Incomplete Immune Tolerance in Pediatric ABO-Incompatible Liver Transplantation: Insights From Donor-Specific Antibody Titers

Introduction

ABO-incompatible liver transplantation has become an essential treatment option for pediatric patients who lack suitable ABO-compatible donors. According to published data, the cumulative survival rates of both recipients and grafts in pediatric ABO-incompatible liver transplantation are comparable to those observed in ABO-compatible liver transplantation [1,2]. Although immunosuppressive therapies have improved clinical outcomes [3], ABO-incompatible liver transplantation presents distinct challenges, particularly the persistence of blood group antibodies (BGAs), especially donor-specific BGAs [4]. The presence of high donor-specific BGA titers after transplantation is correlated with elevated risks of antibody-mediated rejection and graft failure [5,6].

Despite advances in immunosuppressive therapy [7], the mechanisms underlying the persistence of these antibodies in the pediatric population remain poorly understood. This knowledge gap is particularly important because pediatric patients possess an immature immune system that may permit the development of unique immune tolerance mechanisms [8].

The hypothesis that incomplete immune tolerance contributes to the persistence of donor-specific BGAs in pediatric recipients forms the basis of the present study. Previous research has not adequately examined how preoperative immunosuppression, including monoclonal antibody therapy, affects the persistence of these antibodies after transplantation. Clarification regarding these mechanisms may help to refine immunosuppressive strategies for reducing graft failure risk while minimizing the adverse effects of long-term therapy.

This study aimed to evaluate the persistence of donor-specific BGAs in pediatric ABO-incompatible living donor liver transplantation (ABOi-LDLT) recipients, with particular focus on the role of preoperative immunosuppressive therapy, such as rituximab (i.e., an anti-CD20 monoclonal antibody). A clearer understanding of the immunologic mechanisms underlying antibody persistence may help guide the development of optimized immunosuppressive strategies to improve transplant outcomes and long-term graft survival.

Material and Methods

STUDY DESIGN AND PATIENT SELECTION:

A retrospective observational study was conducted involving pediatric patients who underwent ABOi-LDLT between June 2013 and December 2020 at Beijing Friendship Hospital. This study was approved by the Ethical Committee of Beijing Friendship Hospital after rigorous ethical review (No. 2020-P2-119-02). To minimize statistical bias related to blood type variability, only patients with blood type O were included. A formal control group was not established because the primary objective was to examine the dynamics of donor-specific and non-donor-specific BGAs in ABOi-LDLT recipients. All pediatric ABOi-LDLT procedures were conducted in accordance with the Declaration of Helsinki (1964, revised 2013) and approved by the Organ Transplantation Ethics Committee of Beijing Friendship Hospital. Written informed consent for transplantation and inclusion in this study was obtained from the legal guardians of all pediatric patients.

DATA COLLECTION:

Clinical records of all enrolled patients were reviewed to extract epidemiological and clinical data. Variables collected included demographic information, recipient and donor blood types, pre- and post-transplant titers of donor-specific and non-donor-specific BGAs (IgG and IgM), and liver biopsy histopathological findings.

BGA TESTING:

IgM antibody titers were determined using the conventional tube method with serial 2-fold dilutions, and results were interpreted by experienced laboratory technologists. IgG antibody titers were assessed using the microcolumn gel card technique performed on the Grifols WADiana automated blood typing system.

LIVER BIOPSY:

Ultrasound-guided percutaneous liver biopsy was performed either as a protocol biopsy or an event-driven biopsy. Patients with stable liver function were regularly followed and underwent protocol biopsies annually beginning 1 year after transplantation. Event-driven biopsies were performed when patients exhibited abnormal liver function or when rejection was suspected. Biopsy samples were subjected to routine hematoxylin and eosin staining; they were evaluated by immunohistochemistry to assess complement C4d deposition. All liver biopsies were analyzed by an experienced liver pathologist, using the Banff Schema for liver allograft rejection. C4d positivity was defined as positive staining of vascular endothelial cells in more than 50% of the lumen, including hepatic sinusoidal endothelial cells, portal interstitial microvessels, interlobular veins, and central vein endothelial cells [9].

STATISTICAL ANALYSIS:

Graft and recipient survival rates were analyzed using the Kaplan-Meier method, which is appropriate for estimating survival probabilities over time within a cohort. The log-rank test was utilized to compare survival curves between groups. BGA titer data were log₂-transformed prior to statistical analysis. For comparisons of BGA titers, the chi-square test was conducted to evaluate associations between categorical variables, whereas the paired t-test was performed to assess differences in BGA titers within the same group before and after transplantation. This study did not utilize more sophisticated longitudinal data analysis methods (e.g., linear mixed models) due to the relatively small sample size and because the primary study objective comprised characterization of overall BGA titer trajectory. No imputation methods were used for missing data; instead, complete case analysis was performed for all statistical tests. This approach was justified by the low rate of missing data (<5% of total BGA measurements) and the absence of evidence suggesting systematic bias in missingness. All statistical analyses were conducted using SPSS software (version 18.0). Two-tailed P-values <0.05 were considered statistically significant.

PREOPERATIVE PREPARATION AND IMMUNOSUPPRESSION:

Based on our institution’s standard protocol, patients with high pre-transplant donor-specific BGA titers (IgG or IgM >1: 64) received mycophenolate mofetil 10 mg/kg daily beginning 2 weeks before transplantation; a single intravenous dose of rituximab (375 mg/m²) was administered 1 week before transplantation. Peripheral blood samples were collected to assess BGA titers. Therapeutic plasma exchange was considered when BGA titers greater than 1: 64 persisted after immunosuppressive preconditioning.

INTRAOPERATIVE MANAGEMENT:

During the transplantation procedure, intravenous methylprednisolone (10 mg/kg) and intravenous immunoglobulin (400 mg/kg) were administered to all patients. When transfusion was required, AB group plasma and irradiated, washed red blood cells were used to minimize antigenic stimulation.

POSTOPERATIVE IMMUNOSUPPRESSIVE STRATEGY:

A triple immunosuppressive regimen consisting of a calcineurin inhibitor, mycophenolate mofetil, and corticosteroids was initiated postoperatively for all patients. Additionally, intravenous immunoglobulin was administered at 400 mg/kg/day for 7 consecutive days to further reduce humoral immune responses.

Results

CLINICAL CHARACTERISTICS:

In total, 29 pediatric patients underwent ABOi-LDLT at our center, including 17 boys and 12 girls (Table 1). All recipients had blood type O; 10 received liver grafts from blood type A donors, and 19 received grafts from blood type B donors. Graft types included the left lateral section in 26 patients and the left lobe in 3 patients. The median recipient age at transplantation was 10 months (range, 3–114 months), and the median body weight was 8.0 kg (range, 5–30 kg). The median follow-up duration was 41.9 months (range, 10.6–95.7 months). Within the cohort, 5 patients exhibited persistent donor-specific BGA titers greater than 1: 64 (IgG or IgM) and received standard preoperative immunosuppressive treatment.

COMPARISON OF DONOR-SPECIFIC AND NON-DONOR-SPECIFIC BGAS:

Both IgG and IgM titers of donor-specific BGAs remained consistently low throughout the follow-up period. These titers were significantly lower than those of non-donor-specific BGAs (P<0.001) (Figure 2A, 2B). Donor-specific BGA titers were also compared between the A-O and B-O groups. Although the preoperative IgG level was significantly higher in the A-O group than in the B-O group, no significant differences in donor-specific BGA titers (both IgG and IgM) were observed between the 2 groups at any subsequent time point (Figure 3A, 3B).

LIVER BIOPSY:

Of the 29 patients, 23 underwent liver biopsy. Among these 23 patients, protocol liver biopsies were performed in 18, and event-driven biopsies were performed in 5 due to abnormal liver function during follow-up. Based on the protocol biopsies, C4d deposition in vascular endothelium was detected in 3 patients (patients 20, 27, and 29); these patients were diagnosed with mild antibody-mediated rejection. Immunosuppressive therapy was intensified accordingly. Among the 5 patients who underwent event-driven biopsies, 2 were diagnosed with moderate antibody-mediated rejection involving positive vascular C4d staining (patients 9 and 16), and another 2 were diagnosed with biliary complications involving C4d positivity in bile duct epithelium (patients 19 and 26). These diagnoses resolved after adjustment of the immunosuppressive regimen. The remaining patient was diagnosed with anastomotic biliary stricture and underwent surgical biliary reconstruction (patient 10). Antibody titers were monitored in all patients who developed rejection or biliary complications associated with C4d positivity. Donor-specific BGA titers were 1: 4 or 1: 2 in the rejection group and 1: 4 or 1: 16 in the biliary complications group; no significant increases in antibody levels were noted before or after the onset of these events.

Discussion

HOST FACTORS:

First, the degree of immune system maturation exerts a strong influence. The immune system during the embryonic or neonatal period is not fully developed. Compared with mature immune cells, immature immune cells are more likely to develop immune tolerance. In adults with a mature immune system, the antigen dose required to induce tolerance is approximately 30 times higher than that needed in immature cells, making tolerance induction more difficult. Second, immunosuppressive measures can promote immune tolerance. It is difficult to induce immune tolerance in healthy adults using antigens alone; however, the combined use of radiotherapy, antithymocyte globulin, anti-T/B cell monoclonal antibodies, and calcineurin inhibitors can artificially deplete mature immune and lymphatic systems. This process renders the immune system functionally immature, resembling that of the neonatal period, and thus facilitates tolerance induction (Figure 4).

ANTIGENIC FACTORS:

First, antigen specificity plays a crucial role. Small, soluble, non-polymeric molecules such as serum proteins, polysaccharides, and lipopolysaccharides are more likely to induce immune tolerance. ABO antigens, composed of glycoproteins and glycolipids embedded within cell membranes, also possess soluble properties that may enhance their potential to induce tolerance. Second, antigen dose is an important determinant. Extremely high or low antigen doses can induce high-zone or low-zone tolerance, respectively. The continuous presence of tolerogenic antigens is essential to maintain long-term immune tolerance [18]. Because the body continuously generates new immunologically active cells, persistent antigen exposure can sustain these new cells in a tolerant state. In pediatric ABOi-LDLT recipients, the ongoing low-level expression of blood group antigens within the vascular endothelium and bile duct epithelium of the liver graft contributes to both the induction and maintenance of incomplete immune tolerance [19].

These findings suggest that the distinct immunologic environment of pediatric recipients, in combination with strategically applied immunosuppression, creates optimal conditions for developing incomplete tolerance to donor blood group antigens. This mechanism may explain the relatively favorable outcomes observed in pediatric ABOi-LDLT [20].

Although these findings provide valuable clinical insights, the present study has some limitations. First, as a single-center retrospective analysis, the study design may have introduced selection bias, and the duration of follow-up varied among participants. Second, no mechanistic investigations were conducted to characterize the kinetics of B lymphocytes or the biological phenotypes and functional profiles of immune cells. Future research should aim to track the kinetics and functional characteristics of immune cells over time to better define the immune responses and tolerance mechanisms in pediatric ABOi-LDLT recipients. Despite these limitations, the findings have considerable clinical relevance. The persistence of low donor-specific BGA titers in pediatric ABOi-LDLT recipients suggests the potential for optimizing immunosuppressive strategies to reduce adverse effects while maintaining adequate rejection prevention.

In summary, this study underscores the importance of immune tolerance mechanisms in pediatric ABOi-LDLT and provides a theoretical basis for developing more individualized immunosuppressive strategies in pediatric liver transplantation. Future studies should further investigate the phenomenon of incomplete immune tolerance in other transplant types and its impacts on long-term survival and graft function.

Conclusions

In this study, pediatric recipients of ABOi-LDLT demonstrated persistently low or undetectable titers of donor-specific BGAs, which were significantly lower than the titers of non-donor-specific BGAs. These findings indicate a state of incomplete immune tolerance to donor-specific blood group antigens, potentially influenced by the developmental characteristics of the pediatric immune system and preoperative immunosuppressive therapy with monoclonal antibodies (e.g., rituximab). Although these results provide important insights into immune tolerance mechanisms, the hypothesis of incomplete immune tolerance remains speculative and requires further validation.