Utilization of donor-derived Cell-Free DNA in pediatric kidney transplant recipients: A single center study.

High donor-derived cell-free DNA (dd-cfDNA) levels indicate transplant allograft injury and can identify graft rejection in kidney transplant recipients. Here, we evaluated the use of dd-cfDNA in pediatric kidney transplant rejection monitoring and treatment. METHODS: Forty-two pediatric kidney transplant patients were enrolled between February 2020 and August 2021. Dd-cfDNA was tested before and after biopsy/rejection treatment. There was a total of 61 allograft biopsies (44 for-cause, 17 surveillance). RESULTS: Graft rejection was found in 35/61 biopsies. Rejection was more common in basiliximab induction compared to rATG (77.1% vs. 22.9%, p = .0121). Median dd-cfDNA was higher in those with rejection (1.2% [0.34-3.12] vs. 0.24% [0.08-0.78], p < .0001). Dd-cfDNA was highest in biopsies with AMR and mixed AMR/TCMR. In addition, dd-cfDNA in basiliximab induction was higher compared to rATG (0.92% [0.27-1.8] vs. 0.26% [0.08-2], p = .0437). Median change in dd-cfDNA after rejection treatment was -0.57% (-1.67 to 0.05). Median time to dd-cfDNA <1% post-rejection treatment was 8.5 days (3.0-19.5). Dd-cfDNA in AMR was higher compared to TCMR or mixed rejection, and levels remained higher in AMR after treatment. In surveillance biopsies, 4/17 had rejection. Median dd-cfDNA was not different in those with versus without rejection (0.48% vs. 0.28%, p = .2342). Those without rejection all had dd-cfDNA <1%. In those with rejection, only one patient had dd-cfDNA >1%, and all had TCMR. CONCLUSIONS: Our findings support dd-cfDNA as a useful indicator of graft rejection and response to treatment. Additional studies are needed to determine the role of dd-cfDNA in graft health surveillance.

Evaluation of serial monitoring of donor-specific antibodies in pediatric and adult intestinal/multivisceral transplant recipients.

BACKGROUND: The study purpose was to add to limited literature assessing anti-HLA donor-specific antibody (DSA) appearance, clearance, specificity, and impact in intestinal/multivisceral (MV) transplant as well as the value of serial monitoring following an institutional protocol shift implementing serial monitoring. METHODS: This single-center retrospective review included intestinal/MV recipients transplanted 1/1/15-9/31/17 with completed DSA testing. Patients were divided into groups based on DSA presence post-transplant. The primary outcome was biopsy-proven acute rejection (BPAR). Secondary outcomes included graft loss and death. Descriptive analysis of DSA was completed. RESULTS: Of the 35 intestinal/MV recipients (60% pediatric) with DSA testing, 24 patients had post-transplant DSA. Fifteen patients in the DSA(+) group had T-cell-mediated BPAR versus five in the DSA(-) group (63% vs 45%, p = .47). Days to BPAR were 25 [IQR 19-165] (DSA(+) group) versus 232 [IQR 25.5-632.5] (DSA(-) group) (p = .066). There were no differences between groups for graft loss or death. One hundred and five DSA were identified in the DSA(+) group with 63% being class II, and 54% cleared during follow-up. DSA were directed against 50 different HLA alleles, with the most common being directed against HLA- DQ (35%). Time to first DSA and to clearance did not differ between class I and II. CONCLUSION: Findings confirm previous data that suggest post-transplant DSA in this population may lead to increased BPAR or shorter time to BPAR, although not statistically significant. Most DSA were identified within the first month after transplant, and ahead of rejection identification on biopsy. DSA therefore may have utility as an early rejection biomarker and use may be considered in place of early protocol biopsies, particularly in pediatric patients. We identified novel findings of DSA directed against a large breadth of HLA in intestinal/MV patients.

Impact of catch-up vaccinations on anti-HLA antibody response in pediatric kidney transplant candidates.

BACKGROUND: Efforts have been concentrated on improving vaccination administration during the pretransplant evaluation period. However, concern for human leukocyte antigen (HLA) sensitization subsequent to vaccination exists. METHODS: A retrospective review of pediatric kidney transplant candidates (PKTCs) ≤18 years old who had received vaccinations between February 1, 2017 and November 30, 2019 was conducted. Emergence of de novo anti-HLA antibody (HLA-Ab) 3-4 weeks postvaccinations detected by the Luminex single antigen bead assay (SAB) was evaluated. Outcomes assessed included change in the HLA-Ab mean fluorescence intensity (MFI) ≥25% from baseline, and change in preexisting HLA-Ab MFI strength, categorized as weak: 1000-2999; moderate: 3000-9999; and strong: ≥10 000. RESULTS: Sixty vaccinations were administered to 14 patients. Forty-one potential de novo HLA-Ab were detected in five patients. After additional antibody panel testing, 5/41 potential de novo HLA-Ab were determined to be HLA specific; the remaining 36 were deemed nonspecific. The 5 de novo HLA-Ab were observed in three patients and were deemed weak antibody (Ab). Median MFI showed a significant increase for nonspecific Ab, but not de novo HLA-Ab. Median MFI values were deemed transient at 7-10 week follow-up. No HLA-donor-specific Ab developed posttransplant in the patients who developed de novo HLA-Ab. CONCLUSION: Vaccination resulted in a transient increase in non-HLA-specific Ab. The majority of responses were non-HLA specific, hypothesized to be related to denatured antigens on single antigen beads. These data suggest limited clinical impact of vaccinations on the emergence of de novo HLA-Ab.

Impact of active antibody-mediated rejection treatment on donor-specific antibodies in pediatric kidney transplant recipients.

AMR is a major cause of graft loss after kidney transplantation. We evaluated a retrospective cohort of 13 pediatric kidney transplant patients diagnosed with active AMR. All 13 patients were treated with plasmapheresis (PP), IVIg, and rituximab. Anti-HLA DSAs were measured at the time of transplantation, AMR diagnosis, 30 days post-rejection treatment, 90 days post-rejection treatment, and 24 ± 12 months post-AMR. A total of 68 DSAs were identified from 13 patients at the time of active AMR diagnosis. The primary objective of this study was to differentiate treatment response rates between class I and class II anti-HLA DSA post-AMR treatment. Overall, DSAs were significantly reduced at 30 days, and the reduction was sustained at 90 days post-treatment, even for class II anti-HLA and strongly positive DSAs. A significant difference between class I and class II anti-HLA DSA was observed at 30 days; however, between class significance was lost at 90-day follow-up due to continued class II anti-HLA DSA treatment response. Low DSA strength was predictive of treatment response. eGFR demonstrated significant improvement 90 days after AMR diagnosis compared to the initial value at the time of AMR, and the effect was sustained for 12 months. These results suggest that the AMR treatment is effective in pediatric kidney transplant recipients with an early diagnosis of active AMR across both class I and class II anti-HLA DSAs.

Chronic lung allograft dysfunction is associated with an increased number of non-HLA antibodies.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) is the major cause of adverse outcomes in lung transplant recipients. Multiple factors, such as infection, alloimmunity, and autoimmunity, may lead to CLAD. Here, we aim to examine the role of non-human leukocytes antigen (HLA) antibodies in CLAD in a large retrospective cohort. METHODS: We analyzed non-HLA antibodies in the pre- and post-transplant sera of 226 (100 CLAD, 126 stable) lung transplant recipients from 5 centers, and we used a separate cohort to confirm our findings. RESULTS: A panel of 18 non-HLA antibodies was selected for analysis based on their significantly higher positive rates in CLAD vs stable groups. The panel-18 non-HLA antibodies (n > 3) may be positive pre- or post-transplant; the risk for CLAD is higher in the latter. The presence of both non-HLA antibody and HLA donor-specific antibody (DSA) was associated with an augmented risk of CLAD (HR=25.09 [5.52-14.04], p < 0.001), which was higher than that for single-positive patients. In the independent confirmatory cohort of 61 (20 CLAD, 41 stable) lung transplant recipients, the risk for CLAD remained elevated in double-positive patients (HR=10.67 [0.98-115.68], p = 0.052). After adjusting for nonstandard immunosuppression, patients with double-positive DSA/Non-HLA antibodies had an elevated risk for graft loss (HR=2.53 [1.29-4.96], p = 0.007). CONCLUSIONS: Circulating non-HLA antibodies (n > 3) were independently associated with a higher risk for CLAD. Furthermore, when non-HLA antibodies and DSA were detected concomitantly, the risk for CLAD and graft loss was significantly increased. These results show that humoral immunity to HLA and non-HLA antigens may contribute to CLAD development.

COVID-19 infection and vaccination rarely impact HLA antibody profile in waitlisted renal transplant candidates- a multicenter cohort.

Although rare, infection and vaccination can result in antibodies to human leukocyte antigens (HLA). We analyzed the effect of SARS-CoV-2 infection or vaccination on HLA antibodies in waitlisted renal transplant candidates. Specificities were collected and adjudicated if the calculated panel reactive antibodies (cPRA) changed after exposure. Of 409 patients, 285 (69.7 %) had an initial cPRA of 0 %, and 56 (13.7 %) had an initial cPRA > 80 %. The cPRA changed in 26 patients (6.4 %), 16 (3.9 %) increased, and 10 (2.4 %) decreased. Based on cPRA adjudication, cPRA differences generally resulted from a small number of specificities with subtle fluctuations around the borderline of the participating centers' cutoff for unacceptable antigen listing. All five COVID recovered patients with an increased cPRA were female (p = 0.02). In summary, exposure to this virus or vaccine does not increase HLA antibody specificities and their MFI in approximately 99 % of cases and 97 % of sensitized patients. These results have implications for virtual crossmatching at the time of organ offer after SARS-CoV-2 infection or vaccination, and these events of unclear clinical significance should not influence vaccination programs.