Complement-Binding Donor-Specific Anti-HLA Antibodies: Biomarker for Immunologic Risk Stratification in Pediatric Kidney Transplantation Recipients

Antibody-mediated rejection is a common cause of early kidney allograft loss but the specifics of antibody measurement, therapies and endpoints have not been universally defined. In this retrospective study, we assessed the performance of risk stratification using systematic donor-specific antibody (DSA) monitoring. Included in the study were children who underwent kidney transplantation between January 1, 2010 and March 1, 2018 at Stanford, with at least 12-months follow-up. A total of 233 patients were included with a mean follow-up time of 45 (range, 9–108) months. Median age at transplant was 12.3 years, 46.8% were female, and 76% had a deceased donor transplant. Fifty-two (22%) formed C1q-binding de novo donor-specific antibodies (C1q-dnDSA). After a standardized augmented immunosuppressive protocol was implemented, C1q-dnDSA disappeared in 31 (58.5%). Graft failure occurred in 16 patients at a median of 54 (range, 5–83) months, of whom 14 formed dnDSA. The 14 patients who lost their graft due to rejection, all had persistent C1q-dnDSA. C1q-binding status improved the individual risk assessment, with persistent; C1q binding yielding the strongest independent association of graft failure (hazard ratio, 45.5; 95% confidence interval, 11.7–177.4). C1q-dnDSA is more useful than standard dnDSA as a noninvasive biomarker for identifying patients at the highest risk of graft failure.


INTRODUCTION
Kidney allograft survival is typically between 10 and 15 years, reflecting an area of unmet need in pediatric kidney transplantation since these children are destined to require more than one transplant during their lifetime. The most common cause of graft failure is induced alloimmune response and rejection (1). Antibodies formed against polymorphic human leukocyte antigen (HLA) molecules on the donor endothelium are central to the pathogenesis of antibody-mediated rejection (ABMR). In kidney allograft recipients, the presence of donorspecific antibodies (DSA) both before and after transplantation correlates with poor graft survival (2)(3)(4)(5). DSA that activate the complement system appear to cause particularly severe injury to the allograft, and new complement blood tests (C4d, C1q, C3d) have been developed as tools to stratify the immunologic risk (6)(7)(8)(9). Studies link complement-binding de novo DSA (C1q-dnDSA) to inferior graft outcomes (3,4,(10)(11)(12)(13)(14)(15)(16). The value of using complement-activating antibody testing in clinical care is currently a subject of substantial debate (6,(17)(18)(19)(20). In a multicenter study comprising more than 1,000 DSA-positive kidney allograft recipients, Loupy et al. (3) reported that 24% of detected antibodies bound to C1q. More importantly, the glomerular filtration rate (GFR) was lower at 1-year posttransplant, and 5-years graft survival was significantly worse among patients whose DSA-bound C1q as compared to those with DSA that did not demonstrate C1q binding. In a retrospective cohort study of 193 patients at Lucile Packard Children's Hospital (LPCH) who received a kidney transplant in 2000-2008, a C1q solid-phase assay was employed in parallel to the standard immunoglobulin G (IgG) assay to identify C1q-dnDSA (12). Patients with C1q-dnDSA (n 15) were almost 6 times more likely to suffer graft failure than those without such antibodies. In fact, 47% of the C1q-dnDSA-positive patients suffered premature graft failure at a mean of 33.0 ± 17 months posttransplant. A shortcoming of that study was that C1q-dnDSA were analyzed retrospectively in a small cohort using a single blood sample from each case.
A recent study of adult kidney allograft recipients with ABMR showed that persistence of C1q-dnDSA after augmented immunosuppressive treatment was an independent determinant of allograft loss (21). We wanted to better characterize the immunologic risk and tailor the treatment to reduce the risk of allograft complications, such as infection, in our pediatric kidney transplant population (22). After reporting that C1q-dnDSA are associated with increased risk of premature graft failure in this group of patients (12), we implemented a DSA monitoring and treatment protocol after transplant, using the DSA characteristics and kinetics as a biomarker in an individualized risk stratification for graft failure. Intensified immunosuppressive treatment was guided by the C1q kinetics. The purpose of this study was to assess the performance of our individualized risk stratification using systematic DSA monitoring, including their complement-binding capacity, in addition to the standard approach. We hypothesized that the presence, and in particular persistence, of C1q is a key biomarker for risk stratification.

Ethical Approval
The present study was approved by the Stanford University Institutional Review Board (49,338

Kidney Allograft Biopsies, Definition of Antibody-Mediated Rejection and Immunosuppressive Protocols
During the period of the study, protocol biopsies were performed at 6, 12, and 24 months after kidney transplantation and as clinically indicated (i.e., if graft dysfunction and/or dnDSA • Treatment initiated if complement-binding donor-specific antibodies with MFI ≥1,000 and/or if positive C4d staining on biopsy • Concurrent cellular rejection treated with corticosteroid if Banff borderline or 1a. Thymoglobulin considered for Banff 1b or 2 • Management -Tacrolimus target trough levels increased to 7-10 ng/ml and MMF to 4-6 mcg/ml for 2-3 months -Intravenous immunoglobulin (IVIG) 2 g/kg, administered initially and then every month for minimum of 3 months. Discontinued when C1q--dnDSA MFI <1,000 (defined as disappeared) -DSA levels are obtained prior to IVIG infusion (data used in study) and immediately after infusion is complete -If C1q-dnDSA persist, can consider continuing IVIG monthly -If the C1q-dnDSA do not respond to IVIG at all after 8 months, discontinuation of treatment should be considered. If MFI levels are decreasing, treatment is continued until disappearance -Rituximab 500 mg/m 2 administered within 2 weeks of first dose of IVIG.
-Plasmapheresis added if severe graft dysfunction -Bortezomib if graft dysfunction is resistant to IVIG and/or plasmapheresis • After C1q-binding dnDSA detection, DSA levels are monitored at a minimum of every 3 months until eliminated. If they do not disappear, DSA levels are followed at least every 3 months for 12 months. C1q-binding dnDSA then is monitored based on risk, but at least every 12 months

Transplant International | Published by Frontiers
March 2022 | Volume 35 | Article 10158 appearance). To ensure consistency, transplant biopsy specimens were gathered and scored for analysis by an expert in kidney transplant pathology according to the consensus rules of the most recent international Banff Classification criteria (27). Baseline immunosuppression and treatment protocol for ABMR after detection of C1q-dnDSA, with or without biopsy, remained unchanged throughout the study period. With the initiation of standardized DSA monitoring, including complement-biding capacity, we implemented an augmented immunosuppressive treatment protocol for all patients with C1q-dnDSA MFI >1,000 shown in Table 1. This protocol was unchanged throughout the study period. Patients who formed standard dnDSA only were not treated.

Definition of Antibody Status, Risk Factors of C1q--dnDSA Formation and Outcomes
Donor-specific antibody status was considered as the exposure. C1q-dnDSA was a time-fixed variable of C1q status, categorized as negative, eliminated or persistent. If C1q-dnDSA disappeared after detection at some point and the MFI was <1,000 for the remainder of the study, the C1q-dnDSA were considered "eliminated." In those who continued to have C1q-dnDSA throughout the study or at the time of allograft failure, the C1q-dnDSA were categorized as "persistent." Standard dnDSA were either negative, present without ever binding complement or detected together with C1q-dnDSA. The persistence and elimination of standard dnDSA was followed in patients with C1q-dnDSA and defined in the same manner as above.
Decreased immunosuppressive therapy as a risk factor for dnDSA formation was defined as purposeful reduction of immunosuppression in response to side effects, malignancy or infectious concerns. Nonadherence to medical care was defined when documented in the medical chart, when patients had undetectable tacrolimus levels, missed clinic visits, missed blood draws, or by patient report. Allograft failure was defined as return to dialysis or preemptive re-transplantation. Decreased GFR was defined as estimated GFR <60 ml/min/1.73 m 2 that persisted over at least 3 months. The creatinine-based "Bedside Schwartz" equation (2009) and/or Chronic Kidney Disease Epidemiology Collaboration equation were used to calculate GFR. Proteinuria cutoff was set at urine protein/creatinine ratio of 0.5 mg/mg that persisted over 3 months. Patients who did not have adverse graft outcome were censored at their last follow up. No patient died before the endpoints of interest were reached.

Statistical Considerations
Baseline demographic and clinical characteristics of the cohort were summarized descriptively using means (range), medians (interquartile range, IQR), and counts (percentages) as appropriate. Median time-to-allograft failure was reported using Kaplan-Meier estimates. The Kruskal-Wallis, Mann-Whitney, Chi-squared, and Fisher's exact tests were used to compare clinical characteristics of patients with persistent C1q-dnDSA to those of patients with eliminated C1q-dnDSA as appropriate based on underlying statistical assumptions. A conditional inference forest analysis was employed to assess the hierarchy of the characteristics of de novo anti-HLA DSA based on their ability to predict adverse graft outcome, defined by decreased GFR or allograft failure (28). Included in the model were iDSA MFI, RIS of standard DSA, C1q-binding status, and standard dnDSA status. The variables included in the random forest approach were set to zero for patients who did not form anti-HLA dnDSA. The conditional forest was fit to 1,000 trees. Given the collinearity among features, conditional variable importance measures were computed in order to quantify the contribution of each variable, using the integrated Brier score as a risk measure (29). Out-of-bag model performance statistics were expressed for the binary outcome of adverse graft event.
Outcomes of graft failure and proteinuria were analyzed based on time-varying C1q-dnDSA-binding status. An unadjusted Cox proportional hazards regression model was fit to time-to-allograft failure as a function of time-varying C1q binding. The proportional hazards assumption was assessed for each model and found to hold in all cases (30). Hazard ratios (HR), 95% confidence intervals (CI), and model concordance (using the   C-index) are reported (31). Covariates of interest included sex, historic peak panel-reactive antibodies (pPRA), age at transplant, type of donor and HLA match. A p-value <0.05 was considered statistically significant. All analyses were conducted using R version 3.5.2.

Patient Characteristics
Out of 288 patients who underwent kidney transplantation at LPCH, 233 met the inclusion criteria. One patient who experienced graft failure before 12 months posttransplant due to BK nephropathy was included. Mean follow-up time was 45 (range, 9-108) months. The median age at transplant was 12.3 years, and the majority of the transplant recipients were of Hispanic/Latino origin (39%) and male sex (53%) ( Table 2). A little over half (54%) had an HLA match of 0 or 1 out of 6, with only 6% having an HLA match >3/6. The median pPRA was 7%, and 76% of donors were deceased. The most common cause of end-stage kidney disease was renal aplasia/hypoplasia/ dysplasia (22%).

Anti-HLA dnDSA Characteristics
Among the 233 study subjects, 118 formed standard dnDSA and of those, 52 also had complement-binding activity triggering interventions at initial detection of C1q-dnDSA. A flowchart illustrating the study cohort is provided in Figure 1 All 233 patients were included in the random forest analysis. The most important characteristic for an adverse graft outcome (n 46) was the persistence of C1q binding ( Figure 2). Seventeen out of 21 (80.9%) patients with C1q persistence had an adverse graft outcome, 14 of whom lost their graft. In patients without DSA formation, 16 (13.9%) had an adverse graft outcome, including two who lost their graft. Eight (12.1%) of the patients with standard dnDSA without complement binding and 5 (16.1%) of those with standard dnDSA and disappearance of C1q after treatment experienced an adverse graft outcome, although none lost their graft. Adverse graft outcomes were significantly more frequent among patients with C1q persistence compared to others (p <0.0001), but were not significantly different between non-DSA formers, standard dnDSA formers only and C1q-dnDSA eliminated groups (p 0.86). The random forest model correctly classified only 17 of the 46 (37%) patients as having an adverse graft outcome, while 184 of 187 (98.3%) were correctly classified as not having an event. Of 14 patients who lost their graft due to immunological reasons, 13 had HLA class 2 dnDSA. Of those with dnDSA and graft failure12 of 14 had DQor DR-specific iDSA. Maximum iDSA MFI was more useful to predict graft outcome than maximum RIS. Peak iDSA MFI was correlated with adverse outcome. Looking at the peak iDSA MFI among the 118 patients who formed standard dnDSA, patients with adverse graft outcome had significantly higher peak of 10,861 (IQR, 12,394) compared with 2,987 (IQR, 6,891) in those without adverse graft outcomes (p 0.01). If we only analyze the C1q formers, there was no difference between peak

Formation of C1q-dnDSA and Antibody-Mediated Rejection
The 52 patients who formed C1q-dnDSA had a median time of 3.8 years (IQR, 1.3) until their first detection. C1q-dnDSA disappeared in 31 (59.6%) patients and persisted in 21 (40.4%). The time to detection was not different between patients with persistent C1q-dnDSA and those who eliminated the antibodies. Forty-four (84.6%) patients had a kidney allograft biopsy carried out at the time of detection, of those 33 (75%) had ABMR according to histological findings. Eight patients were not biopsied, of these four were treated according to protocol without a biopsy, two had contraindications to biopsy, 1 patient refused to have the biopsy performed, and 1 patient started dialysis shortly after detection of C1q. The clinical characteristics and histological findings of patients who formed C1q-dnDSA are shown in Table 3. The C1q persistent was more likely to have histological signs of ABMR on biopsy at the time of detection, including positive C4d and interstitial inflammation, compared with the C1q disappearance group. They were also older at kidney transplantation and more likely to be nonadherent to immunosuppressive treatment compared with patients who eliminated C1q. The latter group was younger at transplantation and at first detection of C1q; they were also more likely to have purposeful immunosuppressive therapy reduction as a risk factor for C1q formation. The evolution of DSA over time in the cohort is shown in Figure 4. Standard dnDSA RIS and C1q-dnDSA RIS were moderately correlated (Pearson correlation coefficient of 0.61). C1q-binding status varied and sometimes reappeared after a period of being undetectable. In the group where C1q disappeared, five patients had at least one C1q reappearance, whereas this was observed for nine patients in the group where C1q persisted. Two patients with C1q-dnDSA persistence were treated with plasmapheresis. All patients with persistent C1q had documented medication non-compliance. One patient with persistent C1q had both infections and history of noncompliance at the time of C1q detection. Two patients in whom C1q disappeared did not have an identifiable risk for C1q binding. Comparing the evolution of C1q-dnDSA and standard dnDSA, the latter stayed elevated longer and did not respond to enhanced immunosuppressive treatment in the same way as C1q-dnDSA. Patients with purposeful reduction of immunosuppression appeared to have a steeper decrease of C1q-dnDSA. In the patients with C1q-dnDSA, persistence of standard dnDSA was not strongly correlated with graft failure, unlike C1q persistence. dnDSA Characteristics, Graft Failure and Proteinuria In the entire cohort, graft failure occurred in 16 (6.9%) patients after a median of 45 months (IQR, 28). Fourteen of these patients experienced allograft rejection and two had other potential causes of graft failure; one had BK nephropathy and the other cancer chemotherapy nephrotoxicity ( Figure 5). All patients whose grafts failed due to immunological causes had persistent C1q-dnDSA in

DISCUSSION
In this retrospective study of 233 pediatric kidney transplant recipients, we evaluated the value of systematic anti-HLA DSA screening in predicting risk of adverse graft outcomes and show that compliment binding capacity improved individual risk assessment and is potentially a useful guide of ABMR treatment. Detection of new complement-binding DSA simultaneously with standard dnDSA triggered intensified immunosuppression. Patients forming standard dnDSA without C1q binding were not treated and had non-inferior outcomes compared with those who did not form dnDSA. While higher standard dnDSA strength was associated with adverse graft outcomes, no patient experienced graft failure from immunological causes without persistence of C1q-dnDSA which was the strongest independent predictor of kidney allograft failure. Furthermore, there was no difference between peak iDSA MFI levels in patients with complement binding DSA with or without graft failure. The appearance of both new standard dnDSA and complement binding serves as a decision point triggering a diagnostic kidney biopsy or a change in therapeutic immunosuppression. The present study shows that C1q-dnDSA assessment after interventions may reflect treatment effectiveness and may be a promising approach to guide the intensity and duration of immunosuppressive treatment. C1q-dnDSA status after treatment outperformed standard dnDSA as a predictor of adverse graft outcomes. Post-treatment assessment of complement binding guides therapy by informing when to stop interventions in case of C1q-dnDSA disappearance and justifies continued interventions as long as the C1q-dnDSA titer is responding. Additionally, the absence of C1q-dnDSA response is a useful sign that should prompt a change or discontinuation of therapeutic interventions. The disappearance of C1q-dnDSA is reassuring in our cohort as no patient in that group experienced graft failure. This is in contrast to 66.7% graft failure when C1q-dnDSA persists. Our study is in agreement with prior studies demonstrating the association of C1q-dnDSA persistence and allograft survival, irrespective of enhanced immunosuppressive therapy (3,4,12,21). Our results, demonstrating that C1q-dnDSA response to treatment (persistence or disappearance) is a better predictor of outcome than that of standard dnDSA, are similar to those of Ramon et al. in adults treated for severe ABMR with plasmapheresis (16). More severe inflammation and C4d staining at the time of diagnostic biopsy was more common in patients with C1q-dnDSA persistence. Our findings expand the results of past research showing that patients with treatment resistant C1q-dnDSA were more likely to have a histological diagnosis of ABMR, C4d deposition in peritubular capillaries and interstitial inflammation of the allograft (32,33). Patients who have resolved their C1q-dnDSA might still be at risk for premature graft failure and should be monitored closely.
Although this was not apparent in the current study with an average of 45 months of follow-up, graft injury that occurs during the presence of C1q-dnDSA may be progressive. The increased frequency of proteinuria in this group supports this notion ( Figure 6). We hypothesize that there is a cumulative, dose-dependent effect of nonadherence to immunosuppressive medications on graft survival. Late detection of C1-dnDSA or ongoing medication nonadherence after detection may sabotage even the most effective C1q-dnDSA elimination therapy.
The strength of anti-HLA antibodies measured with the Luminex technology in the present study correlated moderately with C1q-binding status like most of studies included in a recent meta-analysis (13). The analysis had sufficient power to show that the association of C1q binding and allograft outcomes were independent of the level of standard anti-HLA DSA MFI (13,32,34), and that incorporation of complement-binding capacity increased the accuracy of risk prediction above that of the standard anti-HLA DSA MFI levels alone (32). These findings are consistent with the results of the current study. Observing the evolution of MFI of standard dnDSA and C1q-dnDSA, we noted that standard dnDSA MFI tends to stay elevated longer after initiation of therapy. In nearly half of patients with C1q-dnDSA disappearance, standard dnDSA were not eliminated with none of the patients losing their graft. It has been hypothesized that a difference in the pathogenicity of IgG subclasses may exist in patients with ABMR, explaining variable outcomes in patients with high MFI and rejection (35,36). IgG is thought to follow a sequential subclass switching after the initial immune response in ABMR (37). The association of persistently elevated titers of standard dnDSA with good outcomes in our cohort possibly represent less injurious subclasses of IgG. Peak iDSA or clearance of standard dnDSA are not as useful for guiding treatment as C1q-dnDSA and would possibly lead to longer and more aggressive treatment regimen, carrying a risk of infection and impacting the cost of therapy.
The study has a several limitations which are largely inherent in the retrospective design. As such, the study was not originally designed for the purpose of addressing biomarker risk prediction or treatment response and therefore weakens the conclusions that can be drawn. Our treatment response has to be interpreted with caution since we did not have a concurrent control group and the two groups might be inherently different. In addition, follow-up allograft biopsy was not obtained in most of the patients. An appropriately powered randomized controlled trial is needed to confirm our observations. One of the strengths of this study is that C1q-dnDSA and standard dnDSA were simultaneously monitored over a long period of time, allowing us to characterize the kinetics of these antibodies.
In conclusion, this retrospective study of children with a kidney transplant demonstrates that systematic anti-HLA DSA screening together with complement-binding status and allograft biopsies improves risk stratification at the individual patient level and may assist the clinician in determining timing and duration of therapeutic interventions of AMBR. Timely treatment of C1q-dnDSA associated ABMR or C1q-dnDSA in the absence of kidney allograft biopsy, with early detection and elimination of C1q-dnDSA, may be associated with improved outcomes, whereas inability to clear C1q-dnDSA identifies the subset of patients with poor graft survival.

DATA AVAILABILITY STATEMENT
The datasets presented in this article are not readily available because the data cannot be shared due to protection of participant privacy. Requests to access the datasets should be directed to vakaks@gmail.com.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by The Stanford University Institutional Review Board (49338). Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.

CONFLICT OF INTEREST
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.