dnDSA Development Research Articles

Combined Analysis of HLA Class II Eplet Mismatch and Tacrolimus Levels for the Prediction of De Novo Donor Specific Antibody Development in Kidney Transplant Recipients.

We investigated whether HLA class II eplet mismatch was related to dnDSA development and analyzed its combined impact with tacrolimus levels for kidney transplantation outcomes. A total of 347 kidney transplants were included. HLA Matchmaker was used for the single molecular eplet, total eplet, antibody (Ab)-verified eplet mismatch analyses, and Ab-verified single molecular analysis to identify HLA-DR/DQ molecular thresholds for the risk of dnDSA development. A time-weighted tacrolimus trough level (TAC-C0) of 5 ng/mL and a TAC-C0 time-weighted coefficient variability (TWCV) of 20% were applied to find the combined effects on dnDSA development. A high level of mismatch for single molecular eplet (DQ ≥ 10), total eplet (DQ ≥ 12), Ab-verified eplet (DQ ≥ 4), and Ab-verified single molecular eplet (DQ ≥ 4) significantly correlated with HLA class II dnDSA development. Class II dnDSA developed mostly in patients with low TAC-C0 and high eplet mismatch. In the multivariable analyses, low TAC-C0 and high eplet mismatch showed the highest hazard ratio for the development of dnDSA. No significant combined effect was observed in dnDSA development according to TWCV. In conclusion, the determination of HLA class II eplet mismatch may improve the risk stratification for dnDSA development, especially in conjunction with tacrolimus trough levels.

Effects of tacrolimus intrapatient variability and CYP3A5 polymorphism on the outcomes of pediatric kidney transplantation.

The intrapatient variability (IPV) of tacrolimus (Tac) is associated with the long-term outcome of kidney transplantation. The CYP3A single-nucleotide polymorphism (SNP) may affect the IPV of Tac. We investigated the impact of IPV and genetic polymorphism in pediatric patients who received kidney transplantation. A total of 202 pediatric renal transplant recipients from 2000 to 2016 were analyzed retrospectively. The IPV was calculated between 6 and 12months after surgery. Among these patients, CYP3A5 polymorphism was analyzed in 67 patients. The group with high IPV had a significantly higher rate of de novo donor-specific human leukocyte antigen antibodies (dnDSA) development (35.7% vs. 16.7%, p=.003). The high IPV group also had a higher incidence of T-cell-mediated rejection (TCMR; p<.001). The high IPV had no significant influence on Epstein-Barr virus, cytomegalovirus, and BK virus viremia but was associated with the incidence of posttransplant lymphoproliferative disorders (p=.003). Overall, the graft survival rate was inferior in the high IPV group (p<.001). The CYP3A5 SNPs did not significantly affect the IPV of Tac. In the CYP3A5 expressor group, however, the IPV was significantly associated with the TCMR-free survival rate (p<.001). The IPV of Tac had a significant impact on dnDSA development, occurrence of acute TCMR, and graft failure in pediatric patients who received renal transplantation. CYP3A5 expressors with high IPV of Tac showed worse outcomes, while the CYP3A5 polymorphism had no impact on IPV of Tac.

Should We Be Comforted by a “Negative” Endomyocardial Biopsy? Risk of Future Events with Donor Derived Cell Free DNA in the Setting of Histologic Quiescence

<h3>Purpose</h3> Endomyocardial biopsy (EMB) is considered the gold standard for screening for rejection in heart transplant (HT) recipients. Lack of histological evidence of rejection on EMB has typically been reassuring to clinicians, but the interpretation of elevated levels of donor-derived cell free DNA (dd-cfDNA) in the setting of a "negative" biopsy is unclear. <h3>Methods</h3> Patients from the multi-center Surveillance Using HeartCare Outcomes Registry (SHORE) database who had dd-cfDNA measured within 30 days of a negative EMB (Grades 2R/AMR1 or higher rejection) within the first 100 days of heart transplant (HT) were reviewed. These patients were followed for the subsequent development of significant rejection and <i>de novo</i> donor-specific antibodies (dnDSA) over the next 365 days. <h3>Results</h3> A total of 648 HT recipients with a mean age 57, 74% male, white 64%, 60% of which had PRA < 1% and had a total of 982 paired biopsies with a median dd-cfDNA of 0.05% for those with a Grade 0R and 0.06% for Grade 1R biopsy (Figure 1a). The dd-cfDNA was measured a median of 112 days post-transplant for Grade 0R and 109 days post-transplant for Grade 1R. Despite negative histology on EMB, those with a cfDNA >= 0.20% were at significantly higher risk for the development of significant rejection (14.3% v 5.2%, p<0.01) and dnDSA (11.3% v. 6.8%, p<0.01) over the subsequent year. <h3>Conclusion</h3> Patients with elevated dd-cfDNA, but without prior rejection and negative histology in the first 100 days post-transplant are at increased risk for significant rejection and dnDSA in the subsequent year. The use of dd-cfDNA may be a better method to determine true quiescence and call into question the utility of the EMB as the gold standard for cardiac allograft monitoring.

Development of De Novo Donor Specific Antibodies Following COVID-19 Vaccination in Cardiac Transplant

PurposeSome transplant patients mount an insufficient response to standard COVID-19 vaccine (COVAX) dosing and a booster dose has now been approved. Vaccination carries the theoretical risk of stimulating antibody production to the donor. There are no prior studies surveying de novo donor specific HLA antibodies (dnDSA) after COVAX. HLA-DQ is the most common dnDSA post cardiac transplant and is associated with worse outcomes. We reviewed the development of dnDSA to HLA-DQ in cardiac transplant patients after COVAX.MethodsDSA testing (luminex single antigen bead) was performed routinely at the time of surveillance endomyocardial biopsy. We retrospectively recorded any dnDSA to HLA-DQ with MFI >1000 after COVAX. We further identified a historical cohort (pre vaccination) of 32 patients who had DSA testing a minimum of 5 months after transplant.Results32 adult patients completed COVAX (16 Pfizer-BioNTech, 8 Moderna, 8 Johnson and Johnson) 3-79 mths after transplantation. 16 patients had DSA testing 1-5 mths post COVAX (4-84 mths post transplant) (Table 1). Three patients (18.8 %) demonstrated dnDSA to HLA-DQ (MFI 6360-25,824) 10-20 weeks post COVAX and all had episodes of high grade acute cellular rejection. One additional patient had rejection post COVAX without dnDSA. All 3 patients with dnDSA had robust response to the vaccine with antibodies to spike protein RBD of > 200 U/ml (Elecsys®). Eight vaccinated patients without dnDSA had anti-RBD testing, half had titres > 200 U/ml. In the historical pre COVAX cohort, 3 patients (9 %) demonstrated dnDSA (2022-25480 MFI), one of these had cellular rejection and one developed antibody mediated rejection.ConclusionOne fifth of cardiac transplant recipients developed dnDSA in the weeks following COVAX, all associated with rejection. While no definitive conclusions may be drawn, we believe these data suggest the need for immunological surveillance after COVAX. This may be even more important after a booster dose.

Sub-classification of borderline changes into diffuse or focal and its impact on long-term renal transplant outcomes

BackgroundBorderline changes (BL) with stable renal function is a controversial category in renal transplantation, given its contradictory outcomes. The aim of this study was to compare the clinical outcomes of BL in patients with stable renal function classified as focal and diffuse according to the extent of tubulitis. MethodsPatients with no history of rejection with a surveillance graft biopsy at 3 or 12 months showing BL (n = 40), acute cellular rejection (n = 20) or normal biopsies (n = 20), were included in this study. Biopsies with BL were divided into diffuse BL (BLD) and focal BL (BLF) according to the extent of tubulitis. Because of the low frequency of subclinical ACR (ACRND) (n = 12), biopsies with ACR and graft dysfunction (ACRD) (n = 8) were also included. A composite outcome that included the presence of rejection in subsequent biopsies, graft loss, patient death, decrease in GFR ≥30% or presence of de novo DSA (dnDSA) during the first year of follow-up was evaluated. ResultsThe primary composite outcome occurred in five patients of each of the Normal, BLF and ACRND, eight patients with BLD and six patients with ACRD (p = 0.105). A trend towards more rejection episodes was observed in the ACRND and ACRD. Also, a shorter time to rejection in the BLD, ACRND and ACRD groups compared to BLF and Normal groups (p = 0.039) was observed. During the first year of follow-up, no patient in the ACRND group developed dnDSA, compared to 15–25% in the other groups. The median time of dnDSA development in the BLF group was 45 months, and in the BLD group was 10 months (p = 0.020). ConclusionClassifying BL biopsies with stable renal function into focal and diffuse categories, is a simple and feasible strategy that helps to differentiate between BLD with a phenotype that shows a trend towards worse outcomes, and BLF that behaves more similar to normal biopsies.

Early Blood Transfusion After Kidney Transplantation Does Not Lead to dnDSA Development: The BloodIm Study.

Outcomes after kidney transplantation are largely driven by the development of de novo donor-specific antibodies (dnDSA), which may be triggered by blood transfusion. In this single-center study, we investigated the link between early blood transfusion and dnDSA development in a mainly anti-thymocyte globulin (ATG)-induced kidney-transplant cohort. We retrospectively included all recipients of a kidney transplant performed between 2004 and 2015, provided they had >3 months graft survival. DSA screening was evaluated with a Luminex assay (Immucor). Early blood transfusion (EBT) was defined as the transfusion of at least one red blood-cell unit over the first 3 months post-transplantation, with an exhaustive report of transfusion. Patients received either anti-thymocyte globulins (ATG) or basiliximab induction, plus tacrolimus and mycophenolic acid maintenance immunosuppression. A total of 1088 patients received a transplant between 2004 and 2015 in our center, of which 981 satisfied our inclusion criteria. EBT was required for 292 patients (29.7%). Most patients received ATG induction (86.1%); the others received basiliximab induction (13.4%). dnDSA-free graft survival (dnDSA-GS) at 1-year post-transplantation was similar between EBT+ (2.4%) and EBT- (3.0%) patients (chi-squared p=0.73). There was no significant association between EBT and dnDSA-GS (univariate Cox’s regression, HR=0.88, p=0.556). In multivariate Cox’s regression, adjusting for potential confounders (showing a univariate association with dnDSA development), early transfusion remained not associated with dnDSA-GS (HR 0.76, p=0.449). However, dnDSA-GS was associated with pretransplantation HLA sensitization (HR=2.25, p=0.004), hemoglobin >10 g/dL (HR=0.39, p=0.029) and the number of HLA mismatches (HR=1.26, p=0.05). Recipient’s age, tacrolimus and mycophenolic-acid exposures, and graft rank were not associated with dnDSA-GS. Early blood transfusion did not induce dnDSAs in our cohort of ATG-induced patients, but low hemoglobin level was associated with dnDSAs-GS. This suggests a protective effect of ATG induction therapy on preventing dnDSA development at an initial stage post-transplantation.

Impact of calcineurin inhibitor-free immunosuppression on de novo donor-specific antibody formation in liver transplant recipients.

Low calcineurin inhibitor (CNI) levels expose liver transplant recipients to rejection episodes and potentially to antibody-mediated rejection. There are little data on the impact of CNI-free immunosuppression on de novo donor-specific HLA antibody (dnDSA) development. Here we evaluated the prevalence of dnDSA in liver transplant recipients on CNI-free maintenance regimens and their associations with histopathological abnormalities of allografts. Seven hundred and twenty-seven liver transplant recipients underwent a first liver transplant between 2000 and 2018 in three French transplant centres and had protocolized follow-up with dnDSA screening and allograft biopsy 1, 5 and 10years after transplantation. CNIs were withdrawn in 166 (22.8%) patients with or without conversion to mammalian target of rapamycin inhibitors and/or maintenance with mycophenolic acid. DSA were present after withdrawal in 30.1% (50/166) patients on CNI-free immunosuppression compared with 16% (90/561) on CNI maintenance therapy (p<0.001). The cumulative incidence of dnDSA 10years after transplant was 20% in the CNI group versus 28% in the CNI-free group (p<0.01). dnDSAs were associated with histological graft abnormalities (significant allograft fibrosis or rejection) (HR 2.24, 95% CI 1.2-4.1; p=0.01). In univariate Cox regression analysis, being on a CNI-free regimen did not impact graft histology. Patients on a CNI-free IS regimen have a higher prevalence of dnDSA than patients on a standard IS regimen. dnDSAs but not CNI-free immunosuppression were associated with abnormal allograft histology.

Association of intraindividual tacrolimus variability with de novo donor-specific HLA antibody development and allograft rejection in pediatric kidney transplant recipients with low immunological risk

BackgroundTacrolimus (Tac) intraindividual variability (TacIPV) in pediatric kidney transplant patients is only poorly understood. We investigated the impact of TacIPV on de novo donor-specific HLA antibodies (dnDSA) development and allograft rejection in Caucasian pediatric recipients of a living or deceased donor kidney with low immunological risk.MethodsThis was a single-center retrospective study including 48 pediatric kidney transplant recipients. TacIPV was calculated based on coefficient of variation (CV%) 6–12 months posttransplant. TacIPV cutoff was set at the median (25%). Outcome parameters were dnDSA development and rejection episodes.ResultsIn total, 566 Tac levels were measured with median 11.0 (6.0–17.0) measurements per patient. The cutoff of 25% corresponded to the median CV% in our study cohort (25%, IQR 18–35%) and was comparable to cutoffs determined by receiver operating characteristic (ROC) curve analysis. High TacIPV was associated with higher risk of dnDSA development (HR 3.4, 95% CI 1.0–11.1, P = 0.047; Kaplan–Meier analysis P = 0.018) and any kind of rejection episodes (HR 4.1, 95% CI 1.1–14.8, P = 0.033; Kaplan–Meier analysis P = 0.010). There was a clear trend towards higher TacIPV below the age of 6 years. TacIPV (CV%) was stable over time. A TacIPV (CV%) cutoff of 30% or IPV quantification by mean absolute deviation (MAD) showed comparable results.ConclusionsHigh TacIPV is associated with an increased risk of dnDSA development and rejection episodes > year 1 posttransplant even in patients with low immunological risk profile. Therefore, in patients with high TacIPV, potential causes should be addressed, and if not resolved, changes in immunosuppressive therapy should be considered.Graphical abstractA higher resolution version of the Graphical abstract is available as Supplementary information.

Human leukocyte antigen epitope mismatch loads and the development of de novo donor-specific antibodies in cardiothoracic organ transplantation.

De novo donor-specific human leucocyte antigen (HLA) antibodies (dnDSA) are associated with increased risk of rejection and mortality in solid organ transplantation. Such dnDSA is produced in some recipients upon allorecognition of mismatched HLA post-transplant. HLA matching is not currently considered in the allocation of deceased donor hearts and lungs and pre-transplant immunological risk stratification is based entirely on the mean fluorescence intensity (MFI) of circulating donor-directed HLA antibodies. HLA epitope-based matching tools predict B-cell or T-cell HLA epitopes that are present in the donor's HLA but absent in the recipient's HLA. We hypothesized that patients with higher epitope mismatch loads would be at increased risk of dnDSA development. We retrospectively analysed 73 heart and/or lung transplant recipients who were tested for DSA between 2015 and 2020. HLAMatchmaker, PIRCHE-II and HLA epitope mismatch algorithm (HLA-EMMA) were used to calculate eplet mismatch (EpMM) loads, T-cell epitope mismatch (TEpMM) loads and solvent accessible amino acid mismatch (SAMM) loads, respectively. Multivariate analyses showed that HLA-EMMA was the only tool with a significant association between the total score for all HLA loci and dnDSA production [odds ratio (OR) 1.021, 95% confidence interval (CI) 1.003-1.042, p=.0225] though this increased risk was marginal. The majority of dnDSA were directed against HLA-DQ and patients with higher HLA-DQ TEpMM loads (OR=1.008, CI=1.002-1.014, p=.007), and HLA-DR+DQ SAMM loads (OR=1.035, CI=1.010-1.064, p=.0077) were most at risk of producing dnDSA. We also showed that patients with a risk epitope within the HLA molecule encoded for by HLA-DQA1*05 + HLA-DQB1*02/03:01 were significantly more likely to produce dnDSA. The use of HLA epitope-based matching tools could be used for cardiothoracic transplant risk stratification to enable early intervention and monitoring of patients at increased risk of producing dnDSA.

Eplet mismatches associated with de novo donor-specific HLA antibody in pediatric kidney transplant recipients.

Optimizing amino acid (eplet) histocompatibility at first transplant decreases the risk of de novo donor-specific antibody (dnDSA) development and may improve long-term graft survival in pediatric kidney transplant recipients (KTR). We performed a retrospective analysis of pediatric KTR and their respective donors to identify eplets most commonly associated with dnDSA formation. Eplet mismatch analysis was performed in a cohort of 125 pediatric KTR-donor pairs (2006-2018). We determined the prevalence of each eplet mismatch and quantified the percentage of exposed patients who developed dnDSA for each mismatched eplet. Recipient median age was 14 (IQR 8-17) years with a racial distribution of 42% Black, 48% Caucasian, and 5.6% Middle-Eastern. Median eplet load varied significantly by recipient race, Black 82 (IQR 58-98), White 60 (IQR 44-81) and Other 66 (IQR 61-76), p = 0.002. Forty-four percent of patients developed dnDSA after median 37.1 months. Compared to dnDSA- patients, dnDSA+ patients had higher median eplet load, 64 (IQR 46-83) vs. 77 (IQR 56-98), p = 0.012. The most common target of dnDSA were eplets expressed in HLA-A*11 and A2 in Class I, and HLA-DQ6 and DQA5 in Class II. The most commonly mismatched eplets were not the most likely to result in dnDSA formation. In a racially diverse population, only a subset of eplets was linked to antibody formation. Eplet load alone is not a sufficient surrogate for eplet immunogenicity. These findings illustrate the need to optimize precision in donor selection and allocation to improve long-term graft outcomes. Graphical Abstract A higher resolution version of the Graphical abstract is available as Supplementary information.

Clinical Implications of Tacrolimus Time in Therapeutic Range and Intrapatient Variability in Urban Renal Transplant Recipients Undergoing Early Corticosteroid Withdrawal.

Background.Tacrolimus demonstrates wide intrapatient and interpatient variability requiring therapeutic drug monitoring. The utility of tacrolimus time in therapeutic range (TTR) after renal transplantation (RT) under an early corticosteroid withdrawal (ECSWD) protocol is unknown. The purpose of this study is to assess the impact of tacrolimus TTR in an ECSWD RT population.Materials.A retrospective analysis of adult RT recipients maintained on tacrolimus was conducted. Patients were excluded if they were on nonstandard protocol immunosuppression agents <12 months post-RT. Tacrolimus TTR was calculated using the Rosendaal method. Patients were divided into high (TTR-H) and low (TTR-L) TTR groups based on cohort median. The primary outcome was to compare the incidence of acute rejection 12 months post-RT. Secondary outcomes included comparing rejection subtypes, incidence of donor-specific antibody (DSA) and de novo DSA (dnDSA), risk factors for acute rejection and dnDSA development, and allograft function (serum creatinine and estimated glomerular filtration rate).Results.A total of 193 patients were analyzed (TTR-H = 98 and TTR-L = 95). There was no difference in the incidence of acute rejection (TTR-H 20.4% versus TTR-L 20.0%; P = 0.944). Positive DSA posttransplant (odds ratio [OR], 3.62; 95% confidence interval [CI], 1.41-9.26; P = 0.007) was associated with a higher acute rejection at 12 months posttransplant. Mycophenolate dose reduction (OR, 2.82; 95% CI, 1.13-6.97; P = 0.025) and acute rejection (OR, 2.99; 95% CI, 1.09-8.18; P = 0.032) were associated with dnDSA formation. No difference in serum creatinine or estimated glomerular filtration rate was observed (P > 0.05).Conclusions.Tacrolimus TTR was not significantly different with regards to acute rejection in an ECSWD population. Future studies are still needed to determine tacrolimus TTR thresholds post-RT and identify populations that may benefit from this intrapatient variability monitoring parameter.

Donor-Derived Cell-Free DNA Predicts De Novo DSA after Heart Transplantation

PurposeDevelopment of de novo donor-specific HLA antibodies (dnDSA) may contribute to allograft injury and dysfunction, antibody-mediated rejection (ABMR), and potential development of cardiac allograft vasculopathy (CAV). Elevated plasma donor-derived cell-free DNA (dd-cfDNA) levels have been demonstrated during acute cellular rejection (ACR) and ABMR events after heart transplant (HT). The purpose of this study is to investigate the relationship between dd-cfDNA and subsequent DSA development.MethodsFrom the 916 HT patients enrolled in the Surveillance Heart Care Outcome Registry (SHORE), we reviewed 2136 samples of dd-cfDNA surveillance monitoring. Patients with ACR or ABMR on biopsy were excluded from analysis. The dnDSAs included HLA Class I and/or Class II epitopes and were analyzed to development of first dnDSA or last negative test, with censoring at 365 days. There were 613 DSA samples paired with dd-cfDNA levels that were obtained within 30 days of the DSA draw, with prior negative endomyocardial biopsy. Cox proportional hazards model fitting was utilized with dd-cfDNA as a covariate (p<0.05).ResultsFrom analysis of 613 paired samples, 127 (20.7%) demonstrated dnDSA with an associated median dd-cfDNA of 1.20% (IQR: 0.100 - 3.10%). 71 (11.6%) were associated with dnDSA Class I and 93 (15.2%) with Class II. When dichotomized for dd-cfDNA > 0.15%, [FIGURE] the positive likelihood ratio for development of Class I and/or Class II dnDSA (+LR) was 4.47 (p=0.03). When analyzed without censoring at 365 days, using dd-cfDNA at this cutoff, the +LR was 5.68 (p=0.02).ConclusionIn the prospective SHORE cohort, elevated dd-cfDNA levels > 0.15% were associated with increased likelihood for the subsequent development of dnDSA within the first year after heart transplantation. Development of de novo donor-specific HLA antibodies (dnDSA) may contribute to allograft injury and dysfunction, antibody-mediated rejection (ABMR), and potential development of cardiac allograft vasculopathy (CAV). Elevated plasma donor-derived cell-free DNA (dd-cfDNA) levels have been demonstrated during acute cellular rejection (ACR) and ABMR events after heart transplant (HT). The purpose of this study is to investigate the relationship between dd-cfDNA and subsequent DSA development. From the 916 HT patients enrolled in the Surveillance Heart Care Outcome Registry (SHORE), we reviewed 2136 samples of dd-cfDNA surveillance monitoring. Patients with ACR or ABMR on biopsy were excluded from analysis. The dnDSAs included HLA Class I and/or Class II epitopes and were analyzed to development of first dnDSA or last negative test, with censoring at 365 days. There were 613 DSA samples paired with dd-cfDNA levels that were obtained within 30 days of the DSA draw, with prior negative endomyocardial biopsy. Cox proportional hazards model fitting was utilized with dd-cfDNA as a covariate (p<0.05). From analysis of 613 paired samples, 127 (20.7%) demonstrated dnDSA with an associated median dd-cfDNA of 1.20% (IQR: 0.100 - 3.10%). 71 (11.6%) were associated with dnDSA Class I and 93 (15.2%) with Class II. When dichotomized for dd-cfDNA > 0.15%, [FIGURE] the positive likelihood ratio for development of Class I and/or Class II dnDSA (+LR) was 4.47 (p=0.03). When analyzed without censoring at 365 days, using dd-cfDNA at this cutoff, the +LR was 5.68 (p=0.02). In the prospective SHORE cohort, elevated dd-cfDNA levels > 0.15% were associated with increased likelihood for the subsequent development of dnDSA within the first year after heart transplantation.

Location, Location, Location - Does Epitope Matching Matter in Pediatric Heart Transplantation?

<h3>Purpose</h3> Recent data in adult solid organ transplantation suggest that epitope-based human leukocyte antigen (HLA) matching may permit better risk assessment of <i>de novo</i> donor‐specific antibody (dnDSA) development compared to antigen-based HLA matching, but data in pediatric heart transplantation (PHTx) are scarce. We evaluated the association between epitope mismatches and dnDSA in PHTx. <h3>Methods</h3> We performed high resolution HLA typing and epitope mismatch analysis (HLA Matchmaker) in children undergoing primary PHTx at a single center from 2014 - 2020. DSA >1000 MFI (Luminex Single Antigen Bead Assay; One Lambda) were considered positive. Data are presented as median (interquartile range). <h3>Results</h3> Epitope and dnDSA data were available in 109 transplants (age 5.8 years [1-12], 66% male, 32% non-Hispanic White, follow-up 2.5 years [1.3 - 4.7]). There was significant diversity in the number of epitope mismatches within each HLA locus (Fig 1A), and the number of mismatches were: A 20 (13-28), B 17 (12-22), C 10 (6-15), DR 16 (8-30), DQ 12 (8-16), and DP 8 (3-13). The number of epitope mismatches did not differ among any donor-recipient race combination (not shown). The development of dnDSA was not associated with a higher number of epitope mismatches at any HLA-loci (Fig 1B). However, patients with antibody mediated rejection (AMR) had more DQ mismatches (15 vs 11; p=0.04) than those without AMR, and there were more anti-DQB1 DSA than any other DSA in the cohort (Figure 1C). <h3>Conclusion</h3> There is significant variability in the number of HLA epitope mismatches within each HLA locus. These data suggest that it may not be the total number of epitope mismatches but rather epitope mismatches at specific loci that vary in location and function that are of immunogenic importance. Further investigation of epitope-matching in PHTx may permit a more personalized immunosuppressive approach based on individualized alloimmune risk assessment in the future.

Time to Therapeutic Tacrolimus Levels and Association with Acute Cellular Rejection and De Novo Donor-Specific Antibody Development in Lung Transplant Recipients

<h3>Purpose</h3> Tacrolimus (FK506) is standard of care for preventing rejection in lung transplant (LTx) for maintenance immunosuppression. Both acute cellular rejection (ACR) and <i>de novo</i> donor-specific antibodies (dnDSAs) are strongly associated with development of chronic lung allograft dysfunction. Time to therapeutic FK506 has not been standardized. We evaluated whether time to therapeutic FK506 impacted ACR and <i>dn</i>DSA rates within 6 months (M) of LTx. <h3>Methods</h3> We reviewed LTx recipients between 5/2016 - 12/2019. We excluded patients who did not receive FK506, received their first dose ≥48 hours post-LTx, were transitioned off FK506 during the study period, or expired within 30 days (d). Primary outcome was a composite of ACR and <i>dn</i>DSA development within 6M for patients achieving therapeutic FK506 (8-15 ng/mL) within 7d, versus >7d. Secondary endpoints included: time within therapeutic range (TTR) at 1M and incidence of acute kidney injury (AKI) within 14d. <h3>Results</h3> Median time to therapeutic was 7d (IQR 5.0-10.0) in 222 patients. There was no difference in ACR [7.6% vs 9.7% (p=0.63)] or dnDSA [35% vs 44% (p=0.20)] between patients achieving therapeutic FK506 ≤7d vs >7d. Recipients therapeutic ≤7d had a longer median time to dnDSA development (44d vs 27d, p=0.043). TTR at 1M was longer in patients therapeutic ≤7d (40% vs 28%, p<0.001). Neither median nor percentage of TTR had an impact on the composite outcome (HR 0.99; 95% CI 0.95-1.03). AKI incidence (≥stage 2) was similar (40.3% vs 38.8%, p=0.82) between groups. Multivariate analysis identified receipt of a double LTx as the only covariate associated with a higher risk of AKI [OR 3.56, 95% CI 1.40-9.05 (p=0.01)]. <h3>Conclusion</h3> There was no significant differences in development of ACR or dnDSA within 6M between LTx recipients achieving therapeutic FK506 ≤7d vs >7d. Increased time to dnDSA development in the ≤7d cohort suggests a potential benefit to minimizing time to therapeutic FK506 level post-LTx.

Non-adherence assessment to immunosuppressant therapy with a self-report questionnaire and intra-patient variability in renal transplantation: risk factors and clinical correlations.

Non-adherence (NA) to immunosuppressive drugs is to date considered a crucial issue in kidney transplanted patients (KTRs), leading to de-novo donor-specific anti-HLA antibodies (dnDSA) development, acute and chronic rejection, and at least graft loss. However, NA assessment is challenging, often leading to underestimation in real-life settings. NA evaluation in all KTRs referred to our post-transplantation clinic in the period between 01/01-15/07/2018 with self-report questionnaire combined to intra-patient variability (IPV) of the pivotal immunosuppressive drug (based on trough levels of tacrolimus/mTOR inhibitor). Based on both questionnaire and IPV, 86 out of the 504 tested KTRs (17%) were classified as NA. Male gender (OR, 2.0; 95% confidence interval [CI], 1.2 to 3.4), high educational level (OR for KTRs with a degree, 1.8 [95% CI, 1.0 to 3.1]), employment (OR, 2.0 [95% CI, 1.2 to 3.3]), young age at transplantation (P=0.017), longer time on the waiting list and after transplantation (P=0.027 and 0.049 respectively) were all associated with NA. High IPV was mostly documented in KTRs treated with the twice-daily formulation of the immunosuppressive drug (OR, 1.5 [95% CI, 1.0 to 2.1]) and better associated with dnDSA appearance (OR, 2.1 [95% CI, 1.1 to 3.9]). NA is a significant problem, difficult to assess, and can lead to dnDSA development also in our population. Identifying risk factors for NA might be an underestimated tool to improve graft and patient outcome in KTRs.

Characterization of follicular T helper cells and donor-specific T helper cells in renal transplant patients with de novo donor-specific HLA-antibodies

T follicular helper (TFH) cells are a heterogeneous subset of immunocompetent T helper (TH) cells capable of augmenting B cell responses in lymphoid tissues. In transplantation, exposure to allogeneic tissue activates TFH cells increasing the risk of the emergence of de novo donor-specific HLA-antibodies (dnDSA). These can cause antibody-mediated rejection (AMR) and allograft loss. Follicular regulatory T (TFR) cells counteract TFH cell activity. Here, we investigated the implications of TFH and TFR cells on dnDSA formation after renal transplantation (RTX). Considering TFH cells to be CXCR5+ and IL-21+, we found by flow cytometry that patients with dnDSA produced IL-21 more abundantly compared to healthy volunteers. In in vitro alloreactivity assays, patients with dnDSA featured an enhanced alloreactive TH cell pool in response to donor-specific HLA antigens. Besides, longitudinal investigations suggested enhanced alloreactivity shortly after transplantation increasing the risk of dnDSA development. Taken together, in spite of continuous immunosuppression we report a strong IL-21 response in TFH cells and an expanded reservoir of donor-specific memory TH cells in patients with dnDSA. This warrants further investigations if aberrant TFH cell activation may precede the formation of dnDSA promoting AMR.

Complex Linkage Disequilibrium Effects in HLA-DPB1 Expression and Molecular Mismatch Analyses of Transplantation Outcomes.

HLA molecular mismatch (MM) is a risk factor for de novo donor-specific antibody (dnDSA) development in solid organ transplantation. HLA expression differences have also been associated with adverse outcomes in hematopoietic cell transplantation. We sought to study both MM and expression in assessing dnDSA risk. One hundred three HLA-DP-mismatched solid organ transplantation pairs were retrospectively analyzed. MM was computed using amino acids (aa), eplets, and, supplementarily, Grantham/Epstein scores. DPB1 alleles were classified as rs9277534-A (low-expression) or rs9277534-G (high-expression) linked. To determine the associations between risk factors and dnDSA, logistic regression, linkage disequilibrium (LD), and population-based analyses were performed. A high-risk AA:GX (recipient:donor) expression combination (X = A or G) demonstrated strong association with HLA-DP dnDSA (P = 0.001). MM was also associated with HLA-DP dnDSA when evaluated by itself (eplet P = 0.007, aa P = 0.003, Grantham P = 0.005, Epstein P = 0.004). When attempting to determine the relative individual effects of the risk factors in multivariable analysis, only AA:GX expression status retained a strong association (relative risk = 18.6, P = 0.007 with eplet; relative risk = 15.8, P = 0.02 with aa), while MM was no longer significant (eplet P = 0.56, aa P = 0.51). Importantly, these risk factors are correlated, due to LD between the expression-tagging single-nucleotide polymorphism and polymorphisms along HLA-DPB1. The MM and expression risk factors each appear to be strong predictors of HLA-DP dnDSA and to possess clinical utility; however, these two risk factors are closely correlated. These metrics may represent distinct ways of characterizing a common overlapping dnDSA risk profile, but they are not independent. Further, we demonstrate the importance and detailed implications of LD effects in dnDSA risk assessment and possibly transplantation overall.

What happens after graft loss? A large, long-term, single-center observation.

The number of patients returning to dialysis after graft failure increases. Surprisingly, little is known about the clinical and immunological outcomes of this cohort. We retrospectively analyzed 254 patients after kidney allograft loss between 1997 and 2017 and report clinical outcomes such as mortality, relisting, retransplantations, transplant nephrectomies, and immunization status. Of the 254 patients, 49% had died 5years after graft loss, while 27% were relisted, 14% were on dialysis and not relisted, and only 11% were retransplanted 5years after graft loss. In the complete observational period, 111/254 (43.7%) patients were relisted. Of these, 72.1% of patients were under 55years of age at time of graft loss and only 13.5% of patients were ≥65years. Age at graft loss was associated with relisting in a logistic regression analysis. In the complete observational period, 42 patients (16.5%) were retransplanted. Only 4 of those (9.5%) were ≥65years at time of graft loss. Nephrectomy had no impact on survival, relisting, or development of dnDSA. Patients after allograft loss have a high overall mortality. Immunization contributes to long waiting times. Only a very limited number of patients are retransplanted especially when ≥65years at time of graft loss.

Risk factors for progression from low level BK dnaemia to unfavorable outcomes after BK management via immunosuppressive reduction.

Effective management of BK viremia (BKPyV-DNAemia) in kidney transplant recipients (KTRs) involves regular monitoring and adjustment of immunosuppression. With this strategy, the majority of patients will clear BK or have ongoing, but non-significant, low-level BKPyV-DNAemia. However, despite adjustments, some will develop more severe sequelae of BK including BKPyV-DNAemia >5 log10 copies/mL and BK nephropathy, and others may develop de novo DSA (dnDSA) or acute rejection (AR). This was a single-center study of KTRs transplanted at the University of Wisconsin-Madison between 01/01/2015 and 12/31/2017. In this study, we sought to elucidate characteristics associated with the progression of BKPyV-DNAemia to unfavorable outcomes after decreasing immunosuppressive medications for the management of BK viremia as described in consensus guidelines. A total of 224 KTRs fulfilled our selection criteria; 118 (53%) resolved or had persistent low DNAemia, 64 (28%) had severe BK/nephropathy, and 42 (19%) developed dnDSA or AR. In multivariable analysis, female gender (HR: 2.05; 95% CI: 1.08-3.90; P=.02); previous rejection (HR: 2.90; 95% CI: 1.04-8.12; P=.04), and early infection (HR: 0.81; 95% CI: 0.72-0.90; P<.001) were associated with the development of severe BK/nephropathy. Conversely, non-depleting induction at transplant (HR: 2.06; 95% CI: 1.03-4.11; P=.03), HLA mismatches >3 (HR: 2.27; HR: 1.01-5.06; P=.04), and delayed graft function (HR: 4.14; 95% CI: 1.12-15.28; P=.03) were associated with development of dnDSA and/or rejection. Our study suggests that almost half of KTRs with BKPyV-DNAemia managed by our immunosuppressant adjustment protocol progress unfavorably. Identification of these risk factors could assist the frontline clinician in creating an individualized immunosuppressive modification plan potentially mitigating negative outcomes.

Eplet mismatch scores and de novo donor-specific antibody development in simultaneous pancreas-kidney transplantation

Antibody-mediated rejection is the principal cause of allotransplant graft failure. Available studies differ on the impact of de novo donor specific antibody (dnDSA) in pancreas transplants but are limited by patient sample size and sera sample collection. High-resolution HLA incompatibility scoring algorithms are able to more accurately predict dnDSA development. We hypothesized that HLA incompatibility scores as determined by the HLA-Matchmaker, HLA-EMMA, and PIRCHE-II algorithms would serve as a predictor of de novo donor specific antibody (dnDSA) development and clarify the role dnDSA as detrimental to simultaneous pancreas-kidney graft survival.Our results show that female sex and race were significantly associated with dnDSA development and dnDSA development resulted in worse kidney and pancreas graft survival. The majority of individuals who developed dnDSA (88%), developed anti-HLA-DQ antibody in some combination with anti-HLA class I or -DR. A multivariate analysis of the incompatibility scores showed that both HLA-Matchmaker and PIRCHE-II scores predicted anti-DQ dnDSA development. An optimal cutoff threshold for incompatibility matching was obtained for these scores and demonstrated statistical significance when predicting freedom from anti-DQ DSA development.In conclusion, increased scores from high-resolution HLA matching predict dnDSA development, and dnDSA is associated with antibody-mediated rejection and worse pancreas and kidney graft outcomes.