
==== Front
Int J Surg
Int J Surg
JS9
International Journal of Surgery (London, England)
1743-9191
1743-9159
Lippincott Williams & Wilkins Hagerstown, MD

38833360
IJS-D-24-00323
10.1097/JS9.0000000000001671
00031
3
Original Research
Establishment and validation of a predictive model of immune tolerance after pediatric liver transplantation: a multicenter cohort study
Wang Bingran MD adr_wangbingran@163.com

Zhou Aiwei MD azhouaiwei97@163.com

Wu Yichi MD awuyichi_0804@163.com

Pan Qi MD ayxcbxxpanqi@sina.com

Wei Xinzhe MD bweixinzhe1215@163.com

Gao Yunmu MD agaoyunmu@163.com

Xiao Wanglong MD awlshaw@163.com

Jin Jing MD ajingjin@renji.com

Zhou Tao MD ataozhou@renji.com

Luo Yi MD ayiluo@renji.com

Zhan Zhenzhen PhD czhanzz@sjtu.edu.cn

Liu Yongbo PhD ac*liuyongbo@renji.com

Gao Wei MD b*gaowei_tjfch@163.com

Liu Yuan MD ad*liuyuanbird@163.com

Xia Qiang MD, PhD xiaqiang@shsmu.edu.cn
ace*
a Department of Liver Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai
b Department of Pediatric Transplantation, Organ Transplantation Center, Tianjin First Central Hospital, Tianjin
c Shanghai Institute of Transplantation
d Shanghai Immune Therapy Institute
e Shanghai Engineering Research Center of Transplantation and Immunology, Shanghai, People’s Republic of China
* Corresponding author. Address: Renji Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai Engineering Research Center of Transplantation and Immunology, Shanghai Institute of Transplantation, 160 Pujian Road, Shanghai 200127, People’s Republic of China. Tel.: +86 21 683 837 75. E-mail: xiaqiang@shsmu.edu.cn (Q. Xia); Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine, 160 Pujian Road, Shanghai 200127, People’s Republic of China. Tel.: +86 150 212 490 46. E-mail: liuyuanbird@163.com (Yuan Liu); Tianjin First Central Hospital, 24 Kangfu Road, Tianjin, People’s Republic of China. Tel.: +86 22 2362 6600. E-mail: gaowei_tjfch@163.com (W. Gao).; Shanghai Institute of Transplantation, 160 Pujian Road, Shanghai 200127, People’s Republic of China. E-mail: liuyongbo@renji.com (Yongbo Liu).
9 2024
4 6 2024
110 9 56155626
25 1 2024
9 5 2024
Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc/4.0/

Background:

Side-effect of life-long immunosuppressants (IS) administration is a major obstacle for the long-term survival of pediatric liver transplantation (LT) recipients. Immunotolerance is the status that recipients discontinued IS with normal liver function and intrahepatic histology. So far, only a few clinical parameters were identified related with tolerance but failed to accurately discriminate tolerant recipients in clinical practice. Here, the authors aimed to provide a comprehensive view of pre-LT and post-LT risk factors associated with the achievement of tolerance after pediatric LT and established a tolerance predictive nomogram (ITPLT) with high accuracy and specificity.

Methods:

The authors enrolled 2228 pediatric recipients who received LT in Renji Hospital between October 2006 and December 2020. All participants survived over 3 years after transplantation with comprehensive and intact medical history and follow-up data. They were randomly assigned to training and validation cohorts in accordance with a ratio of 1:1. Univariate and multivariable Logistic regression were used to identify clinical factors associated with post-LT immune tolerance and establish a predictive model. The model was further validated in an independent external validation cohort from Tianjin First Central Hospital.

Results:

Among all participants, 6% recipients successfully tapered IS with intact allograft function. The most common reason for IS discontinuity was pneumonia. Univariate analysis identified 15 clinical factors associated with tolerance achievement, including age at LT, follow-up time, preoperative total bilirubin, creatinine, INR, CYP polymorphism, types of transplantation, massive postoperative ascites, episodes of acute rejection, and the severity of EBV and CMV infection. Using multivariable Logistic regression, the authors established the predictive ITPLT model for post-LT tolerance, which included seven easily accessible clinical factors (age at LT, CYP3A5 genotype, types of transplantation, post-LT massive ascites, preoperative INR, creatinine, and total bilirubin levels). Then, the authors visualized the model using nomogram. The c-statistics for predicting tolerance achievement in the training, internal validation, and external validation cohorts were 0.854, 0.787, and 0.746, respectively.

Conclusion:

Multiple pre-LT and post-LT clinical factors affected the process of immune remodeling after pediatric LT. The predictive ITPLT model, composed of seven easily accessible clinical factors, could comprehensively reveal the effect of these clinical parameters on immune remodeling and accurately identify tolerant recipients after pediatric LT. The application of ITPLT could facilitate the individualized IS strategy in the future.

Keywords:

immune remodeling
immune tolerance
immunosuppressants
pediatric liver transplantation
predictive model
OPEN-ACCESSTRUE
SDCT
==== Body
pmcIntroduction

Highlights

Our research provided a landscape of clinical factors affected the achievement of immune tolerance after pediatric liver transplantation (LT), including preoperative, intraoperative, and postoperative factors.

We were the first to reveal that preoperative status had relationship with the achievement of immune tolerance.

Based on large-scale multicenter cohorts, a predictive model of tolerance formation after LT with high accuracy and specificity was established, facilitating the individualized immunosuppression strategy in the future.

Liver transplantation (LT) has become the most efficient treatment for pediatric end-stage liver diseases (ESLD). With the development of surgical techniques and perioperative management, survival rates after pediatric LT had witnessed a giant improvement in the last decade. Over 75–90% recipients could survive 5 years after transplantation. However, the long-term side-effect caused by immunosuppressant (IS) usage, like opportunistic infection, developmental retardation, malignant tumor, metabolic disorder, and renal dysfunction has become the dominant obstacle to the long-term survival rates and life quality of pediatrics recipients.

As an ‘immune privilege’ organ, liver is the most prone solid organ to develop tolerance after transplantation due to its special tolerogenic microenvironment. In the 1990s, University of Pittsburgh first reported tolerant state of six patients after pediatric LT1, which can be defined as totally free from IS with retained graft function and no histological evidence of rejection. Then, several attempts have been made to safely minimize or wean IS after LT and to discover biomarkers of tolerance. Feng et al. 2 recruited 20 pediatric LDLT recipients in the WISP-R trial to conduct operational immune tolerance and 12 recipients (60%) successfully withdrawn IS with normal liver function and histology. Another multicenter trial (iWITH) enrolling 88 long-term follow-up recipients revealed that 37.5% of selected pediatric LT recipients could achieve operational tolerance3. The rates of operational tolerance after LT varied among centers due to the difference of inclusion criteria3. The most challenging part is how to discriminate potential tolerant recipients from the population. It was found that younger age at transplantation and living donor graft were predictive factors of successful IS weaning4, which indicated the remarkable prospective of operational tolerance in pediatric living donor liver transplantation (LDLT) recipients. Recent studies also identified several clinical characteristics that related to operational tolerance in pediatric LT recipients, including age at transplantation, living donors, original diseases, and frequency of rejection5,6. However, due to the limited cohort size, integrated analysis to clinical features in predicting post-LT tolerance is still lacking7–9.

In this retrospective study, we systematically analyzed medical records and follow-up data of 2310 cases of pediatric LT and identified clinical features that were related with spontaneous and proper tolerance after pediatric LT. Then, we established and validated a multivariable predictive model of tolerant state (ITPLT) with high accuracy and specificity, which can be used to direct the selection of possible tolerant patients and facilitate individualized IS strategy.

Methods

Study design and participants

This multicenter cohort analysis focuses on immune tolerance formation and clinical factors associated with tolerance achievement after pediatric LT. This study was conducted in Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine, and Tianjin First Central Hospital. From October 2006 to December 2020, a total of 2310 cases of pediatric LT were included in the study. A self-developed follow-up system was used to record recipients’ backgrounds, medical history, laboratory tests, daily dosage of immunosuppressive (IS) agents, developmental information, complications, and treatments. Among all recipients, 2228 patients from Renji Hospital (Ren Ji cohort) and 82 patients from Tianjin First Central Hospital (Tian Jin cohort) with comprehensive and intact follow-up records were involved in this study. All patients were alive and followed-up for at least 3 years. All patients underwent LT at the age younger than 18-year-old. All recipients received calcineurin inhibitor (CNI, tacrolimus, or cyclosporin) based immunosuppressive strategy after transplantation. Participants or their legal guardians were required to provide written informed consent before inclusion. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (IRB) of Renji Hospital Ethical Committee (KY2022-117-B) and Tianjin First Central Hospital Ethical Committee (2023DZX32). The work has been reported in line with the strengthening the reporting of cohort, cross-sectional, and case–control studies in surgery (STROCSS) criteria10 (Supplemental Digital Content 1, http://links.lww.com/JS9/C693).

Complications after LT

Complications after pediatric LT were defined according to the criteria set by the European Association for the Study of the Liver (EASL)11 and British society of Gastroenterology (BSG)12, and the following treatment strategy were recorded in the follow-up system of Renji Hospital. Patients underwent revision surgery for reasons directly related to the primary procedure were analyzed (unscheduled revision surgery). Massive ascites was considered when recipients suffered more than 200 ml daily abdominal drainage 10 days after transplantation. All recipients received nasal and anal swab tests immediately after return to ICU post-LT and bacteria culture would be conducted for the identification of pathogens when infection was suspected.

Immune status (Immunosuppression protocol)

All recipients received steroid and CNI based immunosuppressive strategy after transplantation. Briefly, recipients received infusion of 10 mg/kg methylprednisolone (MP) during the operation and MP tapered from 5 mg/kg per day to oral intake of prednisone in 1 week after transplantation. Steroid will be withdrawn within 3 months after LT in most recipients. The primary choice of CNI drug after LT is tacrolimus, which would be daily taken by recipients from the second day after LT and the trough serum concentration should be maintained among 8–12 ng/ml in the first 3 months. For recipients suffering low FK trough serum concentration or encountering acute rejection, switching to cyclosporine A or the combination of CNI and mycophenolate mofetil would be applied. The last follow-up time point is 28th December 2023. We recommended protocol liver biopsy every one year for every pediatric recipient after 2 years of LT. All of the patients enrolled in this study accepted our suggestion. The inclusion criteria include: 1) followed-up for more than 1 year after LT with stable liver function and histology; 2) Patients had compliance for follow-up protocol liver biopsy; 3) Patients without de novo autoimmune liver diseases (AIH, PSC, PBC). Patient immune status was defined according to their liver function, liver biopsy histology, and IS dosage. Recipients can be categorized into two groups: 1) control group, patients with normal liver function and normal dosage of IS; 2) tolerant group, including spontaneous and proper tolerance, which was defined as normal liver function and no evidence of rejection under liver biopsy for at least 1 year2 after IS withdrawal or IS low dosage (daily IS dosage lower than 0.015 mg/kg for tacrolimus or 0.2 mg/kg for cyclosporine A, accompanied with trough serum tacrolimus concentration lower than 1 ng/ml or cyclosporine A <40 ng/ml). For tolerant patients, after IS withdrawal, they regularly visited our center every 3 months for liver function test, viral load, and ultrasound evaluation. Liver biopsy was also conducted for them every year. If elevated liver enzyme or any histological abnormality of their allograft was identified, including: significant fibrosis (based on LAFSc13), evidence of rejection (based on Banff Schema14), and loss of bile duct, etc. the CNI will be adjusted to normal dosage, and the patient will be incorporated into ‘control group’.

Development and performance of predictive nomogram for tolerance

All enrolled recipients from Ren Ji cohort were randomly grouped into the training cohort (1114 recipients) and validation cohort (1114 recipients), which was used for the development of immune tolerance predictive ITPLT model. Univariate Logistic regression was carried out based on ‘rms [6.3-0]’ R package. Variables that meet the threshold of P<0.1 or found related with tolerance in previous research were selected for multivariable Logistic regression. The predictive model was established using multivariable Logistic regression based on algorithm from ‘glmnet’ R package. To visualize this model, we applied ‘rms [6.3-0]’ R package to establish a nomogram. We used Antolini’s concordance index (C-index), receiver operating characteristic (ROC) curve, and area under ROC curve (AUC) to estimate the prediction accuracy of this model. The Tian Jin cohort was used as external validation cohort to examine the accuracy of ITPLT model.

Statistical analysis

The ROC were analyzed by R package ‘pROC[1.18.0]’, and visualized by R package ‘ggplot2[3.3.6]’. The Wald test and χ 2 test was applied to estimate the differences for continuous and categorical variables, respectively, in logistic regression analysis. We applied R package ‘rms[6.4.0], ResourceSelection[0.3-5]’ to make univariant logistic regression analysis, further by applying glm function, we established multivariant logistic model, which was visualized as nomogram by R package rms[6.4.0]. Hosmer–Lemeshow goodness of fit test was used to test the fitness of our model. The net benefit of our model was evaluated by R package rmda[1.6], and we applied ggplot2[3.3.6] to plot the decision curve analysis (DCA) curve. All statistical tests were two-tailed. p-value less than 0.05 were considered as statistical significance. All analyses were performed on open-source software R version 4.2.1.

Results

General description of immune status of pediatric LT recipients

In this study, 2228 pediatric LT recipients in the Ren Ji cohort were enrolled and followed-up for at least 3 years (median follow-up time: 5.8 year). Patients enrolled in this study received regular liver biopsy and liver function test. Patients who took low dose IS or discontinued IS with no histological rejection and normal liver function were defined as tolerant status (Fig. 1A). According to the criteria, 139 (6%) recipients was defined as tolerant status. Among them, 108 (5%) cases taking low dosage of IS, and 31 (1%) cases were completely free from IS (Fig. 1B). Since our center had not yet carried out large-scale IS withdrawal clinical trials, there were only four patients achieving immune tolerance through operational tolerance, and the remaining patients were spontaneous tolerant due to various infectious disease, including: pneumonia, diarrhea, virial infection, especially EBV and following post-transplant lymphoproliferative disorders (PTLD). Among them, pneumonia (48%) was the major reason for IS discontinuity, followed by PTLD (15%), and diarrhea (10%) (Fig. 1C). There was no significant difference between the reasons for low IS dosage and complete IS withdrawal (Fig. 1D, E).

Figure 1 Overall immune status after pediatric liver transplantation. A, Representative histological structure from two LT recipients who discontinued IS for more than 1 year with normal liver function; B, The ratio of tolerance, and proper tolerance after pediatric LT in Ren Ji Hospital; C, Reasons for IS discontinuity; D. Reasons for IS low dosage; E. Reasons for complete IS withdrawal. LT, liver transplantation; IS, immunosuppressants.

Perioperative clinical factors that were associated with the development of tolerance using univariant analysis

To explore which clinical features could affect the development of tolerance after pediatric LT, we conducted comprehensive univariant logistic analysis to 37 variables between tolerant group and control group, including follow-up time, preoperative laboratory tests, surgical techniques, short-term and long-term complications. For continuous variables, we also conducted ROC cure analysis to determine the predictive ability of each single variable (Fig S1, Supplemental Digital Content 2, http://links.lww.com/JS9/C694). In agreement with previous reports, young age at LT [adjust odds ratio (OR)=0.773, 95% CI (0.666–0.898), p<0.001] and long post-LT follow-up time [OR=1.016, 95% CI (1.011–1.021), P<0.001] were significantly related with the occurrence of tolerance (Table 1). Similar to age, body weight at LT [OR=0.865, 95% CI (0.800–0.934), P<0.001] was negatively associated with tolerance achievement (Table 1). We then interrogated whether preoperative clinical features could affect the achievement of tolerance after LT. Analysis indicated that tolerant group had increased level of preoperative serum total bilirubin (TB) [OR=1.002, 95% CI (1.001–1.003), P=0.001] (Table 1) and elevated international normalized ratio (INR) [OR=3.236, 95% CI (1.899–5.513), P<0.001] (Table 1). In addition, preoperative creatinine (Cr) level was significantly associated with tolerant status after LT as well [OR=0.962, 95% CI (0.936–0.989), P=0.005] (Table 1). Since preoperative TB, INR, and creatinine levels were important parameters in evaluating the severity of primary liver diseases, these results indicated that the general healthy status of recipients before operation may influence the acceptance of allograft after transplantation.

Table 1 Odds ratio of each clinical parameters to immune tolerance formation after pediatric liver transplantation.

		Univariate analysis	
Characteristics	Total (N)	Odds ratio (95% CI)	P value	
Age	2228	0.773 (0.666–0.898)	< 0.001	
Post-LT（Mo）	2228	1.016 (1.011–1.021)	< 0.001	
Weight (kg)	2228	0.865 (0.800–0.934)	< 0.001	
GRWR (%)	2228	1.078 (0.938–1.239)	0.289	
Blood type	2145			
 AB	191	Reference		
 O	708	0.998 (0.487–2.045)	0.996	
 A	660	1.356 (0.671–2.741)	0.396	
 B	586	1.326 (0.650–2.705)	0.438	
ABO mismatch	2228			
 0	1975	Reference		
 2	140	1.425 (0.767–2.648)	0.262	
 1	113	0.704 (0.282–1.758)	0.452	
Recipient CYP3A5	1991			
 *1/*3	818	Reference		
 *3/*3	1006	2.208 (1.434–3.399)	< 0.001	
 *1/*1	167	1.149 (0.496–2.662)	0.746	
Types of transplants	2228			
 LL	1830	Reference		
 L	88	0.144 (0.020–1.044)	0.055	
 W	284	0.044 (0.006–0.318)	0.002	
 R	22	0.598 (0.080–4.476)	0.616	
 PR	4	4.183 (0.432–40.484)	0.217	
Indication for LT	2228			
 Cholestasis	1929	Reference		
 Metabolic	169	0.079 (0.011–0.570)	0.012	
 Liver failure	16	0.887 (0.116–6.765)	0.908	
 Malignancy	20	0.000 (0.000–Inf)	0.979	
 Others	94	0.289 (0.070–1.187)	0.085	
Kassi	2228			
 0	1227	Reference		
 1	1001	0.988 (0.699–1.395)	0.945	
Preoperative WBC	2103	1.006 (0.985–1.028)	0.569	
Preoperative Hb	2104	0.996 (0.987–1.006)	0.471	
Preoperative PLT	2103	1.000 (0.998–1.001)	0.561	
Preoperative ALB	2107	0.981 (0.957–1.005)	0.126	
Preoperative TB	2108	1.002 (1.001–1.003)	0.001	
Preoperative ALT	2110	1.000 (0.999–1.001)	0.881	
Preoperative AST	2110	1.000 (0.999–1.000)	0.870	
Preoperative PT	2024	0.998 (0.984–1.012)	0.784	
Preoperative INR	1376	1.062 (0.961–1.174)	0.235	
Preoperative INR (2 category)	1376			
 Normal	902	Reference		
 Prolonged	474	3.236 (1.899–5.513)	< 0.001	
Preoperative Cr	2109	0.962 (0.936–0.989)	0.005	
Preoperative CMV load	613	1.000 (1.000–1.000)	0.232	
Preoperative HBV	2049			
 0	1720	Reference		
 1	329	1.436 (0.916–2.251)	0.115	
Follow-up EBV load	1940	1.000 (1.000–1.000)	0.366	
Follow-up EBV load (2 category)	1940			
 Low	1175	Reference		
 High	765	2.842 (1.960–4.122)	< 0.001	
Follow-up CMV load	1490	1.000 (1.000–1.000)	0.808	
Follow-up CMV load (2 category)	1490			
 High	1033	Reference		
 Low	457	0.360 (0.193–0.670)	0.001	
Postoperative massive ascites	901			
 0	596	Reference		
 1	305	0.145 (0.034–0.613)	0.009	
Postoperative Tmax	878	0.530 (0.289–0.972)	0.040	
Application of MMF	2207			
 1	1380	Reference		
 0	827	1.959 (1.386–2.768)	< 0.001	
Sputum culture	512			
 0	375	Reference		
 1	137	0.564 (0.188–1.687)	0.305	
Blood culture	897			
 0	844	Reference		
 1	53	0.582 (0.078–4.366)	0.598	
Ascites culture	474			
 0	423	Reference		
 1	51	1.406 (0.400–4.949)	0.595	
Screening culture	611			
 1	319	Reference		
 0	292	2.254 (0.950–5.347)	0.065	
Acute rejection	2228			
 0	1859	Reference		
 1	369	0.594 (0.343–1.026)	0.062	
Frequency of acute rejection	2228	0.649 (0.433–0.973)	0.037	
Chronic rejection	2228			
 0	2218	Reference		
 1	10	1.676 (0.211–13.327)	0.625	
DILI	2228			
 0	2101	Reference		
 1	127	0.113 (0.016–0.815)	0.031	
Meyloid suppression	901			
 0	887	Reference		
 1	14	2.450 (0.309–19.411)	0.396	
Renal damage	901			
 0	853	Reference		
 1	48	0.651 (0.087–4.894)	0.677	
Urological complications	817			
 0	796	Reference		
 1	21	0.000 (0.000–Inf)	0.987	
DILI, drug-induced liver injury; GRWR, Graft to recipient weight ratio; L, left lobe; LL, left lateral lobe; LT, liver transplantation; MMF, mycophenolate mofetil; PR, partial right lobe; R, right lobe; W, whole liver.

Figure 2 Clinical parameters that had significant different between tolerant recipients and nontolerant recipients: The ROC curve of: A, Follow-up maximum EBV load; B, Follow-up maximum CMV load; C, Follow-up maximum EBV load ruling out recipients tapering IS due to uncontrolled viral infection or PTLD; D, Follow-up maximum EBV load ruling out recipients tapering IS due to uncontrolled viral infection or PTLD. LT, liver transplantation, ROC, receiver operating characteristic, EBV, Epstein–Barr virus, CMV, cytomegalovirus, PTLD, post-transplant lymphoproliferative disorders.

In the Ren Ji cohort, 87.3% pediatric ESLD patients received LDLT while 12.7% (284 cases) received deceased donors liver transplantation (DDLT). DDLT had been recognized as a risk factor for intolerance after pediatric LT5. Our analysis also confirmed that DDLT was negatively associated with the development of tolerance after transplantation [OR=0.044, 95% CI (0.006–0.318), P=0.002] (Table 1). For LDLT recipients, types of allografts (left lower lobe, left lobe, right lobe, and partial right lobe) showed no significant difference between control and tolerant groups.

Postoperative immune status was associated with tolerance attainment

Analysis to post-LT clinical parameters indicated that tolerant group exhibited lower postoperative maximum body temperature [OR=0.530, 95% CI (0.289–0.972), P=0.04] and less incidence of massive ascites [OR=0.145, 95% CI (0.034–0.613), P=0.009] than control one. We found that the frequency of acute rejection was significantly lower in tolerant recipients [OR=0.649, 95% CI (0.433–0.973), P=0.037] (Table 1). The metabolism and bioavailability of CNI (calcineurin inhibitor) were impacted by single nucleotide polymorphism of CYP3A5 alle locus, including *1/*1 (fast metabolism), *1/*3 (intermediate metabolism), and *3/*3 (slow metabolism). Our results revealed that CYP3A5*3/*3 carriers were more prone to achieve tolerance compared with *1/*1 and *1/*3 carriers [OR=2.208, 95% CI (1.434–3.399), P<0.001] (Table 1).

Infection was the most important factor that shaped immune status of LT recipients15. By analyzing the long-term follow-up data between tolerant group and control group, we found that post-LT positive findings in sputum culture, blood culture, and ascites culture had scarce difference between two groups (Table 1), while chronic viral infection was significantly associated with the occurrence of tolerance. Patients with higher maximum viral load of EBV or CMV before IS weaning and patients who had PTLD were more possible to achieve tolerance (Fig. 2A, B). However, high viral load and PTLD were also major reasons for IS withdrawal. To clarify the casual link between tolerance and viral infection, we ruled out patients who discontinued IS due to high viral load and PTLD in the tolerant group, and further analysis showed that viral load was consistently higher in the selected tolerant group (Fig. 2C, D) [OREBV=2.570, 95% CI (1.732–3.813), P<0.001; ORCMV=0.274, 95% CI (0.136–0.553), P<0.001], while the incidence of PTLD had no significant difference between two groups [OR=1.389, 95% CI (0.548–3.519), P=0.488] (Table 2). These results confirmed the involvement of viral infection in pediatric LT tolerance.

Table 2 Odds ratio of chronic viral infection to immune tolerance formation among recipients ruling out those who discontinued IS due to high viral load or PTLD.

		Univariate analysis	
Characteristics	Total (N)	Odds ratio (95% CI)	P value	
Follow-up EBV	1922			
 Low	1171	Reference		
 High	751	2.570 (1.732–3.813)	< 0.001	
Follow-up CMV	1486			
 High	1032	Reference		
 Low	454	0.274 (0.136–0.553)	< 0.001	
PTLD/Lymphoma	2210			
 0	2141	Reference		
 1	69	1.389 (0.548–3.519)	0.488	

Establishment and validation of a multivariable model in predicting tolerance after pediatric LT

To establish a predictive model, we split the Ren Ji cohort into a training and an internal validation cohort in a 1:1 ratio, with 1114 cases, respectively, as shown in Table 3 and Fig S2A (Supplemental Digital Content 2, http://links.lww.com/JS9/C695). In the training stage, we applied univariant logistic regression analysis in the training cohort. Variants that had significant difference between tolerance group and control group (P<0.1) or found related with tolerance in previous research were included in the further multivariable logistic regression and model development. Finally, the model was visualized by nomogram with seven variables, including: age at LT, CYP3A5 polymorphism, graft type, post-LT massive ascites, preoperative INR, creatinine, and total bilirubin levels (Fig. 3A). The model showed favorable discrimination for tolerant recipients in both training cohort and validation cohort with the area under curve (AUC) as 0.854 (95% CI: 0.795–0.913) and 0.787 (95% CI: 0.717–0.856), respectively (Fig. 3B). DCA revealed great net benefit when using this model to select tolerant recipients (Fig S2B, Supplemental Digital Content 2, http://links.lww.com/JS9/C695). Satisfactory calibration was also confirmed by calibration curve of training cohort (Hosmer–Lemeshow test χ 2=5.203, P=0.736), and validation cohort (χ 2=5.637, P=0.688) (Fig S2C, Supplemental Digital Content 2, http://links.lww.com/JS9/C695). To test the predictive performance of ITPLT in different patient groups, we stratified our internal validation cohort based on age at LT and primary disease before LT. We found that ITPLT had more predictive value in patients who received LT at the age older than 1-year-old (Fig S2D, Supplemental Digital Content 2, http://links.lww.com/JS9/C695). Whether patients had cholestatic disease before LT did not affect the predictive effectiveness of ITPLT (Fig S2E, Supplemental Digital Content 2, http://links.lww.com/JS9/C695).

Table 3 General clinical characters in the training and internal validation cohorts.

Characteristics	Training	Validation	P value	
n	1114	1114		
Age, median (IQR)	0.71233 (0.54247–1.5973)	0.70822 (0.54521–1.5733)	0.951	
Post-LT (Mo), median (IQR)	46.483 (28.667–65.625)	45.333 (29.233–64.2)	0.998	
Recipient CYP3A5, n (%)			0.597	
 *1/*3	417 (21%)	401 (20.2%)		
 *3/*3	490 (24.6%)	514 (25.8%)		
 *1/*1	86 (4.3%)	81 (4.1%)		
Types of transplants, n (%)			0.180	
 LL	913 (41%)	914 (41.1%)		
 W	142 (6.4%)	142 (6.4%)		
 R	6 (0.3%)	16 (0.7%)		
 L	48 (2.2%)	40 (1.8%)		
 PR	3 (0.1%)	1 (0%)		
Ascites, n (%)			0.212	
 1	161 (15.9%)	144 (14.3%)		
 0	342 (33.9%)	363 (35.9%)		
Preoperative INR, n (%)			0.480	
 Prolonged	241 (17.5%)	232 (16.9%)		
 Low	441 (32.1%)	460 (33.5%)		
Preoperative Cr (μmol/l), median (IQR)	16 (12.85–20)	16 (13–20)	0.957	
Preoperative TB (μmol/l), median (IQR)	177 (62.65–295)	176 (55–306.5)	0.954	
Weight (kg), median (IQR)	7.6 (6.6–9.8)	7.6 (6.6–10)	0.568	
Outcome, n (%)			0.723	
 Control	1044 (46.9%)	1048 (47%)		
 Tolerant	70 (3.1%)	66 (3%)		
IQR, interquartile range; L, left lobe; LL, left lateral lobe; PR, partial right lobe; R, right lobe; W, whole liver.

Figure 3 Establishment and validation of the predictive nomogram: ITPLT. A, visualized predictive nomogram; B, ROC of the ITPLT model for predicting tolerance formation in the training cohort and internal validation cohort; C, ROC of the predictive model for predicting tolerance formation in the external validation cohort; D, Frequency distribution of tolerance formation in patients with high or low ITPLT score in the internal (left) and external validation cohort (right); E, DCA of the predictive model for predicting tolerance formation in the external validation cohort. ROC, receiver operating characteristic; DCA, decision curve analysis; W, whole liver; R, right lobe; PR, partial right lobe; L, left lobe; LL, left lateral lobe; INR, international normalized ratio; Cr, creatinine; TB, total bilirubin.

Validation of ITPLT in an external independent cohort

To further validate the efficiency of ITPLT model in discriminating tolerant recipients, we carried out an external validation in an independent cohort of 82 pediatric LT cases with follow-up longer than 2 years from Tianjin First Central Hospital (Table 4). In this cohort, 10 recipients (12.2%) reached tolerance during follow-up. The ITPLT model showed satisfactory discrimination for tolerant pediatric LT recipients with AUC of 0.769 (95% CI: 0.612–0.927, Fig. 3C). To test the generalizability of cut-off value derived from the training cohort, we stratified the two validation cohorts into a two-category manner based on the ITPLT cut-off value of −1.224. Patients with ITPLT score higher than −1.224 had higher tolerant rates in both internal and external validation cohort (Fig. 3D). DCA also revealed favorable benefit when applying ITPLT to guide the dosage adjustment of IS in pediatric LT recipients (Fig. 3E).

Table 4 General clinical characters in the external validation cohort.

Characteristics	Control (n=72)	Tolerant (n=10)	
Age, median (IQR)	0.55 (0.46–0.81)	0.51(0.46–0.60)	
Types of Transplantation, n (%)	
 W	3 (3.7%)	0 (0%)	
 LL	65 (79.3%)	10 (12.2%)	
 L	4 (4.9%)	0 (0%)	
Recipient CYP3A5, n (%)	
 *3/*3	30 (36.6%)	6 (7.3%)	
 *1/*3	37 (45.1%)	4 (4.9%)	
 *1/*1	5 (6.1%)	0 (0%)	
Preoperative INR, n (%)	
 Normal	54 (65.9%)	6 (7.3%)	
 Prolonged	18 (22%)	4 (4.9%)	
Preoperative TB (μmol/l), median (IQR)	280.19 (165.12–357)	338.41 (268.07–381.27)	
Preoperative Cr, median (IQR)	15 (13–18.36)	12.91 (12.05–14)	
Postoperative massive ascites, n (%)	
 1	60 (73.2%)	10 (12.2%)	
 0	12 (14.6%)	0 (0%)	
IQR, interquartile range; L, left lobe; LL, left lateral lobe; W, whole liver.

Discussion

Immune tolerant pediatric LT recipients maintained normal liver function without taking any ISs, which could significantly minimize the side-effects of long-term IS agents. However, the selection of possible tolerant recipients still remains empirical. In this large-scale multicenter cohort study, we found that spontaneous tolerance occurred in 6% of pediatric LT recipients, who owning unique perioperative clinical features like preoperative hepatorenal function, type of transplantation, and postoperative status. Based on that, we developed a predictive model (ITPLT) for immune tolerance in pediatric LT recipients and validated in both internal and external cohorts. The ITPLT model containing seven perioperative clinical factors could efficiently distinguish tolerant LT recipients with follow-up time over 2 years with the accuracy over 0.8. This could facilitate the personalized immunosuppressive strategy in pediatric LT recipients and reduce chances of severe infection, metabolic disorders and malignant tumor in long-term follow-up, which significantly improves the life quality of LT recipients.

Among all solid organ, the liver exhibited unique and complex immunoregulatory properties, which rendered liver allografts more resistant to immune assaults after transplantation16. The ratio of spontaneous tolerance in long-term follow-up LT recipients varied between 1 and 15%, while operational tolerance ratio could reach up to 60% in selected pediatric recipients2,3,17–19. Different from adults, pediatric recipients had immature immune system with higher plasticity, increasing the propensity of tolerance achievement after LT20. Retrospective analysis indicated that the time between transplantation and IS withdrawal was the most dominant clinical factor relating with the achievement of tolerance, which was consistent with our study18,21. The age at transplantation, type of transplant and cold ischemia time could also affect the result of IS weaning. Studies to invasive biomarkers of tolerance after LT identified that inflammatory status of portal areas, intrahepatic APC/lymphocyte pairs and C4d score, and iron metabolism-related gene expression signature could be used to discriminate potential tolerant recipients3,7,22,23. However, the requirement of repeated protocol biopsy and the lack of large-scale cohort verification limited their application in clinical practice. A model using noninvasive clinical index to discriminate tolerant recipients is required. Here, the ITPLT model with seven easily accessible clinical factors could efficiently stratify pediatric LT recipients into tolerant and nontolerant groups. External validation in an independent clinical cohort also confirmed good discrimination and predictive value, strengthening its generalizability. All tolerant recipients in this study were confirmed with integrated allograft histology and normal liver function by biopsy and blood examination. Thus, the application of ITPLT could facilitate the individualized IS strategy in long-term follow-up recipients, which could further minimize the side-effects of IS regimen.

The immune status of recipients was profoundly shaped by the primary disease and its severity. Our findings demonstrated for the first time that elevated preoperative TB level and prolonged INR were more frequent in tolerant recipients, while high preoperative serum creatinine level was inversely associated with tolerance attainment. Biliary atresia was the dominant pre-LT diagnosis in our center. Consequently, serum TB reflected the degree of cholestasis and was proportional to serum bile acid metabolite level. Recently, increasing evidence has pointed out that bile acid metabolites had immunosuppressive function through regulating the differentiation of T cells, indicating that there existed immune remodeling process before LT as the result of cholestasis24,25. In addition, coagulation factors have long been considered as initiators of innate immune system by activating complement system and priming immune cells through protease-activated receptors (PARs)26,27. Accordingly, we found that patients with prolonged INR before LT were more possible to attain tolerance. Impaired coagulation factor synthesis under fulminant liver damage led to increased bleeding propensity, and meanwhile shaped a more tolerogenic host immune environment as the result of insufficient immune cell activation. Moreover, we also revealed the relationship between preoperative creatinine and tolerance achievement. Actually, increased creatinine level indicated the occurrence of kidney injury or hepatorenal syndrome (HRS). Inflammatory response has been considered as a crucial mechanism of HRS pathogenesis28, and impaired renal function will also ignite systemic immune responses29, imposing a responsive phenotype on immune environment. To sum up, tolerant patients had formed a quite unique regulatory immune repertoire before receiving LT, featured with high serum TB, prolonged INR, and low serum creatinine.

The type of transplantation and graft were found related with immune remodeling and affecting the achievement of tolerance5,30,31. Different from living donor, deceased donor experienced a series of metabolic change, cellular injury and inflammatory cascade, resulting in increased graft immunogenicity, including activated complement system, enhanced antigen presenting and T cell activation32. In addition, as the result of ischemia-reperfusion injury, liver grafts procured from deceased donors had increased immune cell infiltration and inflammatory cytokine secretion comparing with those from living donors33, and thus, the proinflammatory microenvironment of deceased allograft ignited alloimmune response and hampered the achievement of immune tolerance. Previous study had suggested that enhanced T cell priming resulted from the crosstalk between innate and adaptive immune system in deceased liver graft promoted allogenic recognition and acute rejection34. In agreement with previous findings, our study also found that LDLT recipients were more likely to develop tolerance in long-term follow-up, which confirmed the tolerogenic effect of living donor allograft.

Postoperative complications could also affect the process of immune remodeling after transplantation and the development of tolerance. As revealed by our univariant analysis, the episodes of acute rejection were negatively related with the achievement of tolerance, which was consistent with previous studies18,35. Meanwhile, massive ascites after transplantation was firstly found as an independent predictor of immune tolerance by our study. Reasons for postoperative massive ascites include abdominal infection, portal vein or hepatic vein obstruction, hypoalbuminemia, small for size syndrome, chyle leakage, and so on36–38. Analysis to ascites fluid from decompensated cirrhotic patients revealed the enrichment of mucosal-associated invariant T (MAIT) cells, with the decrease of peripheral MAIT39. As MAIT cells in the liver exhibited tissue residency characteristics by expression CD69, CD103, and CD49a, it played a key role in mediating antibacterial defense and alloreactive response39–42. Relocation of intrahepatic MAIT cells from allograft to ascites may exist in the process of postoperative immune remodeling and ameliorate the intensiveness of immune response to allograft, which contributes to the development of tolerance43. Meanwhile, NK (natural killer) cells from uninfected ascites expressed increased inhibitory receptor NKG2A and decreased activating receptor NKG2D, which may also function as regulatory immune cells during the development of tolerance44. Consistently, monocyte-like dendritic cells in ascites from patients with gastric cancer also exhibited reduced antigen-presenting capacity45. Since refractory ascites could contribute to higher mortality after transplantation, detailed analysis to the ecosystem of ascites in LT recipients and illumination to the role of diverse peritoneal immune cells in the development of tolerance are required.

Chronic virus infection plays a key role in shaping the immune status of LT recipients and significantly affects the development of tolerance. Studies to adult LT recipients had revealed that postoperative persistent HCV infection could facilitate the achievement of tolerance, which was associated with exhausted circulating CD8+ T cells46,47. In pediatric LT recipient, EBV and CMV were most common infected pathogens in long-term follow-up48,49. Retrospective analysis to tolerant pediatric LT recipients indicated that recipients suffering from EBV-related PTLD exhibited higher proportion of successful IS weaning than recipients in other circumstances50,51. It has been extensively studied that chronic viral infection drives immune energy, senescence, and exhaustion. Unlike in healthy individuals, where dendritic cells (DC) loaded with MHC class I EBV peptides preferentially activate cytotoxic CD8+ T cells, DC generated from transplant patients primarily activate EBV-specific CD8+ T cells producing both IFN-γ and IL-10, with up-regulation of FoxP3, which generated ‘regulatory-like’ CD8+ T cells52. Furthermore, the increased ratio of peripheral CD27-CD28-CD8+ T cells in EBV infected patients indicates the skewing to senescence of cytotoxic T cells, which could reduce the incidence of rejection53. Studies to the postoperative CMV infection found that CMV infection before transplantation could induce terminally differentiated T cells and alleviate acute rejection after LT54. The expansion of CD244+CD8+ T cells in chronic CMV infected recipients exhibited lower proliferative responses to alloantigen, which further contributed to the development of immune tolerance55. Therefore, the relationship and underlying mechanism of chronic viral infection and immune reshaping in LT recipients required more intensive study in the future.

A limitation of this study is that we did not carry out large-scale IS withdrawal clinical trial and not all of post-LT recipients underwent IS weaning test. Consequently, there existed ‘emerging tolerant recipients’ in patients who received high dosage IS and these patients were classified into control group. Second, since immune remodeling is a dynamic process and continues after IS weaning, we did not analyze the dynamic changes of immune status after IS withdrawal. Previous reports had indicated that tolerant recipients for more than 10-year IS discontinuation could suffer chronic rejection, even with normal liver function56. Therefore, more effort should be made to reveal the dynamic changes of immune status after IS withdrawal in the future. Finally, the nomogram in our study was established and validated in a Chinese population. To expand its generalizability, validation in populations with different ethnic backgrounds is necessary. In the past 10 years, IS withdrawal trails for White and Black race were carried out in several skillful LT centers, including: Pittsburgh (US)57, UCSF (US)3,58, and Barcelona (EU)7, etc. However, cohort size is restricted due to the lack of easily accessible screening criteria for possible tolerant recipients. Our ITPLT model predicted tolerance only based on clinical parameters, which can serve as an effective screening tool for IS withdrawal candidates. In the future, we suggest the routine application of our model before IS withdrawal trail, not only guaranteed the safety of IS withdrawal trail and expanded the cohort size, but further extended the validation our model to broader and ethnically diverse populations.

In summary, this large-scale multicenter retrospective study identified clinical parameters related with immune tolerance after pediatric LT by analyzing comprehensive follow-up data of more than 2000 recipients. Age at LT, types of transplantation, CYP3A5 polymorphism, pre-LT Cr, TB, INR, and post-LT massive ascites were key predictors for tolerance development. Our predictive ITPLT model integrated above clinical factors and provided a useful tool for the prediction of tolerance, which could further guide individualized IS strategy in pediatric LT recipients.

Ethical approval

The ethical approval of this research has been given by the Institutional Review Board (IRB) of Renji Hospital Ethical Committee (KY2022-117-B) and Tianjin First Central Hospital Ethical Committee (2023DZX32).

Consent

Participants or their legal guardians were required to provide written informed consent before inclusion. The study was conducted according to the guidelines of the Declaration of Helsinki.

Source of funding

This study was funded by the National Natural Science Foundation of China (82000586, 82241221), Innovative research team of high-level local universities in Shanghai (SHSMUZLCX20211600), Science and Technology Innovation Plan of Shanghai Science and Technology Commission and Shanghai Immune Therapy Institute (21410750400). All of these sponsors had no involvement in the writing of the manuscript, or in the decision to submit the manuscript for publication.

Author contribution

Y.L., Q.P., B.W., A.Z., J.J., X.W., and Y.L.: conduct data collection and primary analysis; Y.L., B.W., C.C., and Y.L.: conduct deep data analysis and article draft; T.Z., Z.Z., and Q.X.: conduct most of the LT operations and are responsible for long-term follow-up management; W.G., Y.L., and Q.X.: participated in article revision and supervision.

Conflicts of interest disclosure

The authors declare no conflicts of interest.

Research registration unique identifying number (UIN)

This research was registered in ‘ClinicalTrails.gov Protocol Registration and Results System (PRS)’. Our registration ID is NCT05501301, which is public accessible through the following hyperlink: https://classic.clinicaltrials.gov/ct2/show/NCT05501301?cond=Immune+Tolerance&draw=2&rank=2.

Guarantor

Prof. Qiang Xia, Dr Bingran Wang, and Dr Yuan Liu are the guarantors who accept full responsibility for the work and the conduct of the study, had access to the data, and controlled the decision to publish.

Data availability statement

All of the data in the current research are available from the leading corresponding author: Prof. Qiang Xia on reasonable request. E-mail: xiaqiang@shsmu.edu.cn, Phone: 86 021 68383775, President, Renji Hospital affiliated to SJTUSM, Dean, Shanghai Institute of Transplantation, Dean, Shanghai Engineering Research Center of Transplantation and Immunology, 160# Pujian Road, 200127 Shanghai, People’s Republic of China.

Data availability statement

Data will be shared with bona fide researchers who submit a research proposal approved by the independent review board. Individual recipient data will be shared in data sets in a de-identified and anonymized format. Any researchers requiring original data could contact corresponding author.

Provenance and peer review

Not commissioned, externally peer-reviewed.

Supplementary Material

Acknowledgements

The authors are grateful to Figdraw for figure drawing support.

Bingran Wang, Aiwei Zhou, Yichi Wu, and Qi Pan contribute equally to this article.

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Supplemental Digital Content is available for this article. Direct URL citations are provided in the HTML and PDF versions of this article on the journal’s website, www.lww.com/international-journal-of-surgery.

Published online 4 June 2024
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References

1 Tzakis AG Reyes J Zeevi A . Early tolerance in pediatric liver allograft recipients. J Pediatr Surg 1994;29 :754–756.8078013
2 Feng S Ekong UD Lobritto SJ . Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants. Jama 2012;307 :283–293.22253395
3 Feng S Bucuvalas JC Mazariegos GV . Efficacy and safety of immunosuppression withdrawal in pediatric liver transplant recipients: moving toward personalized management. Hepatology 2021;73 :1985–2004.32786149
4 Montano-Loza AJ Rodríguez-Perálvarez ML Pageaux GP . Liver transplantation immunology: immunosuppression, rejection, and immunomodulation. J Hepatol 2023;78 :1199–1215.37208106
5 Tang Y Chen J Chen B . Clinical characteristics of immune tolerance after pediatric liver transplantation. BMC Surg 2022;22 :102.35305597
6 Shamsaeefar A Kazemi K Nikoupour H . Prope tolerance after pediatric liver transplantation: experience at Shiraz Organ Transplant Center. Transpl Immunol 2023;78 :101827.37003498
7 Bohne F Martínez-Llordella M Lozano JJ . Intra-graft expression of genes involved in iron homeostasis predicts the development of operational tolerance in human liver transplantation. J Clin Invest 2012;122 :368–382.22156196
8 Thomson AW Vionnet J Sanchez-Fueyo A . Understanding, predicting and achieving liver transplant tolerance: from bench to bedside. Nat Rev Gastroenterol Hepatol 2020;17 :719–739.32759983
9 Li L Wozniak LJ Rodder S . A common peripheral blood gene set for diagnosis of operational tolerance in pediatric and adult liver transplantation. Am J Transplant 2012;12 :1218–1228.22300520
10 Mathew G Agha R Albrecht J . STROCSS 2021: strengthening the reporting of cohort, cross-sectional and case-control studies in surgery. Int J Surg 2021;96 :106165.34774726
11 European Association for the Study of the Liver . Electronic address eee. EASL clinical practice guidelines: liver transplantation. J Hepatol 2016;64 :433–485.26597456
12 Millson C Considine A Cramp ME . Adult liver transplantation: UK clinical guideline - part 2: surgery and post-operation. Frontline Gastroenterol 2020;11 :385–396.32879722
13 Venturi C Sempoux C Bueno J . Novel histologic scoring system for long-term allograft fibrosis after liver transplantation in children. Am J Transplant 2012;12 :2986–2996.22882699
14 Anthony JD Kenneth PB Amar PD . Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 1997;25 :658–663.9049215
15 Sanchez-Fueyo A Markmann JF . Immune exhaustion and transplantation. Am J Transplant 2016;16 :1953–1957.26729653
16 Heymann F Tacke F . Immunology in the liver–from homeostasis to disease. Nature Rev Gastroenterol Hepatol 2016;13 :88–110.26758786
17 Feng S . Spontaneous and induced tolerance for liver transplant recipients. Curr Opin Organ Transplant 2016;21 :53–58.26709575
18 de la Garza RG Sarobe P Merino J . Trial of complete weaning from immunosuppression for liver transplant recipients: factors predictive of tolerance. Liver Transpl 2013;19 :937–944.23784747
19 Miloh T Barton A Wheeler J . Immunosuppression in pediatric liver transplant recipients: unique aspects. Liver Transplant 2017;23 :244–256.
20 Sánchez-Fueyo A . Hot-topic debate on tolerance: immunosuppression withdrawal. Liver Transpl 2011;17 (Suppl 3 ):S69–S73.21850680
21 Appenzeller-Herzog C Hartleif S Vionnet J . Clinical parameters and biomarkers predicting spontaneous operational tolerance after liver transplantation: a scoping review. Am J Transplant 2021;21 :3312–3323.33783969
22 Vionnet J Miquel R Abraldes JG . Non-invasive alloimmune risk stratification of long-term liver transplant recipients. J Hepatol 2021;75 :1409–1419.34437910
23 Wood-Trageser MA Lesniak D Gambella A . Next-generation pathology detection of T cell-antigen-presenting cell immune synapses in human liver allografts. Hepatology 2023;77 :355–366.35819312
24 Hang S Paik D Yao L . Bile acid metabolites control T(H)17 and T(reg) cell differentiation. Nature 2019;576 :143–148.31776512
25 Fuchs CD Trauner M . Role of bile acids and their receptors in gastrointestinal and hepatic pathophysiology. Nat Rev Gastroenterol Hepatol 2022;19 :432–450.35165436
26 Stallone G Pontrelli P Rascio F . Coagulation and fibrinolysis in kidney graft rejection. Front Immunol 2020;11 :1807.32983089
27 de Bont CM Boelens WC Pruijn GJM . NETosis, complement, and coagulation: a triangular relationship. Cell Mol Immunol 2019;16 :19–27.29572545
28 Bernardi M Moreau R Angeli P . Mechanisms of decompensation and organ failure in cirrhosis: From peripheral arterial vasodilation to systemic inflammation hypothesis. J Hepatol 2015;63 :1272–1284.26192220
29 Rabb H Griffin MD McKay DB . Inflammation in AKI: current understanding, key questions, and knowledge gaps. J Am Soc Nephrol 2016;27 :371–379.26561643
30 Kim MH Akbari O Genyk Y . Immunologic benefit of maternal donors in pediatric living donor liver transplantation. Pediatr Transplant 2019;23 :e13560.31402535
31 Adams DH Sanchez-Fueyo A Samuel D . From immunosuppression to tolerance. J Hepatol 2015;62 (1 Suppl ):S170–S185.25920086
32 Hann A Osei-Bordom DC Neil DAH . The human immune response to cadaveric and living donor liver allografts. Front Immunol 2020;11 :1227.32655558
33 Guo Y Mei Z Li D . Ischemia reperfusion injury facilitates lung allograft acceptance through IL-33-mediated activation of donor-derived IL-5 producing group 2 innate lymphoid cells. Am J Transplant 2022;22 :1963–1975.35510760
34 Martins PN Chandraker A Tullius SG . Modifying graft immunogenicity and immune response prior to transplantation: potential clinical applications of donor and graft treatment. Transpl Int 2006;19 :351–359.16623870
35 Sanchez-Fueyo A Strom TB . Immunologic basis of graft rejection and tolerance following transplantation of liver or other solid organs. Gastroenterology 2011;140 :51–64.21073873
36 Al-Zoubi M Alarabiyat M Hann A . Management of ascites following deceased donor liver transplantation: a case series. Transplant Direct 2022;8 :e1350.35923811
37 Bloom PP Gilbert T Santos-Parker K . The incidence and natural history of ascites after liver transplantation. Hepatol Commun 2023;7 :e0158.37219847
38 Matsudaira S Ishizaki Y Yoshimoto J . Risk factors for intractable ascites after adult-to-adult living donor liver transplantation using left lobe. Transplant Direct 2017;3 :e138.28361122
39 Niehaus CE Strunz B Cornillet M . MAIT cells are enriched and highly functional in ascites of patients with decompensated liver cirrhosis. Hepatology 2020;72 :1378–1393.32012321
40 Jeffery HC van Wilgenburg B Kurioka A . Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1. J Hepatol 2016;64 :1118–1127.26743076
41 Lett MJ Mehta H Keogh A . Stimulatory MAIT cell antigens reach the circulation and are efficiently metabolised and presented by human liver cells. Gut 2022;71 :2526–2538.35058274
42 Hengst J Strunz B Deterding K . Nonreversible MAIT cell-dysfunction in chronic hepatitis C virus infection despite successful interferon-free therapy. Eur J Immunol 2016;46 :2204–2210.27296288
43 Sattler A Thiel LG Ruhm AH . Mucosal associated invariant T cells are differentially impaired in tolerant and immunosuppressed liver transplant recipients. Am J Transplant 2021;21 :87–102.32515136
44 Lutz P Jeffery HC Jones N . NK cells in ascites from liver disease patients display a particular phenotype and take part in antibacterial immune response. Front Immunol 2019;10 :1838.31440239
45 Huang XZ Pang MJ Li JY . Single-cell sequencing of ascites fluid illustrates heterogeneity and therapy-induced evolution during gastric cancer peritoneal metastasis. Nature Comm 2023;14 :822.
46 Bohne F Londono MC Benitez C . HCV-induced immune responses influence the development of operational tolerance after liver transplantation in humans. Sci Translat Med 2014;6 :242ra281.
47 Martinez-Llordella M Lozano JJ Puig-Pey I . Using transcriptional profiling to develop a diagnostic test of operational tolerance in liver transplant recipients. J Clin Invest 2008;118 :2845–2857.18654667
48 Yamada M Fukuda A Ogura M . Early detection of Epstein-Barr virus as a risk factor for chronic high Epstein-Barr viral load carriage at a living-donor-dominant pediatric liver transplantation center. Transplantation 2023;107 :1322–1329.36476718
49 Chanburanavah N Boonsathorn S Apiwattanakul N . Risk factors of cytomegalovirus infection after pediatric liver transplantation and effectiveness of preemptive therapy. Transplant Infect Dis 2023;25 :e14057.
50 Talisetti A Hurwitz M Sarwal M . Analysis of clinical variables associated with tolerance in pediatric liver transplant recipients. Pediatr Transplant 2010;14 :976–979.21108705
51 Lerut J Sanchez-Fueyo A . An appraisal of tolerance in liver transplantation. Am J Transplant 2006;6 :1774–1780.16889539
52 Popescu I Macedo C Abu-Elmagd K . EBV-specific CD8+ T cell reactivation in transplant patients results in expansion of CD8+ type-1 regulatory T cells. Am J Transplant 2007;7 :1215–1223.17331111
53 Deng Y Chatterjee B Zens K . CD27 is required for protective lytic EBV antigen-specific CD8+ T-cell expansion. Blood 2021;137 :3225–3236.33827115
54 Shi XL de Mare-Bredemeijer EL Tapirdamaz Ö . CMV primary infection is associated with donor-specific T cell hyporesponsiveness and fewer late acute rejections after liver transplantation. Am J Transplant 2015;15 :2431–2442.25943855
55 de Mare-Bredemeijer EL Shi XL Mancham S . Cytomegalovirus-induced expression of CD244 after liver transplantation is associated with CD8+ T cell hyporesponsiveness to alloantigen. J Immunol 2015;195 :1838–1848.26170387
56 Yoshitomi M Koshiba T Haga H . Requirement of protocol biopsy before and after complete cessation of immunosuppression after liver transplantation. Transplantation 2009;87 :606–614.19307800
57 Levitsky J Burrell BE Kanaparthi S . Immunosuppression withdrawal in liver transplant recipients on sirolimus. Hepatology 2020;72 :569–583.31721246
58 Feng S Demetris AJ Spain KM . Five-year histological and serological follow-up of operationally tolerant pediatric liver transplant recipients enrolled in WISP-R. Hepatology 2017;65 :647–660.27302659
