial intrahepatic cholestasis (PFIC2)] is the most common genetic cause of PFIC and is associated with pruritus and progressive liver disease. Surgical biliary diversion or pharmacological [ileal bile acid transporter inhibitor (IBATi)] approaches can be used to block the recirculation of bile acids to the liver. There is a paucity of detailed data on the natural history and, in particular, the longitudinal evolution of bile acid levels to predict treatment response. Cross-sectional data from large international consortia suggested a maximum cutoff value of bile acids after the intervention to predict a successful outcome. Methods: This retrospective, single-center, cohort study included all patients with confirmed biallelic pathogenic ABCB11 genotype PFIC2 treated at our institution with ≥2 years follow-up. The outcomes of interventions and predictors of long-term health were analyzed. Results: Forty-eight cases were identified with PFIC2. Eighteen received partial external biliary diversion (PEBD) surgery, and 22 patients underwent liver transplantation. Two patients developed HCC and 2 died. Improved survival with native liver was closely associated with genotype, complete normalization of serum bile acids following PEBD, and alleviation of pruritus. Persistence of mild-to-moderate elevation of bile acids or a secondary rise following normalization was associated with liver disease progression and led to transplantation, suggesting that any prolonged elevation of bile acids worsens the chance of native liver survival. Higher-grade fibrosis at the time of PEBD was not associated with reduced long-term native liver survival. Patients with PFIC2 benefit from PEBD even at a stage of advanced fibrosis. Conclusion: Serum bile acid levels are an early predictor of treatment response and might serve as the gold standard in the evaluation of novel therapies including IBATi.

Leukocyte telomere length (LTL) is a marker for biological age. Pediatric liver transplant recipients show a high rate of subclinical atherosclerosis, indicated by elevated intima-media thickness (IMT). We hypothesized that atherosclerosis is associated with biological age in these patients and investigated the course of LTL over time. We measured LTL from peripheral blood leukocytes by quantitative polymerase chain reaction and IMT from 97 pediatric patients after liver transplantation in a prospective cohort study. Of the patients, 71% (n = 69) had two or more assessments (total, 228 observations; median follow-up, 1.1 years). Lower LTL was associated with higher IMT (β = -0.701, p = 0.01) and higher aspartate aminotransferase (β = -0.001, p = 0.02), adjusted for age, sex, and age at transplantation. Of the patients, 45% showed decreasing LTL over time, whereas 55% exhibited stable LTL. Patients with stable LTL showed a decrease in IMT (median, -0.02 mm/year) and a decrease of tacrolimus trough levels (median, -0.08 μg/L/year). LTL is associated with IMT independent of age in pediatric liver transplant patients, suggesting that early aging contributes to the high burden of subclinical cardiovascular damage and may furthermore negatively affect the graft.

Objective: Structured education programs have been shown to improve somatic outcome and health-related quality of life (HRQOL) in a variety of chronic childhood diseases. Similar data are scarce in paediatric liver transplantation (pLTx). The purpose of this study was to examine the relationship of parental disease-specific knowledge and psychosocial disease outcome in patients after pLTx. Methods: Parents of 113 children (chronic liver disease n = 25, after pLTx n = 88) completed the transplant module of the HRQOL questionnaire PedsQL, the “Ulm quality of life inventory for parents of children with chronic diseases” ULQUI, and a tailor-made questionnaire to test disease-specific knowledge. Results: Parental knowledge was highest on the topic of “liver transplantation” and lowest in “basic background knowledge” (76% and 56% correct answers respectively). Knowledge performance was only marginally associated with HRQOL scores, with better knowledge being related to worse HRQOL outcomes. In contrast, self-estimation of knowledge performance showed significant positive correlations with both PedsQL and ULQUI results. Conclusion: Patient HRQOL and parental emotional wellbeing after pLTx are associated with positive self-estimation of parental disease-specific knowledge. Objective disease-specific knowledge has little impact on HRQOL. Parental education programs need to overcome language barriers and address self-efficacy in order to improve HRQOL after pLTx.

Familial intrahepatic cholestasis 1 (FIC1) disease is a genetic disorder characterized by hepatic and gastrointestinal disease due to ATP8B1 deficiency, often requiring liver transplantation (LT). Extrahepatic symptoms, such as diarrhea, malabsorption, and failure to thrive, do not improve and instead may be aggravated after LT. We describe a patient with FIC1 disease who underwent LT at 2 years, 8 months of age. After LT, the child developed severe refractory diarrhea and failed to thrive. The response to bile acid resins was unsatisfactory, and the parents declined our recommendation for partial external biliary diversion (PEBD). Quality of life was extremely impaired, especially due to severe diarrhea, making school attendance impossible. Attempting to reduce the total bile acids, we initiated off-label use of the ileal bile acid transporter (IBAT) inhibitor Elobixibat (Goofice™), later converted to Odevixibat (Bylvay™). After six months of treatment, the patient showed less stool output, increased weight and height, and improved physical energy levels. The child could now pursue higher undergraduate education. In our patient with FIC1 disease, the use of IBAT inhibitors was effective in treating chronic diarrhea and failure to thrive. This approach is novel; further investigations are needed to clarify the exact mode of action in this condition.

Follow-up care of liver-transplanted children includes both monitoring of graft functioning as well as monitoring for associated comorbidities. Monitoring the allograft is comprised of a number of features, ranging from the innate hepatic metabolic and synthetic functions to late surgical complications, such as biliary and vascular obstruction, and to transplantation-specific, immune-mediated complications such as graft fibrosis, graft hepatitis, and acute or chronic rejection. The use of protocol biopsies at regular intervals after pediatric liver transplantation has not yet been universally endorsed by pediatric liver transplantation centers. Biomarkers that could potentially obviate the need for liver biopsy in (pediatric) liver transplantation include both serological markers and imaging techniques. Serological biomarkers for the diagnosis of hepatic graft fibrosis include FibroSure, enhanced liver fibrosis (ELF) score, hyaluronic acid, and the aspartate aminotransferase (AST) platelet ratio index (APRI). Of these, only hyaluronic acid and APRI appear to have some diagnostic value in the context of pediatric liver transplantation (pLTx). With regard to imaging techniques, transient elastography by FibroScan and acoustic radiation force imaging (ARFI) have been explored in children after liver transplantation. FibroScan has some value in longitudinal follow-up, and ARFI can be used to rule in or rule out significant fibrosis. Magnetic resonance imaging (MRI) elastography is a promising technique, but data for children after pLTx are lacking. In summary, serological markers and imaging techniques exist that might guide patient selection for liver biopsy, but the need for liver biopsy to assess graft fibrosis has not yet been obviated.

We aimed to assess executive functioning in children after liver transplantation compared with healthy controls and in relation to real-life school performance using the PedsQLTM Cognitive Functioning Scale (CogPedsQL) and the Childrens’ Color Trail Test (CCTT). One hundred and fifty five children (78f, median age 10.4 (1.2–18.3) years) underwent testing with CogPedsQL and/or CCTT 4.9 (0.1–17.0) years after transplantation. Results were compared to those of 296 healthy children (165f, median age 10.0 (2.0–18.0) years). Liver transplanted children displayed significantly reduced scores for cogPedsQL and CCTT1&2 compared to healthy controls. Overall, school performance was lower in patients compared to controls. In both patients and controls, results of CCTT2 and CogPedsQL correlated strongly with school performance. In contrast to controls, school performance in patients correlated with the level of maternal but not paternal primary education degree (r = −0.21, p = 0.03). None of the patient CCTT or CogPedsQL test results correlated with parental school education. Conclusion: CogPedsQL and CCTT 1&2 were easily applicable in children after OLT and revealed reduced executive functioning compared to controls. Results reflect real life school performance. The association of parental education with school performance is reduced in transplanted children, which possibly indicates the overriding impact of transplant-associated morbidity on cognitive outcomes.