Pretransplant Donor-specific Antibodies Research Articles

Exploring the immunological relevance of pre-transplant donor-specific antibody in intestinal transplantation, with special consideration to the liver.

Despite recent advances that have improved outcomes following intestinal transplantation (ITx), achieving long-term patient survival and rejection-free survival is still challenging. Understanding the relevance of pre-transplant human leukocyte antigen (HLA) donor-specific antibody (DSA) in ITx and the immunomodulatory potential of the liver within the allograft is crucial to providing an accurate assessment of pre-transplant immunological risk, which could influence and improve post-transplant outcomes further. This was the primary objective of this retrospective study of 95 adult ITx transplants which took place at Cambridge University Hospitals (United Kingdom) between 2007 and 2019. Two novel programs were developed and validated to identify DSA (tested by Luminex single antigen beads) in this dataset. Data analysis utilised Kaplan-Meier survival methods, and statistical analysis was performed using log-rank tests and adjusted Cox models. Fifty-four (57%) ITx cases contained a liver, and 36 (38%) were sensitised to HLA. Pre-transplant DSA >500 mean fluorescent intensity appeared to negatively affect post-ITx patient survival and rejection outcomes. Additionally, liver-inclusive allografts seemed to show particular resistance to HLA class I DSA. Our data hints towards consistency with other ITx studies where deleterious effects of DSA have been demonstrated, and where liver inclusion is protective from HLA class I DSA. This is in line with current national guidelines for immunological risk. Our publicly available research programs could support future large or multicentre studies where statistically relevant data might be gained.

Pre-transplant donor specific antibodies in ABO incompatible kidney transplantation - data from the Swiss transplant cohort study.

Living donor (LD) kidney transplantation in the setting of ABO blood group incompatibility (ABOi) has been previously reported to be associated with increased risk for antibody-mediated rejection (ABMR). It is however unclear if the presence of pre-transplant donor specific antibodies (DSA) works as an additive risk factor in the setting of ABOi and if DSA positive ABOi transplants have a significantly worse long-term outcome as compared with ABO compatible (ABOc) DSA positive transplants. We investigated the effect of pre-transplant DSA in the ABOi and ABOc setting on the risk of antibody-mediated rejection (ABMR) and graft loss in a cohort of 952 LD kidney transplants. We found a higher incidence of ABMR in ABOi transplants as compared to ABOc transplants but this did not significantly affect graft survival or overall survival which was similar in both groups. The presence of pre-transplant DSA was associated with a significantly increased risk of ABMR and graft loss both in the ABOi and ABOc setting. We could not detect an additional risk of DSA in the ABOi setting and outcomes were comparable between DSA positive ABOi and ABOc recipients. Furthermore, a combination of DSA directed at both Class I and Class II, as well as DSA with a high mean fluorescence intensity (MFI) showed the strongest relation to ABMR development and graft loss. The presence of pre-transplant DSA was associated with a significantly worse long-term outcome in both ABOi and ABOc LD kidney transplants and our results suggests that the risk associated with pre-transplant DSA is perhaps not augmented in the ABOi setting. Our study is the first to investigate the long-term effects of DSA in the ABOi setting and argues that pre-transplant DSA risk could potentially be evaluated similarly regardless of ABO compatibility status.

Superiority of Single-Antigen Bead Study in Donor-Specific Antibodies: Determination in Highly Sensitized Patients.

The presence of donor-specific antibodies against HLA before kidney transplant has been variably associated with decreased long-term graft survival. Data on the association between pretransplant donor-specific antibodies and rejection and cause of graft failure in recipients of donor kidneys are scarce. For this study of HLA antibody levels, we analyzed serum samples from 76 patients (48 women and 28 men) who were prepared for kidney transplant at the Baskent University İstanbul Hospital between 2017 and 2022. Levels were determined by using Lifecodes panel reactive antibody class I and II identification kits and Lifecodes LSA class I and II identification kits by the Luminex assay method. Multiple antigen tests showed more than 70% sensitization detected against both class I and class II antigens in our patient group. When some samples were reevaluated with the single-antigen bead method, desensitization values were shown to be considerably reduced compared with values from multiple antigen methods. The single-antigen-coated bead method can be useful in determining the risk of donor-specific antibodies in highly sensitized patients.

What Do Donor-Specific Antibody Changes Mean in Kidney Transplant Patients?

Objective: The role of immunological evaluation is significant in selecting a suitable donor to reduce post-transplant complications in kidney transplantation (KTx). It is unknown how often donor-specific antibody (DSA) positivity causes rejection or how often rejection will develop in patients who do not develop DSA positivity. We aimed to evaluate the relationship between the DSA changes and the KTx patients' biochemical parameters.
 Methods: The study was a cross-sectional study evaluating 45 KTx patients. Demographic and clinical characteristics of the patients, pre-transplant DSA values, post-transplant DSA values, and biochemical parameters were retrospectively scanned from the hospital system. The patients' data were divided into three groups according to DSA changes.
 Results: DSA was negative in 21 (46%) patients and positive in 24 (54%) before transplantation. In the post-transplant follow-up, it was observed that the DSA value became positive in 7 patients and turned negative in 9 patients. Rejection developed in 22% of 9 patients whose DSA was positive before transplantation and turned negative after transplantation, and in 28% of 7 patients turned positive from negative. Estimated glomerular filtration rate (e-GFR) and creatinine levels in the post-transplant period were associated with the change in DSA. Also, e-GFR and neutrophil values were independently associated with rejection.
 Conclusions: Although DSA change affects kidney functions, we found that DSA positivity alone cannot predict rejection, and rejection may occur in the DSA-negative group. Neutrophil count and e-GFR changes were closely related to rejection. Therefore, DSA levels should be monitored regularly, but DSA change alone is insufficient for rejection evaluation.

Recurrent Mild Acute Rejections and Donor-specific Antibodies as Risk Factors for Cardiac Allograft Vasculopathy in a National Pediatric Heart Transplant Cohort.

Immune-mediated factors such as acute cellular rejections and donor-specific antibodies (DSAs) are risk factors for cardiac allograft vasculopathy (CAV). We studied a national cohort with a unified setting and thorough protocol endomyocardial biopsy (EMB) data for an association between cellular rejections, especially when mild and recurrent, and DSAs with CAV in pediatric heart transplant (HTx) patients. This is a retrospective, national cohort study of 94 pediatric HTxs performed between 1991 and 2019 and followed until December 31, 2020. Diagnosis of CAV was based on reevaluation of angiographies. Protocol and indication EMB findings with other patient data were collected from medical records. Associations between nonimmune and immune-mediated factors and CAV were analyzed with univariable and multivariable Cox regression analyses. Angiographies performed on 76 patients revealed CAV in 23 patients (30%). Altogether 1138 EMBs (92% protocol biopsies) were performed on 78 patients (83%). During the first posttransplant year, grade 1 rejection (G1R) appeared in 45 patients (58%), and recurrent (≥2) G1R findings in 14 patients (18%). Pretransplant DSAs occurred in 13 patients (17%) and posttransplant DSAs in 37 patients (39%). In univariable analysis, pretransplant DSAs, appearance and recurrence of G1R findings, and total rejection score during the first posttransplant year, as well as recurrent G1R during follow-up, were all associated with CAV. In multivariable analysis, pretransplant DSAs and recurrent G1R during the first posttransplant year were found to be associated with CAV. Our results indicate that pretransplant DSA and recurrent G1R findings, especially during the first posttransplant year, are associated with CAV after pediatric HTx.

#3962 BALANCING THE RISK OF ALLOIMMUNITY WHILE MINIMIZING IMMUNOSUPPRESSIVE THERAPY: A RETROSPECTIVE STUDY OF PAEDIATRIC KIDNEY TRANSPLANT RECIPIENTS

Abstract Background and Aims Kidney transplantation is the best treatment for end-stage kidney disease, but life with a kidney allograft comes with significant challenges. Many paediatric kidney transplant recipients (KTRs) experience side effects from immunosuppressive medication. Reducing the medication is often necessary, but the reduction comes with a risk of inducing alloimmunity. We aimed to describe the alteration in immunosuppressive medication after kidney transplantation, the reasons for the reductions, and the possible impact on alloimmunity. Method 49 paediatric KTRs aged 1-16 years, receiving a kidney allograft from 2009 to 2020 in a Danish single centre were retrospectively studied. The standard protocol for immunologically low risk KTRs was steroid-free immunosuppression with basiliximab as induction, tacrolimus and mycophenolate mofetil (MMF) as maintenance therapy. KTRs were screened for anti-HLA antibodies with mixed and single bead antigen kits. We defined reduced immunosuppression as a KTR receiving one; OR two immunosuppressive medications AND MMF below 600 mg/m2. Results 49 KTRs received a kidney allograft, two were ABO incompatible transplantations and six KTRs had pretransplant donor specific antibodies. The median age of the KTRs was 11 years (IQR 8), with a median follow-up time of 5 years (IQR 5). In total, 78% KTRs received standard immunosuppressive therapy (steroid free). 5-year death censored (one KTR died) graft survival was 88%, and the cumulative incidence of rejection was 4% (95% CI: -1.5; 5.5) within the first year. None of the six graft losses were related to alloimmunity. 11.4% developed de novo donor specific antibodies (dnDSA) within a median of 3.91 years (IQR 2.28; range: 2.22-9.20) from transplantation. At transplantation, 6% received reduced immunosuppressive medication increasing to 74% at year one. Immunosuppression (primarily MMF) remained reduced in most of the KTRs during follow-up (Figure 1). Side effects predominantly to MMF led to the reduction. Leukopenia caused a reduction in 77%, gastrointestinal side effects in 34%, EBV in 19% (two KTRs developed PTLD), CMV in 15%, and BK polyomavirus in 15%. The reduction was significantly more pronounced in KTRs under 11 years (87% vs. 61%, p-0.044). Tacrolimus seemed well tolerated and was sufficiently regulated according to a target through of five microg/L two months after transplantation (Figure 2). Conclusion Despite 74% of the KTRs receiving reduced immunosuppressive medication according to our protocol, the rejection rate was low, the graft survival was comparable to European reports, and only 11% developed dnDSA during follow-up. Scheduled reduction of MMF shortly after transplantation could decrease the adverse side effects and improve the treatment of paediatric KTRs.

#5884 HLA COMPATIBILITY DOES NOT INFLUENCE KIDNEY GRAFT SURVIVAL AND DOES NOT IMPACT THE DEVELOPMENT OF POST-TRANSPLANT NEOPLASIA

Abstract Background and Aims HLA compatibility is a key issue in deceased and living kidney allocation, although the continuous improvement of immunosuppressive protocols might have reduced its relevance. However, an increased immunosuppression is associated with several adverse events, including the development of post-transplant neoplasia. Indeed, several reports demonstrated that the improvement in immunosuppressive drug efficiency corresponded to an increase incidence of malignancies after kidney transplantation. Thus, the aim of our study was to investigate the effect of HLA compatibility on kidney graft survival and incidence of post-transplant neoplasia. Method This is an observational, retrospective, multicenter center study including 1506 patients receiving a single kidney transplant at the Gemelli and Policlinico “Consorziale” kidney transplant centers from 1/1/2000 to 1/2/2022. The primary outcomes were death-censored graft survival and the development of post-transplant neoplasia. HLA compatibility was measured as the number of HLA mismatches (MM) at the three main class I (A and B) and class II (DR) loci. To this purpose, both donor and recipients were genotyped for HLA A, B and DR. We included in the analysis all deceased and living kidney donor transplants and excluded recipients from AB0 incompatible living kidney donors and with pre-transplant donor specific antibodies. Results Based on HLA compatibility, we divided our patients’ population in three groups (0-2 MM, 369 patients 3-4 MM, 856 patients and 5-6 MM, 281 patients). At univariate analysis (Kaplan-Meyer) number of MM were not associate with death-censored graft survival (Logrank p = 0.6) whereas we observed a significant association with the development of post-transplant neoplasia (Logrank p = 0.01). However, multivariate analysis (Cox model including recipient age, induction and maintenance immunosuppressive therapy) demonstrated that patients with 3-4 (HR 1.033, 95% CI 0.676-1.580, p = 0.9) and 5-6 MM (HR 1.342, 95% CI 0.853-2.111, p = 0.2) did not have any statistically significant increase in the risk to develop a post-transplant malignancy. Conclusion In this observational retrospective multicenter analysis, we demonstrated that HLA incompatibility, as indicated by the number of HLA A, B and DR MM, does not predict death-censored graft survival and does not impact the development of post-transplant neoplasia. Our observation would suggest that the missing effect of HLA compatibility on graft outcome is not associated with an increased long-term over-immunosuppression.

Impact of pre-transplant donor specific antibodies detected by Luminex with negative microlymphocytotoxicity assay and flow crossmatch in live-related renal transplant recipients.

The Luminex assay, where beads are coated with a single HLA antigen, has been shown to detect HLA antibodies with more sensitivity and specificity as compared to microlymphocytotoxicity (CDC) assay and flow cross match (FCXM). We report the impact of low Mean Flourescence intensity (MFI) pre-transplant DSA by Luminex with negative CDC and FCXM on acute rejection, graft function, and survival. In this retrospective study between January 2015 to December 2021, 45 recipients had pre-transplant anti-HLA donor-specific antibodies (DSAs) detected by Luminex. Two control groups of 45 patients each matched for age and gender, first with non-DSA HLA antibodies and second with no antibodies by Luminex were selected to compare outcomes with DSA group. In the DSA group of 45, 22 (48.8%) had class I (MFI mean 4043±1909, range: 1096-7111), 20 (44.4%) class II (MFI mean 3601±2310, range: 1031-9259), and 3 (6.6%) both class I (MFI mean 4746±1922) and class II (MFI mean 3940±2312) antibodies. Acute rejection episodes were reported in 15.6%, DSA group, 17.8% in non-DSA, and 24.4% in no antibody group (p=.538). Death censored graft survival at 1 and 5 years was 98% and 93% in DSA group, 100% and 95% in non-DSA and 93% and 85% in the no antibody group (p=.254). Patients with low MFI DSA pre-transplant, with a negative CDC and FCXM under ATG induction, have similar graft outcomes at 1 and 5 years when compared to non-DSA and no antibody groups.

Donation type and the effect of pre-transplant donor specific antibodies - Data from the Swiss Transplant Cohort Study.

The type of donation may affect how susceptible a donor kidney is to injury from pre-existing alloimmunity. Many centers are, therefore, reluctant to perform donor specific antibody (DSA) positive transplantations in the setting of donation after circulatory death (DCD). There are, however, no large studies comparing the impact of pre-transplant DSA stratified on donation type in a cohort with a complete virtual cross-match and long-term follow-up of transplant outcome. We investigated the effect of pre-transplant DSA on the risk of rejection, graft loss, and the rate of eGFR decline in 1282 donation after brain death (DBD) transplants and compared it to 130 (DCD) and 803 living donor (LD) transplants. There was a significant worse outcome associated with pre-transplant DSA in all of the studied donation types. DSA directed against Class II HLA antigens as well as a high cumulative mean fluorescent intensity (MFI) of the detected DSA showed the strongest association with worse transplant outcome. We could not detect a significant additive negative effect of DSA in DCD transplantations in our cohort. Conversely, DSA positive DCD transplants appeared to have a slightly better outcome, possibly in part due to the lower mean fluorescent intensity (MFI) of the pre-transplant DSA. Indeed when DCD transplants were compared to DBD transplants with similar MFI (<6.5k), graft survival was not significantly different. Our results suggest that the negative impact of pre-transplant DSA on graft outcome could be similar between all donation types. This suggests that immunological risk assessment could be performed in a similar way regardless of the type of donor kidney transplantation.

The detrimental effect of donor-specific antibodies is irrespective of its level in highly-immunized living donor kidney transplant recipients: A case-control series.

The impact of donor-specific antibodies (DSA) in (highly-) immunized living donor kidney transplant recipients is reported differentially in various patient cohorts. We have performed a retrospective analysis of all consecutive HLA-incompatible living donor kidney transplant recipients in our center between 2010-2019. Recipients who underwent plasmafiltration for a positive CDC-crossmatch were excluded. For each DSA+ recipient (DSA+), one immunized recipient without DSA (pPRA+) and two non-immunized recipients (pPRA-) were included. Patient and graft survival were analyzed and a subgroup analysis of DSA+ recipients was performed. For 63 DSA+ recipients, 63 PRA+ and 126 PRA- recipients were included. 26 (41%) had class I, 24 (38%) class II and 13 (21%) combined HLA class I and II DSA. Death-censored graft survival was inferior in DSA+ recipients compared to pPRA+ (HR 2.38 [95% CI 1.00-5.70]) as well as to pPRA- (HR 3.91 [1.86-8.22]). In multivariate analysis, DSA remained of negative influence on death-censored graft survival. Flowcytometric crossmatch, MFI value, HLA class and origin of DSA were not of significant impact. In our cohort of (highly-) immunized recipients, pretransplant DSA led to inferior death-censored graft survival. There were no "safe" DSA characteristics since only DSA per se impacted death-censored graft survival.

De novo Donor-specific Anti-human Leukocyte Antigen Antibody and Its Outcome in Pediatric Renal Transplant Recipients: A Single-center Experience in India

Development of de novo donor-specific anti-HLA antibody (dnDSA) is associated with poor graft survival in adults. However, there is a paucity of data about its prevalence and outcome in Indian children. We retrospectively assessed the proportion and spectrum of dnDSA and its outcome on antibody-mediated rejection (ABMR) and graft function. Children ≤18 years who were transplanted between November 2016 and October 2019 were included in this study. Pretransplant donor-specific antibody (DSA) was screened by complement-dependent cytotoxicity, flow cytometry crossmatch, and single antigen bead (SAB) class I and II by Luminex platform. Either antithymocyte globulin or basiliximab was used as induction. Tacrolimus, mycophenolate, and prednisolone were used for the maintenance of immunosuppression. SAB screening was done at 1, 3, 6 months, and yearly in seven children and at the time of acute graft dysfunction in eight. Mean fluorescence intensity ≥1000 was considered positive. Protocol biopsies were done at 3, 6, and 12 months and annually thereafter in seven children. Fifteen children, all males with a median age (interquartile range) of 13 years (11; 15.5) were analyzed. Only one child had pretransplant DSA who developed dnDSA posttransplant. Overall, 8 (53%) developed dnDSA over a median follow-up of 18 months. Seven (87%) had Class II, one Class I and 3 (37%) both Class I and II. Six had dQ and two had DR. All children with dnDSA had ABMR, of these two had subclinical rejection. DSAs persisted despite treatment, though graft function improved. Children with DSA and ABMR had lower graft function than those without DSA. The proportion of dnDSA was high in our study, majority against DQ. The detection of dnDSA prompted early diagnosis and treatment of ABMR.

Clinical relevance and characteristics of pretransplant donor-specific anti-human leukocyte antigen-DQ antibodies in kidney transplantation

Clinical relevance and characteristics of pretransplant donor-specific anti-human leukocyte antigen-DQ antibodies in kidney transplantation

On the clinical relevance of using complete high-resolution HLA typing for an accurate interpretation of posttransplant immune-mediated graft outcomes.

Complete and high-resolution (HR) HLA typing improves the accurate assessment of donor–recipient compatibility and pre-transplant donor-specific antibodies (DSA). However, the value of this information to identify de novo immune-mediated graft events and its impact on outcomes has not been assessed. In 241 donor/recipient kidney transplant pairs, DNA samples were re-evaluated for six-locus (A/B/C/DRB1/DQB1+A1/DPB1) HR HLA typing. De novo anti-HLA antibodies were assessed using solid-phase assays, and dnDSA were classified either (1) as per current clinical practice according to three-locus (A/B/DRB1) low-resolution (LR) typing, estimating donor HLA-C/DQ typing with frequency tables, or (2) according to complete six-locus HR typing. The impact on graft outcomes was compared between groups. According to LR HLA typing, 36 (15%) patients developed dnDSA (LR_dnDSA+). Twenty-nine out of 36 (80%) were confirmed to have dnDSA by HR typing (LR_dnDSA+/HR_dnDSA+), whereas 7 (20%) did not (LR_dnDSA+/HR_dnDSA−). Out of 49 LR_dnDSA specificities, 34 (69%) were confirmed by HR typing whereas 15 (31%) LR specificities were not confirmed. LR_dnDSA+/HR_dnDSA+ patients were at higher risk of ABMR as compared to dnDSA− and LR_dnDSA+/HR_dnDSA− (logRank < 0.001), and higher risk of death-censored graft loss (logRank = 0.001). Both LR_dnDSA+ (HR: 3.51, 95% CI = 1.25–9.85) and LR_dnDSA+/HR_dnDSA+ (HR: 4.09, 95% CI = 1.45–11.54), but not LR_dnDSA+/HR_dnDSA− independently predicted graft loss. The implementation of HR HLA typing improves the characterization of biologically relevant de novo anti-HLA DSA and discriminates patients with poorer graft outcomes.

The impact of pre-transplant donor specific antibodies on the outcome of kidney transplantation – Data from the Swiss transplant cohort study

BackgroundPre-transplant donor specific antibodies (DSA), directed at non-self human leukocyte antigen (HLA) protein variants present in the donor organ, have been associated with worse outcomes in kidney transplantation. The impact of the mean fluorescence intensity (MFI) and the target HLA antigen of the detected DSA has, however, not been conclusively studied in a large cohort with a complete virtual cross-match (vXM).MethodsWe investigated the effect of pre-transplant DSA on the risk of antibody-mediated rejection (ABMR), graft loss, and the rate of eGFR decline in 411 DSA positive transplants and 1804 DSA negative controls.ResultsPre-transplant DSA were associated with a significantly increased risk of ABMR, graft loss, and accelerated eGFR decline. DSA directed at Class I and Class II HLA antigens were strongly associated with increased risk of ABMR, but only DSA directed at Class II associated with graft loss. DSA MFI markedly affected outcome, and Class II DSA were associated with ABMR already at 500-1000 MFI, whereas Class I DSA did not affect outcome at similar low MFI values. Furthermore, isolated DSA against HLA-DP carried comparable risks for ABMR, accelerated eGFR decline, and graft loss as DSA against HLA-DR.ConclusionOur results have important implications for the construction and optimization of vXM algorithms used within organ allocation systems. Our data suggest that both the HLA antigen target of the detected DSA as well as the cumulative MFI should be considered and that different MFI cut-offs could be considered for Class I and Class II directed DSA.

216.15: Natural History of CXCR5+ t Follicular-like Helper (TFH) Cells in Kidney Transplant Recipients With Rejection

Introduction: T follicular-like helper cells (Tfh) have been associated with donor-specific antibody (DSA) and antibody-mediated rejection (ABMR) in kidney transplant recipients. However, changes in Tfh according to rejection type are less clear. We sought to describe the natural history of Tfh by rejection type. Methods: A pilot study of 4 recipients with ABMR were age-sex matched to recipients with TCMR, and recipients with no rejection. Prospectively collected blood samples were analysed retrospectively by cytometry by time of flight and manually gated to identify CD3+CD4+CXCR5+ Tfh (Figure 1). Pre-transplant Tfh frequency of CD3+ T cells was compared between rejection groups, pre-transplant DSA and de-novo DSA using student’s T-test. Change in Tfh frequency post-transplant was tested between rejection groups using linear mixed regression. Results: Pre-transplant Tfh frequency was highly variable between individuals (median=3.3%, IQR=1.1-5.9) and not significantly different between rejection groups (p=0.7), those with and without pre-transplant DSA (p=0.4) or those that developed and did not develop de-novo DSA (p=0.7). Early post-transplant Tfh frequency decreased in recipients with subsequent TCMR or stable function but increased in those who developed ABMR (Figure 2). Of the four recipients with AMBR, three had a Tfh frequency increase within 60 days prior to ABMR. Change in Tfh frequency over time was 2.7% (95%CI=0.3-5.2) higher in ABMR than stable recipients, adjusting for pre-transplant DSA and de-novo DSA (p<0.001). There was no difference in the change of Tfh frequency between TCMR and stable recipients. Conclusion: In the early post-transplant period and immediately before rejection, Tfh frequency increased above pre-transplant levels in patients who subsequently developed ABMR. This was not observed in stable patients or those with TCMR. These results will inform timepoints appropriate for screening in larger cohort studies. Kidney Health Australia. Ramaciotti Facility for Human Systems Biology. RPAH Transplant Insititute.

From bench to bedside: reversing established antibody responses and desensitization.

Basic transplant immunology has primarily focused on the definition of mechanisms, but an often-stated aspirational goal is to translate basic mechanistic research into future therapy. Pretransplant donor-specific antibodies (DSA) mediate hyperacute as well as early antibody-mediated rejection (AMR), whereas DSA developing late posttransplantation may additionally mediate chronic rejection. Although contemporary immunosuppression effectively prevents early cellular rejection after transplant in nonsensitized patients, it is less effective at controlling preexisting HLA antibody responses or reversing DSA once established, thus underscoring a need for better therapies. We here review the development of a bench-to-bedside approach involving transient proteasome inhibition to deplete plasma cells, combined with maintenance co-stimulation blockade, with CTLA-4Ig or belatacept, to prevent the generation of new antibody-secreting cells (ASCs). This review discusses how this treatment regimen, which was rationally designed and validated to reverse established DSA responses in mouse models, translated into reversing active AMR in the clinic, as well as desensitizing highly sensitized patients on the transplant waitlist.

Clinical significance of low pre-transplant donor specific antibodies (DSA) in living donor kidney recipients with negative complement-dependent cytotoxicity crossmatches (CDCXM), and negative flow cytometry crossmatches (FLXM) – A single-center experience

BackgroundIt is controversial whether all donor-specific antibodies (DSA) detected by the solid-phase single antigen bead (SAB) assay negatively affect kidney transplantation outcomes. The study aimed to evaluate the possible clinical significance of low pre-transplant DSA in living donor kidney recipients. We analyzed a group of patients with HLA-A, B, and -DR DSA reactivities below a virtual crossmatch (VXM) value of 5000 MFI but with all VXM DSA reactivities at HLA-DQ, -DP, and -Cw, which were not typed routinely for donors prior to transplantation. We also investigated the incidence of persistent and de novo DSAs in available posttransplant SAB assays. MethodsFrom the historical cohort of living donor recipients transplanted between 2014 and 2018 at our center (n = 82), 55 patients met the inclusion criteria, namely: these patients were > 18 years old with non-HLA identical sibling donors, who were not desensitized, who had available pre-transplant SAB results, and who had negative both complement-dependent cytotoxicity crossmatch (CDCXM) and flow cytometry crossmatch (FLXM) results. An additional donor HLA typing, performed for all 55 recipients, identified donor additional HLA-DQ, -DP, and -Cw DSA reactivities. These patients were then divided by SAB reactivity into three groups: 1) those with DSA-positive reactivities; 2) those with non-donor-specific anti-HLA reactivities (NDSA); and, 3) those who were anti-HLA-negative. All these recipients were followed for three years and checked for their de novo or persistent DSA. ResultsIn the studied cohort, DSA-positive, NDSA reactive, and anti-HLA negative recipients constituted 33%, 36%, and 31% of 55 patients, respectively. Non-routinely considered pre-transplant HLA-DQ, -DP, and -Cw DSA-positive reactivities were shown in as many as 78% of DSA-positive cases (group 1) with the lowest MFI value of 319 to DP4 and the highest MFI of 5767 to DQ2. Of the pre-transplant HLA-A, B, and -DR DSA reactivities, only -DR52 DSA reactivity reached the highest MFI value of 2191. These detected DSAs did not reduce the mean estimated glomerular filtration rate (eGFR) values and did not increase the incidence of proteinuria in recipients. While the 3-year graft survival was lower in the DSA-positive group (94.4%) with one recipient who lost kidney transplant, the difference was not significantly different (p = 0.7) from the NDSA (100%) and negative (100%) groups. In terms of the incidence of de novo acute antibody-mediated rejection (AMR) at three years after transplantation, no case has been reported in the cohort. This may suggest that low DSA-positive recipients do not experience higher rejection rate. However, DSA-positive recipients had a tendency for a higher frequency of C4d deposits in peritubular capillaries (PTC) and de novo DSA. ConclusionOur 3-year follow-up of patients with low pre-transplant DSA found no association with a deterioration in graft function and worse graft survival. Furthermore, we did not observe an increase in AMR in our patients with low DSA. A larger cohort and a longer follow-up period may be needed to evaluate the tendency of low DSA-positive recipients towards the higher incidence of C4d deposits in PTC and/or de novo DSA.

The Accuracy of Sequence-Specific Oligonucleotide and Real-Time Polymerase Chain Reaction HLA Typing in Determining the Presence of Pre-Transplant Donor-Specific Anti-HLA Antibodies and Total Eplet Mismatches for Deceased Donor Kidney Transplantation

High resolution human leukocyte antigen (HLA) typing is important in establishing eplet compatibility and the specificity of donor-specific anti-HLA antibodies (DSA). In deceased donor kidney transplantation, high resolution donor HLA typing may not be immediately available, leading to inaccuracies during the organ allocation process. We aimed to determine the concordance and agreement of HLA-Class I and II eplet mismatches calculated using population frequency based allelic haplotype association (linkage disequilibrium, LD) from sequence-specific oligonucleotide (SSO) and real-time polymerase chain reaction (rtPCR) donor HLA typing (available at time of donor kidney allocation) compared to high-resolution Next Generation Sequencing (NGS) donor typing. NGS high resolution HLA typing were available for all recipients prior to donor kidney allocation. A cohort of 94 deceased donor-recipient pairs from a single Western Australian center were included (77 individual donors typed, 55 local and 22 interstate). The number of class I (HLA-A+B+C) and class II (HLA-DRB1+DRB3/4/5+DQB1+DQA1+DPB1+DPA1) eplet mismatches were calculated using HLAMatchmaker, comparing LD- and NGS-HLA typing. The accuracy in assigning pre-transplant DSA was compared between methods. The concordance correlation coefficient (95%CI) for HLA-class I and II eplet mismatches were 0.994 (0.992 to 0.996) and 0.991 (0.986 to 0.993), respectively. The 95% limits of agreement for class I were -1.3 (-1.6 to -1.1) to 1.4 (1.2 to 1.7) and -4.8 (-5.7 to -3.9) to 5.0 (4.1 to 5.9) for Class II. Disagreement between the two methods were present for 11 and 37 of the Class I and II donor/recipient pairs. Of which, 5 had a difference of ≥5 class II eplet mismatches. There were 34 (36%) recipients with potential pre-transplant DSA, of which 8 (24% of recipients with DSA) had indeterminate and ultimately false positive DSA assigned by donor LD-typing. While the concordance between NGS- and LD-typing was high, the limits of agreement suggest meaningful differences between these two techniques. The inaccurate assignment of DSA from donor LD-typing may result in associated HLA being considered unacceptable mismatches, inappropriately precluding candidates’ access to transplantation. Accurate imputation of two-field HLA alleles based on LD from SSO and rtPCR HLA typing remains a substantial challenge in clinical practice in-lieu of widely available, rapid, high-resolution methods.

Causes of Kidney Graft Failure in a Cohort of Recipients With a Very Long-Time Follow-Up After Transplantation.

BackgroundBiopsy-proven causes of graft loss many years after kidney transplantation are scarcely documented.MethodsPatients transplanted between 1995 and 2005 (n = 737) in a single center were followed on a regular basis until 2021. The recipients were divided according to age at transplantation into 3 groups; 18–39 years (young), 40–55 years (middle age), and older than 55 years (elderly). For cause biopsies of renal transplants were clustered into the categories, rejection, IFTA, return original disease, and diagnosis of de novo kidney disease.ResultsRejection was the main cause of graft failure censored for death at every time period after transplantation. The incidence of T cell-mediated rejection (TCMR) became rare 6 years after transplantation while the cumulative incidence of antibody-mediated rejection (ABMR) increased over time (1.1% per year). ABMR was not diagnosed anymore beyond 15 years of follow-up in recipients without pre-transplant donor-specific antibodies (DSA). An episode of TCMR was associated with an increased incidence of ABMR diagnosis in the short-term but did not increase the overall incidence of AMBR not in the long-term. Death as a cause of graft failure was an important competitive risk factor long after transplantation and resulted in a significantly lower frequency of rejection-related graft loss in the elderly group (11 vs. 23% in the young group at 15 year follow-up).ConclusionRejection is a major cause of graft loss but recipient’s age, time after transplantation, and the presence of DSA before transplantation determine the relative contribution to overall graft loss and the type of rejection involved.

The clinical utility and thresholds of virtual and Halifaster flow crossmatches in lung transplantation

Immune sensitization, defined as the presence of alloreactive donor-specific antibodies (DSA), is associated with increased wait-times and inferior transplant outcomes. Identifying pretransplant DSA with a physical cell-based assay is critical in defining immunological risk. However, improved solid phase antibody detection has provided the potential to forgo this physical assay. Here, we evaluated the association between DSA mean fluorescence intensity (MFI) and the recently introduced Halifaster Flow cytometry crossmatch (FXM) to determine if MFI could predict the outcome of FXM and whether a virtual crossmatch (VXM) would provide an accurate risk assessment. Sera from 134 waitlisted lung patients was retrospectively assessed by Halifaster FXM against lymphocytes preparations from 32 donors, resulting in 265 FXMs. HLA typing was performed to 2-field allelic level and Luminex single antigen beads (SAB) used to identify DSA. The association between FXM and Luminex MFI was calculated using ROC analysis. MFI threshold accuracy was confirmed using a separate validation cohort (174 recipient sera and 34 donors), whereby both VXM and FXMs were compared. From the 265 FXM performed, 48 (18%) T-cell (TFXM) and 56 (21%) B-cell (BFXM) were positive. In the evaluation cohort, MFI thresholds of 2000 for HLA-A, B, DRB1, and > 4000 for DQB1, were predictive of a positive FXM. The validation cohort of 233 paired FXM and VXM confirmed these MFI thresholds for both TFXM and BFXM with an accuracy of 91.4% and 89.3%, respectively. A positive VXM, defined with HLA-specific MFI thresholds predicts Halifaster FXM reactivity, and can potentially expedite organ allocation, by minimizing the need for the more time-consuming FXM.