non-HLA Antibodies Research Articles

Comprehensive Characterization of Anti-HLA and Non-HLA Antibodies in Patients on Kidney Transplant Waiting List and Evaluation of Their Impact on Alloimmunization Risk and Dialysis Treatment

Alloimmunization remains a major obstacle to successful kidney transplantation, mainly due to the formation of anti-HLA antibodies. In recent years, non-HLA antibodies have emerged as additional immunologic factors that can potentially contribute to graft rejection. The aim of this study was to investigate the prevalence and specificity of both anti-HLA and non-HLA antibodies in patients with end-stage renal disease on a waiting list for kidney transplantation. Serum samples from 74 patients were analyzed using complement-dependent cytotoxicity and solid-phase assays. IgG anti-HLA antibodies were identified in 43.2% of participants, while IgG non-HLA antibodies were detected in 91.9%. The most frequent non-HLA antibodies included anti-ENO1 (28.4%), anti-FIBR1 (23.0%) and anti-PRKCZ (23.0%). A significant difference was found between the number of distinct IgG anti-HLA and IgG non-HLA antibody specificities. However, no significant correlation was found between the number of IgG non-HLA antibody specificities and previous alloimmunization events or dialysis treatments. These results suggest that non-HLA antibodies, although often overlooked, can sometimes play a critical role in transplant outcomes. Routine testing for non-HLA antibodies, in addition to mandatory anti-HLA antibody screening and identification, could improve immunologic risk assessment in transplant patients and post-transplant care.

De novo granulocyte–macrophage colony-stimulating factor antibody production has been linked to acute graft-versus-host disease following hematopoietic stem cell transplantation from an HLA-matched, unrelated donor

The role of non-HLA antibodies in hematopoietic stem cell transplantation (HSCT) and acute graft-versus-host disease (aGVHD) is not established. Serum samples collected from 58 adult patients before and after HSCT were examined for non-HLA antibodies. Following HSCT, 47 out of 58 patients (81.0 %) had various antibody patterns, with 23 of them (39.7 %) producing denovo antibodies. The most prevalent antibodies were directed against granulocyte–macrophage colony-stimulating factor (GM-CSF). The Fisher exact test revealed a statistically significant correlation between the incidence of acute graft-versus-host disease and denovo production of GM-CSF antibodies in patients fully HLA-matched with their donors (p = 0.001). There were no cases of denovo GM-CSF antibody production seen in non-permissively HLA-mismatched patients. Consequently, we hypothesize that the development of aGVHD after HLA-matched HSCT may differ from that following HLA-mismatched HSCT.

Distinct Non-Human Leukocyte Antigen Antibody Signatures Correlate with Endothelial Crossmatch Status in Lung and Renal Transplant Recipients

Non-HLA antibodies against heterogeneous targets on endothelial cells have been associated with allograft injuries. The endothelial cell crossmatch (ECXM) is used in the detection of non-HLA antibodies but remains non-discriminatory for specific antibody identification. The primary objective of this study was to delineate the specific non-HLA antibody signatures associated with ECXM positivity and to determine the correlation of ECXM status and non-HLA antibody signatures on allograft health. Serum specimens from 25 lung transplant recipients (LTRs) and 13 renal transplant recipients (RTRs) were collected as part of clinical evaluation, and testing for angiotensin II receptor type 1 (AT1R) and donor-specific MHC class I chain-related gene A (MICA) antibodies and ECXM was performed. Remnant sera were tested for non-HLA antibodies using the LABScreen™ Autoantibody (LSAUT) Group 1, 2, and 3 kits (One Lambda, Inc., Los Angeles, CA, USA). In both cohorts, the concordance of AT1R and MICA together or individually with ECXM+ status was poor (<0.7), suggesting the presence of other unaccounted antibodies. Autoantibody profiling revealed three distinct clusters targeting fibrotic products, cytoskeletal proteins, and cell signaling molecules. A comparative analysis of ECXM+ and ECXM− specimens identified nine and five differentially expressed antibodies in the LTR and RTR cohorts, respectively. Employing machine learning techniques (variable importance, feature selection, ROC-AUC), we derived a five-antibody panel (TNFα, collagen V, CXCL11, GDNF, GAPDH) and a two-antibody panel (TNFα, CXCL9) that effectively discriminated between ECXM+ and ECXM− status in the LTR and RTR cohorts, respectively. Distinct antibody signatures were identified in LTR and RTR cohorts that correlated with ECXM+ status and were associated with allograft dysfunction.

Transplantation Genetics: The Importance of non-HLA Antibodies, Genetic Insights and Future Perspectives

Solid organ transplantation is an effective and life-saving treatment method for patients with end-stage organ failure. Immunological evaluation is of great importance in the achievement of transplantation, and the determination of HLA incompatibility forms the basis of immunological transplantation success. The presence of donor HLA-related antibodies is known to be asssociated with graft loss and decreased survival rates. However, recent studies have shown clinical and experimental findings related to the important role of non-HLA antibodies in the antibody-mediated rejection and chronic rejection process. The mechanisms triggering auto-antibody production in organ transplantation are an important area of research. These mechanisms stimulate auto-antibody production in the patient causing the expression of organ-origin autoantigens such as ischaemia-reperfusion damage, surgical trauma, alloimmune responses, soluble antigens, extracellular vesicles and apoptotic bodies. In patients with a risk of non-HLA antibody-mediated rejection, there is an urgent need to determine the recipient immunological phenotypes, both for the development of targetted treatments and to increase both graft and patient survival. The aim of this review was to evaluate non-HLA antibody types and their effects on transplantation.

Synergistic effect of DSA and non-HLA antibodies on T cell-mediated rejection of adult liver allografts

Synergistic effect of DSA and non-HLA antibodies on T cell-mediated rejection of adult liver allografts

Confounding variables such as previous transplants, gender, ethnicity, and primary disease complicate the diagnostic validity of single-antigen bead-based non-HLA antibody assays

Confounding variables such as previous transplants, gender, ethnicity, and primary disease complicate the diagnostic validity of single-antigen bead-based non-HLA antibody assays

Absence of the GSTT1 gene in a recipient can cause anti-GSTT1 non-HLA antibody development contributing to AMR

Absence of the GSTT1 gene in a recipient can cause anti-GSTT1 non-HLA antibody development contributing to AMR

Effect of angiotensin II type 1 receptor antibodies on graft function and survival in paediatric kidney transplant recipients.

HLA donor specific antibodies (DSA) are implicated in antibody-mediated rejection (AMR), graft dysfunction and failure in kidney transplant (KT) recipients. Non-HLA antibodies including angiotensin II type 1 receptor (AT1R) may also play a role in AMR, impact graft function and survival. Data is limited in paediatric KT cohorts. We aimed to assess the prevalence and effect of pre-transplant AT1R antibodies on rejection, graft function and survival in paediatric KT recipients. This was a retrospective cohort study conducted across two paediatric centres including KT recipients with a pre-transplant AT1R antibody level. Outcomes included rejection, de novo DSA formation, graft function, failure, proteinuria and hypertension. Of 71 individuals, 72% recorded a positive pre-transplant AT1R Ab level (≥17 U/mL). Over a median follow-up of 4.7 years, AT1R Ab positivity demonstrated a trend towards increased risk of rejection however was not statistically significant (HR 3.45, 95% CI 0.97-12.35, p-value 0.06). Sensitivity analysis with AT1R Ab levels of ≥25 U/mL (HR 2.05 95% CI 0.78-5.39, p-value 0.14) and ≥40 U/mL (HR 1.32, CI 95% 0.55-3.17, p-value 0.53) validated this. De novo DSA formation occurred more frequently with AT1R Ab positivity (41% vs. 20%, p-value 0.9). AT1R Ab was not associated with hypertension, proteinuria, graft failure or dysfunction. In conclusion, this cohort study demonstrated a high prevalence of pre-transplant AT1R Ab positivity (72%). AT1R Ab positivity demonstrated a trend towards increased risk of rejection and de novo DSA formation however did not meet statistical significance. There was no association between AT1R Ab and hypertension, proteinuria, graft failure or dysfunction.

P.173: The impact of post-transplant non-HLA antibody burden on the occurrence of antibody-mediated rejection and graft loss in non-sensitized pediatric kidney recipients.

P.173: The impact of post-transplant non-HLA antibody burden on the occurrence of antibody-mediated rejection and graft loss in non-sensitized pediatric kidney recipients.

Screening for Non-HLA Antibodies – the very first results

Screening for Non-HLA Antibodies – the very first results

Use of Tocilizumab in the treatment of chronic active antibody-mediated rejection in pediatric kidney transplant recipients

Chronic active antibody-mediated rejection is one of the leading causes of graft failure and traditional therapies have unclear efficacy. Recent studies suggested that Tocilizumab could stabilize renal function and improve microvascular inflammation. Here we report the outcomes of Tocilizumab therapy in 6 pediatric kidney transplant recipients with biopsy-proven chronic active antibody-mediated rejection resistant to standard treatments. All patients received monthly Tocilizumab infusions for 6 months and were monitored for renal function (creatinine, estimated glomerular filtration rate (eGFR), proteinuria) and Human Leukocyte Antigens (HLA) and non-HLA antibodies at baseline and 3 and 6 months after Tocilizumab initiation. For each patient, a follow-up biopsy was scheduled at the end of the treatment. Renal function did not show stabilization or improvement (mean eGFR 37 ml/min/1.73 m2 pre-Tocilizumab and 27 ml/min/1.73 m2 3 months after-Tocilizumab) and proteinuria remained stable. Moreover, Tocilizumab had no impact on HLA and non-HLA antibodies. Graft loss was observed in 3 patients (50 %) and 4 patients who underwent post-treatment biopsy showed a worsening in overall chronicity scores. In our pediatric series, rescue therapy with Tocilizumab did not appear to be effective in modifying the natural history of chronic active antibody-mediated rejection.

Local intragraft humoral immune responses in chronic lung allograft dysfunction

IntroductionDonor human leukocyte antigen (HLA)-specific antibodies (DSA) and non-HLA antibodies can cause allograft injury, possibly leading to chronic lung allograft dysfunction (CLAD) after lung transplantation (LTx). It remains unclear whether these antibodies are produced locally in the graft or derived solely from circulating plasma cells. We hypothesized that DSA and non-HLA antibodies are produced in CLAD lungs. MethodsLung tissue was prospectively collected from 15 CLAD patients undergoing re-LTx or autopsy. 0.3g of fresh lung tissue was cultured for 4 days without or with lipopolysaccharide or with CD40L: resulting lung culture supernatant (LCS) was sampled. Protein eluate was obtained from 0.3g of frozen lung tissue. Mean fluorescence intensity (MFI) of DSA and non-HLA antibodies was measured by Luminex and antigen microarray, respectively. ResultsLCS from all 4 patients who had serum DSA at lung isolation were positive for DSA, with higher levels measured after lung tissue stimulation with CD40L (CD40L+LCS). Of these, only 2 had detectable DSA in the lung eluate. MFI of non-HLA antibodies from CD40L+LCS correlated with those from lung eluate, but not with those from sera. Flow cytometry showed higher frequencies of activated lung B cells for patients whose CD40L+LCS was positive for DSA (n=4) or high non-HLA antibodies (n=6) compared to those with low local antibodies (n=5). Immunofluorescence staining showed CLAD lung lymphoid aggregates with local antibodies contained larger numbers of IgG+ plasma cells and greater IL-21 expression. ConclusionsWe show that DSA and non-HLA antibodies can be produced within activated B cell-rich lung allografts.

Evaluation of non-invasive biomarkers of kidney allograft rejection in a prospective multicenter unselected cohort study (EU-TRAIN)

Non-invasive biomarkers are promising tools for improving kidney allograft rejection monitoring, but their clinical adoption requires more evidence in specifically designed studies. To address this unmet need, we designed the EU-TRAIN study, a large prospective multicentric unselected cohort funded by the European Commission. Here, we included consecutive adult patients who received a kidney allograft in nine European transplant centers between November 2018 and June 2020. We prospectively assessed gene expression levels of 19 blood messenger RNAs, four antibodies targeting non-human leukocyte antigen (HLA) endothelial antigens, together with circulating anti-HLA donor-specific antibodies (DSA). The primary outcome was allograft rejection (antibody-mediated, T cell-mediated, or mixed) in the first year post-transplantation. Overall, 412 patients were included, with 812 biopsies paired with a blood sample. CD4 gene expression was significantly associated with rejection, while circulating anti-HLA DSA had a significant association with allograft rejection and a strong association with antibody-mediated rejection. All other tested biomarkers, including AKR1C3, CD3E, CD40, CD8A, CD9, CTLA4, ENTPD1, FOXP3, GZMB, ID3, IL7R, MS4A1, MZB1, POU2AF1, POU2F1, TCL1A, TLR4, and TRIB1, as well as antibodies against angiotensin II type 1 receptor, endothelin 1 type A receptor, C3a and C5a receptors, did not show significant associations with allograft rejection. The blood messenger RNAs and non-HLA antibodies did not show an additional value beyond standard of care monitoring parameters and circulating anti-HLA DSA to predict allograft rejection in the first year post-transplantation. Thus, our results open avenues for specifically designed studies to demonstrate the clinical relevance and implementation of other candidate non-invasive biomarkers in kidney transplantation practice.

Evaluating the Clinical Relevance of Antibodies against Non-Human Leukocyte Antigen in Kidney Transplantation.

Introduction: Kidney transplantation is the preferred modality of kidney replacement therapy for eligible patients with end-stage kidney disease (ESKD), given that it has been found to reduce mortality rates, improve quality of life, and is cost-effective compared to dialysis. Recent advancements in human leukocyte antigen (HLA) typing and donor-specific antibody (DSA) detection have helped to reduce the risk of rejection, but antibody-mediated rejection (AMR) can still occur without DSA. Previous studies suggest that rejection can be attributed to antibodies against Non-Human Leucocyte Antigens (non-HLAs). We aimed to acquire further understanding of the prevalence and distribution of non-HLA antibodies in our local population and attempt to correlate these findings with graft outcomes, as well as assess whether non-HLA antibodies can be utilized to determine graft impairment and dysfunction. Methods: We conducted a retrospective study involving kidney transplant recipients between January 2010 and December 2020. All included individuals were aged over 18 and underwent kidney-alone transplants; were ABO- and HLA-compatible; and were matched at A, B, and DR loci (mismatch 0:0:0). HLA testing was negative at the time of transplantation. The samples from both cases of early graft rejection and the control group were tested for non-HLA antibodies using One Lambda LABScreenTM, Autoantibody kit groups 1, 2, and 3, as well as the Immucor LIFECODES non-HLA autoantibody assay. Results: A total of 850 kidney transplant recipients were included, in which 12 patients experienced early graft rejection within the first month post transplant and 18 patients who did not experience graft rejection were selected as study controls. Our study reported no correlation between the total burden of non-HLA antibodies and early rejection, most likely as the result of a small sample size. Nevertheless, a sub-analysis revealed that specific high-frequency pre-transplant non-HLA antibodies such as GSTT, CXCL11, CXCL10, and HNR, detected by LIFECODES, were associated with rejection (Fisher's exact test with Bonferroni correction, p < 0.001). Most pre-transplant non-HLA antibody levels were reduced after transplantation, which was attributed to immunosuppression. Conclusion: The 'high frequency' non-HLA antibodies displayed an association with graft rejection, though the overall associations between the burden of non-HLA antibodies and rejection episodes remain inconclusive. Further work is needed to establish the rebound phenomenon of non-HLA antibodies, the development of de novo non-HLA antibodies in the long run, and their implications on graft survival.

Outcome-based Risk Assessment of Non-HLA Antibodies in Heart Transplantation: A Systematic Review

Current monitoring after heart transplantation (HT) employs repeated invasive endomyocardial biopsies (EMB). Although positive EMB confirms rejection, EMB fails to predict impending, subclinical, or EMB-negative rejection events. While non-human leukocyte antigen (non-HLA) antibodies have emerged as important risk factors for antibody-mediated rejection after HT, their use in clinical risk stratification has been limited. A systematic review of the role of non-HLA antibodies in rejection pathologies has the potential to guide efforts to overcome deficiencies of EMB in rejection monitoring. Databases were searched to include studies on non-HLA antibodies in HT recipients. Data collected included the number of patients, type of rejection, non-HLA antigen studied, association of non-HLA antibodies with rejection, and evidence for synergistic interaction between non-HLA antibodies and donor-specific anti-human leukocyte antigen antibody (HLA-DSA) responses. A total of 56 studies met the inclusion criteria. Strength of evidence for each non-HLA antibody was evaluated based on the number of articles and patients in support versus against their role in mediating rejection. Importantly, despite previous intense focus on the role of anti-major histocompatibility complex class I chain-related gene A (MICA) and anti-angiotensin II type I receptor antibodies (AT1R) in HT rejection, evidence for their involvement was equivocal. Conversely, the strength of evidence for other non-HLA antibodies supports that differing rejection pathologies are driven by differing non-HLA antibodies. This systematic review underscores the importance of identifying peri-HT non-HLA antibodies. Current evidence supports the role of non-HLA antibodies in all forms of HT rejection. Further investigations are required to define the mechanisms of action of non-HLA antibodies in HT rejection.

#2806 Role of non-HLA antibodies in pre-transplant evaluation of antibody-mediated damage risk

Abstract Background and Aims Donor-specific HLA antibodies (HLA-DSA) contribute to antibody-mediated rejection (ABMR) after kidney transplantation (KT) and worse graft-survival. Non-HLA antibodies may play a role in the development of ABMR in the presence of HLA-DSA or in the development of microvascular inflammation (MVI) in the absence of HLA-DSA. Considering both antibodies in potential recipients before or after KT could enhance the prediction of post-KT ABMR/MVI. Method Retrospective study of a cohort of 169 KT recipients with: a) pre- and post-KT sera; b) follow-up HLA antibody monitoring; and c) follow-up biopsies. We determined the presence of 60 non-HLA antibodies in sera with a multiplex test: the Single Non-HLA Beads kit (LIFECODES®) on a Luminex® platform. We obtained an absolute number of positive antibodies and a sample MFI ratio sum according to which we stratified results into quartiles. Results Pre-KT, we detected a 15.4% of KT recipients with high HLA sensitization (cPRA &amp;gt;50%), being 8.3% HLA-DSA, and non-HLA antibodies in 85% of KT recipients, with a median of 3 (2-5) antibodies and a median MFI ratio sum of 10.1 (2.9-18.5). During a median follow-up of 60 (18.5-89) months 73 patients developed ABMR/MVI. We observed that a high burden of pre-KT non-HLA antibodies was associated with a higher risk of ABMR/MVI, regardless of HLA sensitization and pre-KT HLA-DSA. Notably, 57% of patients with ABMR/MVI had a high non-HLA burden before KT and 24.6% had a HLA cPRA &amp;gt;50%. Altogether, 68.5% of these patients showed either a high HLA sensitization or a high non-HLA burden before KT (Figure). Conclusion A high non-HLA preTR non-HLA antibody burden detected with a multiplex panel allows identification of patients more likely to develop ABMR/MVI, irrespective of the presence of anti-HLA antibodies.

#2239 Identification of non-HLA antibodies associated with the development of antibody-mediated damage after kidney transplantation

Abstract Background and Aims Non-HLA antibodies may play a role in the development of antibody-mediated rejection (ABMR) in the presence of donor-specific anti-HLA antibodies (HLA-DSA) or microvascular inflammation (MVI) without HLA-DSA in kidney transplantation (KT). The development of multiplex panels allowed the analysis of multiple antibodies simultaneously and new antibodies potentially related to ABMR/MVI have been identified. Method Retrospective study of a cohort of 169 KT recipients with: a) pre- and post-KT sera; b) follow-up HLA antibody monitoring; and c) follow-up biopsies. We determined the presence of 60 non-HLA antibodies in sera using a multiplex test: the Single Non-HLA Beads kit (LIFECODES®) suitable for the SAB assay performed using Luminex® technology. Results Pre-KT, we detected non-HLA antibodies in 85% of the recipients, with a median of 3 (2-5) positive antibodies. During follow-up, 73 patients developed ABMR/MVI. Pre-KT, in a univariate analysis including the presence of HLA-DSA (8.3% in the cohort) we detected 7 antibodies individually associated with ABMR/MVI. In multivariate analysis (Figure) GSTT1, NCL, PLA2R1 and Thyroglobulin antibodies together with HLA DSA were significantly associated with the development of ABMR/MVI, although only GSST1 and Thyroglobulin appear in a relevant number of patients (&amp;gt;10 patients) involved in 32 cases of ABMR and 6 cases of MVI. Post-KT, only the detection of anti-GSTT1 antibodies in sera 1- and 3-years post-TR was associated with ABMR/MVI in 3-year biopsies. Conclusion Pre-KT the detection of antibodies against GSST1 and Thyroglobulin using a multiplex non-HLA antibody detection panel is independently associated with the development of ABMR/MVI during follow-up. Post-KT detection of GSST1 is also associated with ABMR/MVI in 3-year biopsies.

Non-HLA angiotensin-type-1 receptor autoantibodies mediate the long-term loss of grafted neurons in Parkinson’s disease models

BackgroundClinical trials have provided evidence that transplants of dopaminergic precursors, which may be replaced by new in vitro stem cell sources, can integrate into the host tissue, and alleviate motor symptoms in Parkinson´s disease (PD). In some patients, deterioration of graft function occurred several months after observing a graft-derived functional improvement. Rejection of peripheral organs was initially related to HLA-specific antibodies. However, the role of non-HLA antibodies is now considered also relevant for rejection. Angiotensin-II type-1 receptor autoantibodies (AT1-AA) act as agonists of the AT1 receptors. AT1-AA are the non-HLA antibodies most widely associated with graft dysfunction or rejection after transplantation of different solid organs and hematopoietic stem cells. However, it is not known about the presence and possible functional effects of AT1-AA in dopaminergic grafts, and the effects of treatment with AT1 receptor blockers (ARBs) such as candesartan on graft survival.MethodsIn a 6-hydroxydopamine PD rat model, we studied the short-term (10 days)- and long-term (3 months) effects of chronic treatment with the ARB candesartan on survival of grafted dopaminergic neurons and microglial graft infiltration, as well as the effects of dopaminergic denervation and grafting on serum and CSF AT1-AA levels. The expression of AT1 receptors in grafted neurons was determined by laser capture microdissection.ResultsAt the early period post-grafting, the number of grafted dopaminergic neurons that survived was not significantly different between treated and untreated hosts (i.e., control rats and rats treated with candesartan), probably because, just after grafting, other deleterious factors are predominant for dopaminergic cell death, such as mechanical trauma, lack of growth factors/nutrients and ischemia. However, several months post-grafting, we observed a significantly higher number of surviving dopaminergic neurons and a higher density of striatal dopaminergic terminals in the candesartan-treated group. For several months, grafted rats showed blood and cerebrospinal fluid levels of AT1-AA higher than normal controls, and also higher AT1-AA levels than non-grafted parkinsonian rats.ConclusionsThe results suggest the use of ARBs such as candesartan in PD patients, particularly before and after dopaminergic grafts, and the need to monitor AT1-AA levels in PD patients, particularly in those candidates for dopaminergic grafting.

Clinical conundrums in pediatric kidney transplantation: What we know about the role of angiotensin II type I receptor antibodies in pediatric kidney transplantation and the path forward.

Antibodies to angiotensin II type 1 receptor (AT1R-Abs) are among the most well-studied non-HLA antibodies in renal transplantation. These antibodies have been shown to be common in pediatric kidney transplantation and associated with antibody-mediated rejection (AMR), vascular inflammation, development of human leukocyte donor-specific antibodies (HLA DSA), and allograft loss. As AT1R-Ab testing becomes more readily accessible, evidence to guide clinical practice for testing and treating AT1R-Ab positivity in pediatric kidney transplant recipients remains limited. This review discusses the clinical complexities of evaluating AT1R-Abs given the current available evidence.

(255) - Non-HLA Antibodies Are Associated with Chronic Lung Allograft Dysfunction in Lung Transplant Recipients

(255) - Non-HLA Antibodies Are Associated with Chronic Lung Allograft Dysfunction in Lung Transplant Recipients