Objectives: De novo donor-specific antibodies are recognized mediators of alloimmune injury after kidney transplant, yet their clinical significance in pediatric recipients remains incompletely defined. We evaluated the incidence of de novo donor-specific antibodies and the associated rejection patterns and graft outcomes in a pediatric kidney transplant cohort.
Materials and Methods: This retrospective single-center study included pediatric kidney transplant recipients who underwent transplants between 2009 and June 2024. Donor-specific antibody positivity was determined using Luminex-based assays (mean fluorescence intensity >1000). Clinical, immunologic, and functional outcomes were compared between patients positive for donor-specific antibodies and patients negative for donor-specific antibodies, including rejection phenotype, estimated glomerular filtration rate, graft loss, and duration of follow-up.
Results: Among 78 recipients, 15 recipients (19.2%) developed de novo donor-specific antibodies during a mean follow-up of approximately 4 years. Acute rejection occurred more frequently in patients positive for donor-specific antibodies versus patients negative for donor-specific antibodies (80% vs 32%, respectively; P = .⁰⁵³), predominantly antibody-mediated or mixed phenotypes. Graft loss rates were comparable between groups (13.3% vs 14.2%; P = 1.000). Although not statistically significant, recipients positive for donor-specific antibodies demonstrated numerically lower eGFR values at 1 year and at last follow-up (49.6 ± 22.2 vs 57.1 ± 29.8 mL/min/1.73 m² at last visit, respectively; P = .⁴⁴⁵).
Conclusions: In this pediatric cohort, de novo development of donor-specific antibodies was associated with a higher tendency toward antibody-mediated rejection, without a significant difference in short-term to mid-term graft survival. Continued surveillance of donor-specific antibodies and longer follow-up are warranted to clarify the long-term clinical effect of donor-specific antibodies in pediatric kidney transplants.

Key words : Antibody-mediated rejection, Human leukocyte antigen antibodies, Immunologic monitoring, Kidney transplantation

Introduction
Kidney transplant is the preferred treatment for children with end-stage kidney disease (ESKD), offering improved survival and quality of life. Despite advances in immunosuppression therapy, acute and chronic rejection remain significant causes of graft dysfunction and loss, particularly in the pediatric population.¹ Donor-specific antibodies (DSA), directed against human leukocyte antigens (HLA), are increasingly recognized as major contributors to adverse outcomes following kidney transplants. Donor-specific antibodies can mediate graft injury via humoral mechanisms, leading to antibody-mediated rejection (AMR), chronic allograft nephropathy, and progressive loss of graft function.^2-4 The pathogenic role of DSA is well characterized in adult transplant recipients,⁵ but data in pediatric patients remain limited. Recent pediatric registry analyses report de novo DSA incidence ranging from 10% to 30%, with associations to humoral rejection and early declines in graft function.^6-8 Early detection of DSA may allow timely interventions to mitigate ongoing graft injury, highlighting the importance of routine posttransplant monitoring in children.⁶ In addition, factors such as medication nonadherence, viral infections, and immunologic mismatch have been implicated as triggers for de novo DSA formation, particularly in adolescent recipients.^6,9,10 In this study, we aimed to evaluate the rate and risk factors for de novo DSA development in pediatric kidney transplant recipients at a single center and to investigate its association with acute rejection patterns and graft outcomes. By identifying early immunologic risk markers, we hope to inform strategies to preserve long-term graft function in this population.

Materials and Methods
This retrospective cohort study included pediatric kidney transplant recipients who underwent transplant between January 2009 and June 2024 at a single tertiary care center. Demographic characteristics, etiology of ESKD, type and duration of kidney replacement therapy, dialysis duration prior to transplant, donor type, immunosuppression regimens, rejection episodes, DSA, estimated glomerular filtration rate (eGFR), graft loss status, and follow-up duration were analyzed. Patients aged <18 years at the time of transplant with available data were included in the study. Those with less than 12 months of follow-up were excluded from outcome analyses. All recipients had a negative complement-dependent cytotoxicity crossmatch and no detectable DSA prior to transplant. Induction therapy consisted of the anti-interleukin-2 receptor antagonist basiliximab, administered intravenously in 2 doses on day 0 and day 4 following transplant (10-20 mg according to body weight). Maintenance immunosuppression therapy included tacrolimus or cyclosporine in combination with mycophenolate mofetil or mycophenolic acid and prednisone. All patients received valganciclovir prophylaxis for 3 to 6 months after transplant. Graft function was evaluated using the eGFR, calculated according to the updated Schwartz formula as follows: eGFR (mL/min/1.73 m²) = 0.413 × [height (cm)/serum creatinine (mg/dL)].^11,12 Graft dysfunction was defined as either a reduction in eGFR to below 30 mL/min/1.73 m² or a decrease of ≥50% versus baseline renal function measured at the first month after transplant. Indication biopsies were performed in cases of an increase in serum creatinine ≥20% from baseline when acute rejection or transplant nephropathy was suspected. Rejection episodes were classified as cellular, antibody-mediated (humoral), or mixed according to histopathology findings. During follow-up, DSA testing was performed in patients with positive panel reactive antibody results detected at posttransplant months 1, 3, 6, 9, and 12, and every 6 months thereafter routinely or after a rise in serum creatinine levels. Most detected de novo DSAs were directed against HLA class II antigens. Detection of DSA was performed using Luminex-based assays, and positivity was defined as a mean fluorescence intensity >1000 according to our center. In most studies, a threshold of ≥1400 is applied to define HLA-DSA positivity.¹³ For comparative analyses, patients were categorized into DSA-positive and DSA-negative groups. These groups were compared in terms of demographic and clinical characteristics, including sex, age at transplant, primary diagnosis of ESKD, type and duration of KRT, dialysis duration prior to transplant, and donor type. Clinical outcomes evaluated included acute rejection episodes, eGFR, and graft loss.

Statistical analyses
We used appropriate statistical software for statistical analyses. We presented continuous variables as mean values (±SD) or median values (with IQR), depending on distribution, and categorical variables as frequencies (with percentage). We compared groups by using the Student t test or the Mann-Whitney U test for continuous variables and the chi-square test or the Fisher exact test for categorical variables. P < .⁰⁵ was considered statistically significant.

Results
The study included 78 pediatric kidney transplant recipients. Baseline demographic and clinical characteristics of the study population are summarized in Table 1. Most patients were male, and mean age at transplant was in early adolescence. Congenital anomalies of the kidney and urinary tract (known as CAKUT) constituted the most common cause of ESKD, followed by glomerulopathies. Most patients underwent dialysis prior to transplant, predominantly hemodialysis, whereas a smaller proportion received preemptive transplants. Living donor transplant was more frequent than deceased donor transplant. The median follow-up period extended over several years, and overall graft function at last follow-up reflected moderate renal function. De novo DSA details were detected in 15 patients, corresponding to a DSA positivity rate of 19.2%. The mean time to DSA development was 21 months after transplant. In 8 patients, DSA emerged within the first posttransplant year, with a mean onset time of 4 months. In the remaining 7 patients, DSA developed after the first year, with a mean onset of 40 months. Among DSA-positive patients, AMR was the predominant rejection type. Only 1 patient experienced cellular rejection, which occurred within the first month after transplant. Long-term follow-up >5 years was available for 3 DSA-positive patients. During the study period, graft loss occurred in 2 patients; however, neither case was attributed to rejection. The causes of graft loss were viral infection and treatment nonadherence. The comparison of demographic and clinical characteristics between DSA-positive recipients and DSA-negative recipients is presented in Table 2. No significant differences were shown between the 2 groups with respect to age at transplant, sex distribution, duration of follow-up, primary diagnosis of ESKD, type of kidney replacement therapy, duration of dialysis prior to transplant, or donor type (all P > .⁰⁵). Although CAKUT was more frequent in the DSA-positive group and glomerulopathy appeared more common in the DSA-negative group, these differences did not reach significance. Similarly, the distribution of living and deceased donors was comparable between groups. Clinical outcomes according to DSA status are summarized in Table 3. The overall rejection rate was significantly higher in the DSA-positive group compared with DSA-negative recipients (80% vs. 32%), approaching significance (P = .⁰⁵³). Antibody-mediated (humoral) rejection was more frequent among DSA-positive patients, whereas cellular rejection was more commonly observed in DSA-negative recipients. Posttransplant graft function, assessed by eGFR at 1 year, 5 years, and at last follow-up, did not significantly differ between the 2 groups (all P > .⁰⁵). Similarly, graft loss rates were comparable between DSA-positive patients and DSA-negative patients, with no significant difference observed.

Discussion
In this single-center retrospective study, we evaluated the incidence of de novo DSA and the associated with rejection patterns and graft outcomes in pediatric kidney transplant recipients. De novo DSA developed in 19.2% of patients during follow-up, a rate consistent with previously reported pediatric cohorts.^6,8,14,15 Positivity for DSA was associated with a higher frequency of acute rejection, predominantly antibody-mediated or mixed phenotypes.^6,14,16,17 Although graft loss was not observed among DSA-positive recipients during the follow-up period, these patients exhibited lower eGFR values at both the first year after transplant and at last assessment, suggesting subclinical and progressive allograft injury.^2,3 Together, these findings reinforce growing evidence that de novo DSA represents a clinically meaningful immunologic event in pediatric kidney transplants. An additional important observation was the temporal variability of antibody emergence. More than half of DSA-positive patients developed antibodies within the first year after transplant, whereas the remainder developed antibodies later during follow-up. Early DSA formation has been associated with insufficient immunosuppression and medication nonadherence, particularly relevant in adolescent recipients, whereas late onset DSA is more often linked to chronic alloimmune stimulation and class II-dominant immune responses.^3,6,18 The predominance of AMR in our cohort supports the pathogenic role of DSA as an active driver of graft injury rather than merely a serology marker.¹⁹ Despite the higher rejection rate, graft loss did not significantly differ between groups. This discrepancy may reflect the relatively limited follow-up duration, the effectiveness of prompt rejection treatment, and the generally favorable short-term and mid-term graft survival observed in pediatric recipients. Importantly, the decline in renal function among DSA-positive patients suggests that functional impairment may precede overt graft failure.¹⁸ Thus, absence of early graft loss should not be interpreted as absence of immunologic injury, but rather as a potential window for therapeutic intervention. In our cohort, traditional demographic and transplant-related variables such as recipient age, dialysis duration, and donor type were not independently associated with de novo DSA development. Previous studies have highlighted the importance of immunologic mismatch, medication adherence, and viral infections to promote antibody formation.^6,20 The lack of significant associations in our analysis likely reflects the multifactorial nature of DSA development as well as limitations inherent to retrospective registry-based data, particularly regarding adherence assessment and detailed immunologic risk stratification. Collectively, our findings suggest that de novo DSA development reflects an ongoing and dynamic alloimmune response in pediatric kidney transplants. Given its association with AMR and functional decline, structured posttransplant DSA surveillance may facilitate early identification of at-risk recipients.^6,19 Integration of immunologic monitoring with functional assessment could improve long-term graft preservation in this population, where graft longevity is of critical importance. This study has several limitations. Its retrospective, single-center design and relatively small sample size limit generalizability and statistical power. Differences in follow-up duration between groups may have influenced outcome comparisons, and detailed data on medication adherence and viral infections were not consistently available. Nevertheless, the study provides clinically relevant insight into the effect of de novo DSA on rejection patterns and graft function in pediatric kidney transplant recipients. In conclusion, de novo DSA development was associated with increased rejection and inferior renal function in our pediatric cohort, although graft loss was not significantly different during follow-up. These findings support the clinical relevance of systematic DSA monitoring and early immunologic risk assessment to preserve long-term graft function in pediatric kidney transplants.