DQ Antibodies Research Articles

Functional epitope analysis of serum samples from transplant patients using LABScreen™, MagSort™, and cell-based adsorption elution reveals a deeper understanding of DQ antibody specificities

Functional epitope analysis of serum samples from transplant patients using LABScreen™, MagSort™, and cell-based adsorption elution reveals a deeper understanding of DQ antibody specificities

The efficacy and safety of protein A immunoadsorption combined with rituximab treatment for highly sensitized patients undergoing haplo-hematopoietic stem cell transplantation

Objective: To investigate the efficacy and safety of protein A immunoadsorption (PAIA) combined with rituximab (RTX) in highly sensitized patients who underwent haplo-hematopoietic stem cell transplantation (haplo-HSCT) . Methods: The clinical data of 56 highly sensitized patients treated with PAIA and RTX before haplo-HSCT at the First Affiliated Hospital of Soochow University and Soochow Hopes Hematonosis Hospital between March 2021 and June 2023 were retrospectively analyzed. The number of human leukocyte antigen (HLA) antibody types and the mean fluorescence intensity (MFI), humoral immunity, adverse reactions during adsorption, and survival within 100 days before and after adsorption were measured. Results: After receiving the PAIA treatment, the median MFI of patients containing only HLA Ⅰ antibodies decreased from 7 859 (3 209-12 444) to 3 719 (0-8 275) (P<0.001), and the median MFI of HLA Ⅰ+Ⅱ antibodies decreased from 5 476 (1 977-12 382) to 3 714 (0-11 074) (P=0.035). The median MFI of patients with positive anti-donor-specific antibodies decreased from 8 779 (2 697-18 659) to 4 524 (0-15 989) (P<0.001). The number of HLA-A, B, C, DR, and DQ antibodies in all patients decreased after the PAIA treatment, and the differences were statistically significant (A, B, C, DR: P<0.001, DQ: P<0.01). The humoral immune monitoring before and after the PAIA treatment showed a significant decrease in the number of IgG and complement C3 (P<0.001 and P=0.002, respectively). Forty-four patients underwent HLA antibody monitoring after transplantation, and the overall MFI and number of antibody types decreased. However, five patients developed new antibodies with low MFI, and nine patients continued to have high MFI. The overall survival, disease-free survival, non-recurrent mortality, and cumulative recurrence rates at 100 days post-transplantation were 83.8%, 80.2%, 16.1%, and 4.5%, respectively. Conclusions: The combination of PAIA and RTX has a certain therapeutic effect and good safety in the desensitization treatment of highly sensitive patients before haplo-HSCT.

Late graft failure of pig-to-rhesus renal xenografts has features of glomerulopathy and recipients have anti-swine leukocyte antigen class I and class II antibodies.

Prolonged survival in preclinical renal xenotransplantation demonstrates that early antibody mediated rejection (AMR) can be overcome. It is now critical to evaluate and understand the pathobiology of late graft failure and devise new means to improve post xenograft outcomes. In renal allotransplantation the most common cause of late renal graft failure is transplant glomerulopathy-largely due to anti-donor MHC antibodies, particularly anti-HLA DQ antibodies. We evaluated the pig renal xenograft pathology of four long-surviving (>300 days) rhesus monkeys. We also evaluated the terminal serum for the presence of anti-SLA class I and specifically anti-SLA DQ antibodies. All four recipients had transplant glomerulopathy and expressed anti-SLA DQ antibodies. In one recipient tested for anti-SLA I antibodies, the recipient had antibodies specifically reacting with two of three SLA I alleles tested. These results suggest that similar to allotransplantation, anti-MHC antibodies, particularly anti-SLA DQ, may be a barrier to improved long-term xenograft outcomes.

(1068) Successful Lung Re-Transplantation with Perioperative Desensitization for Sensitized Recipient with Donor Specific DQ Antibody

(1068) Successful Lung Re-Transplantation with Perioperative Desensitization for Sensitized Recipient with Donor Specific DQ Antibody

Iguratimod reduces panel reactive antibody in high mismatched renal transplant recipients: One single‐center experience

To evaluate the clinical efficacy and safety of iguratimod (IGU) for reducing panel reactive antibody (PRA) in high-mismatched renal transplant recipients. Eligible recipients positive for PRAs who received or did not receive IGU treatment were enrolled. We retrospectively reviewed, collected, and analyzed statistically the clinical data of the recipients. A total of 80 recipients were included for further analysis. After IGU was administered for 9months, no significant difference was found in the change rates of donor specific antibodies between two groups. Meanwhile, the reduction in the PRAs in the IGU group was greater than that in the non-IGU group in anti-human leukocyte antigen (HLA) class I and class II, anti-HLA class I, anti-HLA class II, anti-HLA A, and anti-HLA DR antibodies. However, no differences were found in the anti-HLA B, anti-HLA Cw, anti-HLA DP, and anti-HLA DQ antibodies between the two groups. No serious adverse events were reported, and the incidence of adverse events was comparable between the two groups. PRA levels in high-mismatched renal transplant recipients were significantly reduced after the administration of IGU. The high safety of IGU was also determined.

Prediction of HLA-DQ in Deceased Donors and its Clinical Significance in Kidney Transplantation.

BackgroundHLA-DQ typing in deceased donors is not mandatory in Korea. Therefore, when patients develop DQ antibodies after kidney transplantation (KT) from deceased donor, it is impossible to determine whether they are donor-specific antibodies (DSA). We developed DQ prediction programs for the HLA gene and evaluated their clinical utility.MethodsTwo HLA-DQ prediction programs were developed one based on Lewontin’s linkage disequilibrium (LD) and haplotype frequency and the other on an artificial neural network (ANN). Low-resolution HLA-A, -B, -DR, and -DQ typing data of 5,603 Korean patients were analyzed in terms of haplotype frequency and used to develop an ANN DQ prediction program. Predicted DQ (pDQ) genotype accuracy was analyzed using the typed DQ data of 403 patients. pDQ DSA agreement, sensitivity, specificity, and false-negative rate was evaluated using 1,970 single-antigen bead assays performed on 885 KT recipients. The clinical significance of DQ and pDQ DSA was evaluated in 411 KT recipients.ResultspDQ genotype accuracies were 75.4% (LD algorithm) and 75.7% (ANN). When the second most likely pDQ (LD algorithm) was also considered, the genotype accuracy increased to 92.6%. pDQ DSA (LD algorithm) agreement, sensitivity, specificity, and falsenegative rate were 97.5%, 97.3%, 98.6%, and 2.4%, respectively. The antibody-mediated rejection treatment frequency was significantly higher in DQ or pDQ DSA-positive patients than in DQ or pDQ DSA-negative patients (P < 0.001).ConclusionsOur DQ prediction programs showed good accuracy and could aid DQ DSA detection in patients who had undergone deceased donor KT without donor HLA-DQ typing.

P1604SIGNIFICANT DETECTION OF UNEXPLAINED HLA ANTIBODIES WITH SINGLE-ANTIGEN BEADS IN PATIENTS ON THE WAITING-LIST WITHOUT PREVIOUS SENSITIZING EVENTS

Abstract Background and Aims The introduction of more sensitive and specific tools as solid phase immunoasssays has improved the ability to detect HLA antibodies. The techniques based on solid phase immunoassays in Luminex system can be performed either as screening assays (with pooled antigen panels that use bead populations coated in affinity-purified class I or class II HLA molecules) or to assess the specificity with single-antigen bead (SAB) assays (single cloned allelic HLA antigen).The sensitivity of each assay Is different, and their results may be discordant. Our objective was to evaluate the potential discrepancies between tests in a cohort of patients on our waiting list with no previous sensitizing events, for their potential influence in the access to transplantation. Method Observational study of 184 patients included in the kidney-transplant waiting list on March 31st, 2019. All possible sensitizing events (pregnancy, transfusion and previous transplants) were registered. HLA antibodies where analyzed both by screening and SAB tests. Results We limited the observations to males (64.7% of the cohort), as most women (95.4%) had a known sensitizing event, not being possible to discard possible pregnancies in the remaining. Of men, 46.2% had a sensitizing event and 63.8% did not. All 63 unsensitized patients showed a negative screening test, and 49 (77,78%) had at least one SAB done being positive in 73.4% (36/49). The positive SAB yielded a cPRA &amp;gt;10% in 30 cases (between 21-85%), mostly (27/30) due to HLA class II antibodies (88.8% with at least one DQ antibody) with a median MFI of 1571 (range:752-17650). A thorough review of specificities showed that 81% DQB1* and 89.6% DQA1* antibodies did not show consistency in all beads included for the same allele. Conclusion Screening and SAB assays show a significant discordancy in unsensitized men included in the waiting list for kidney transplantation. Automated detection of antibodies by SAB without a proper specialized interpretation may wrongly limit access to transplantation.

SUN-301 DENOVO DONOR SPECIFIC ANTIBODY AND ITS OUTCOME IN PEDIATRIC RENAL TRANSPLANT RECIPIENTS AT A SINGLE CENTRE IN INDIA

Donor specific anti HLA antibody (DSA) is associated with poor graft survival in adults. However there is paucity of data about its prevalence and outcome in Indian children. This study assessed the prevalence and spectrum of DSA and its outcome on allograft function and antibody mediated rejection (ABMR). Children ≤ 18 years who were transplanted between Nov 2016 and June 2019 were included. HLA typing was done for loci A, B, Cw, DQ and DR. Pre-transplant DSA was screened by complement dependent cytotoxicity, flow cytometry crossmatch and single antigen bead (SAB) class I and II by luminex platform. When SAB calss I or II was positive SAB against specific Antigen was done. Either anti thymocyte globulin or basiliximab was used as induction and tacrolimus, mycophenolate and prednisolone for maintenance immunosuppression. SAB assay was done at episode of rejection or at 3, 6 months, 1 and 2 year when graft function was normal. Mean fluorescent intensity ≥ 1000 was considered positive. 15 children, all males with median age 13 years (8, 17) were analyzed. Only one had deceased donor. One child had pre-transplant DSA. Eight (53%) developed denovo DSA. Seven (87%) had Class II, 3 (37%) had Class I and 2 had both. Six had DQ and 2 had DR. DSA developed as early as 1.5 month post-transplant. Seven developed ABMR and all of them had denovo DSA. One of two DSA positive Children with normal graft function had subclinical ABMR. DSAs persisted despite all possible treatment though there was improvement in graft function. However children developing DSA had similar graft function than who did not. Prevalence of denovo DSA was high in our cohort, majority being DQ antibody. HLA typing should include DQ antigen and SAB assay should be done post transplant to identify and treat DSA.

HLA-DQ alloantibodies directly activate the endothelium and compromise differentiation of FoxP3high regulatory T lymphocytes

Development of donor-specific antibodies is associated with reduced allograft survival in renal transplantation. Recent clinical studies highlight the prevalence of human leukocyte antigen (HLA)-DQ antibodies amongst de novo donor-specific antibodies (DSAs), yet the specific contribution of these DSAs to rejection has not been examined. Antibody-mediated rejection primarily targets the microvasculature, so this study explored how patient HLA-DQ alloantibodies can modulate endothelial activation and so immunoregulation. HLA-DQ antibodies phosphorylated Akt and S6 kinase in microvascular endothelial cells. This activation prior to culture with alloreactive lymphocytes increased IL-6 and RANTES secretion. The antibody-mediated upregulation of IL-6 was indeed Akt-dependent. The binding of HLA-DQ antibodies to endothelial cells selectively reduced T cell alloproliferation and FoxP3high Treg differentiation. In clinical studies, detection of HLA-DQ DSAs with other DSAs is associated with worse graft survival than either alone. Endothelial cells stimulated with HLA-DR and HLA-DQ antibodies showed a synergistic increase in pro-inflammatory cytokine secretion and a decrease in Treg expansion. HLA-DQ antibodies strongly promote pro-inflammatory responses in isolation and in combination with other HLA antibodies. Thus, our data give new insights into the pathogenicity of HLA-DQ DSAs.

Markers of graft microvascular endothelial injury may identify harmful donor-specific anti-HLA antibodies and predict kidney allograft loss.

Graft microvasculature is a major target of donor-specific antibodies (DSA) and endothelial damage is direct evidence of antibody-mediated rejection (ABMR). Using immunohistochemistry, we analyzed the expression of three microvascular endothelial activation markers (fascin, vimentin, and hsp47), suggestive of endothelial-to-mesenchymal transition (EndMT) in 351 graft biopsies from 248 kidney recipients, with concomitant screening of circulating antihuman leukocyte antigen (HLA) DSA at the time of the biopsy. The factors associated with EndMT marker expression were DSA and the presence of microvascular inflammation (MI). EndMT expressing grafts had significantly more allograft loss compared to EndMT negative grafts (P<.0001). The expression of EndMT markers positively correlated with anti-HLA DSA class II mean fluorescence intensity (MFI) levels and especially identified DQ and DR antibodies as being more closely associated with microvascular injury. Moreover, only DSA linked to positive EndMT score affected allograft survival, regardless of DSA MFI levels or presence of C4d deposition. Thus, EndMT markers could represent a clinically relevant tool for early identification of ongoing endothelial injury, harmful DSA, and patients at high risk for allograft failure.

Relationship of distribution frequency of HLA antigen/antibody and PIRCHE score with DSA production and AMR occurrence

Objective: To analyze the distribution frequency of HLA antigen gene and antibody, and explore the relationship of PIRCHE score with DSA production and AMR occurrence. Methods: Kidney transplantation cases of department Kidney transplantation, the First Hospital of Xi'an Jiaotong University from November 2013 to June 2017 were included in our study. HLA high resolution typing were detected with LAB Type (TM) SSO method. HLA classⅠ & Ⅱ antibody detection were tested by LAB screen Single Antigen beads with Luminex 200 technology. PIRCHE score were graded with PIRCHE score system. Results: HLA high resolution classification data of 798 recipients and 409 donors showed that HLA A2, A11, A24 were common in HLA A locus; HLA B46, B51 B60, B35, B62, B61 were common in HLA B locus; HLA DR9, DR4, DR15, DR12, DR7, DR11 were common in HLA DR locus; HLA DQ7, DQ6, DQ5, DQ9, DQ2 were common in HLA DQ locus. The positive cases of HLA class Ⅰ & Ⅱ antibody and DSA were 105, 40 and 32, respectively. The most common of HLA class Ⅰ antibody were A24 and B7 antibody; the most common of HLA class Ⅱ antibody was DQ antibody, including DQ2, DQ9, DQ4, DQ6, DQ7 and DQ8. PIRCHE scores of living donor transplantation recipients were significantly lower than DCD group (P<0.01). PIRCHE score of DSA(+) and DSA(+)AMR(+) group were markedly higher than that of DSA(-)and DSA(+)AMR(-)group, respectively(P<0.05); analysis of ROC curve on PIRCHE scores could predict DSA production and AMR occurrence. The AUC for prediction DSA production was 0.80, and the critical value was 115.5. For prediction of AMR, the AUC was 0.89 and the critical value was 133.5. Conclusions: The common HLA antigens have stronger immunogenicity, and easy to stimulate the body to produce HLA antibodies; matching of common antigen sites and HLA class Ⅱ antigen should be attached importance before kidney transplantation. PIRCHE score can predict the generation of DSA and the occurrence of AMR effectively. PIRCHE score for HLA match, is more sensitive than traditional methods, and contains more information.

Investigation of Anti-HLA Antibodies of Highly Sensitized Patients by Single Antigen Bead and C1q Tests

PurposeThe single antigen bead (SAB) test contributes to conventional cellular and solid phase crossmatch tests in renal transplantation. However, the determination of anti-HLA antibodies of the patients may not reflect the pathologic features of these antibodies. Highly sensitized patients produce antibodies against a number of HLAs; therefore, their transplantation chance decreases. In this study, we aimed to evaluate SAB and C1q test results of highly sensitized patients. MethodIn this study, 33 end-stage renal failure patients with >80% panel reactive antibody were included. Of the patients, 58% (n = 19) were female, and 42% (n = 14) were male. The mean age was 46.2 ± 12.4. All of the serum samples were inactivated by heat before use. SAB and C1q tests were performed according to the manufacturer's instructions. ResultsWe obtained statistically significant results between the positive bead counts and raw mean fluorescence intensity (MFI) values of 2 tests (P < .01 for class I and II). There was a statistically significant difference between the 2 tests in terms HLA-A, -C, -DR, and -DP MFI values, whereas HLA-B and -DQ MFI values were similar for the 2 tests. ConclusionThe difference of raw MFI values between the 2 tests may be due to the fact that the C1q test detects only IgG1 and IgG3 antibodies, whereas the SAB test can detect all IgG subtypes. We considered that anti-HLA-B and -DQ antibodies have high complement-fixing features; these antibodies should be investigated selectively due to the similarity of anti-HLA-B and -DQ antibody MFI values in the 2 tests.

P048 Persistent detection of anti-hla b8 by igm and c1q testing in the post-transplant setting

A female patient presented for follow-up care approximately ten years post-cardiac transplantation. HLA antibody testing revealed multiple class I and class II donor specific antibodies (DSA), including anti-HLA A1 (MFI > 2500), DQ6, and DQ7/DQ8/DQ9 antibodies (DQ antibodies have MFIs >15,000). Per our current testing protocols, the sample was reflexed to C1q to determine if any DSAs were complement- fixing. As expected, based on antibody strength, the class II DSAs were C1q positive, while anti-HLA A1 was C1q negative. Interestingly, C1q testing revealed reactivity for anti-HLA B8 which was not previously detected by our standard IgG testing methodology. While anti-HLA B8 was not a DSA to the previous transplant, we wanted to determine the underlying cause for the positive reactivity seen with C1q testing: prozone effect or IgM? Since our laboratory routinely treats all sera tested for HLA antibodies with EDTA prior to testing, we thought prozone was unlikely as previous studies performed in our lab demonstrated the removal of this effect. Nonetheless, to rule out prozone with another method, IgG testing was repeated using a series of dilutions (Neat, 1:4, 1:16, 1:64, and 1:256). This testing did not yield positive reactivity for anti-HLA B8, and therefore prozone effect was excluded, indicating that EDTA methodology is robust to overcome prozone effect. To confirm the isotype of this antibody, IgM testing was conducted in three sera using the same assays as the IgG testing which produced positive results. In conclusion, anti-HLA B8 was determined to be complement-fixing and of the IgM isotype. Unexpectedly, our results supported the absence of an IgM-IgG isotype class switch for the period of testing performed in our lab spanning 17 months. *RLU and JJX contributed equally to this work.

OR51 To treat or not to treat: Long term stable kidney allograft function in presence of strong complement-fixing donor-specific antibody (DSA) to HLA-DQ alpha chain

The clinical significance of HLA antibody specific to the DQ alpha chain is controversial. Here we present a case of long term stable kidney allograft function in presence of strong de novo complement-fixing DQA DSA. A 27-year-old Caucasian male (non-sensitized; 0% CPRA) with ESRD to IgA nephropathy received a living donor kidney transplant (TX) 10+ years ago with a negative pre-TX CDC crossmatch (XM). Mismatched donor HLA were A1, A11, B8, DR13, DR17, DR52, DQ2, DQ6. Beginning 6 years post-TX, routine antibody screening by single antigen beads (SAB) revealed the emergence of de novo DQ antibody, with the reactivity to donor DQ2 and DQ6 initially detected at 700–10400 and 100–2400 MFI, respectively. Subsequent SAB profiles indicated that the DSA had progressively converted to be primarily directed against DQA1∗04/05/06, likely a result of sensitization from donor DQA1∗05:01. Epitope analysis suggested that the DSA was directed against a well-defined eplet 40GR3 shared by all DQA1∗04/05/06 alleles and may represent three distinct eplets 40G, 47C, 50V51L53Q, all located in the alpha-1 domain accessible to antibody binding. Testing by phenotype beads confirmed this strong DQA reactivity (5900–14100 MFI) and flow XM with surrogate donors expressing DQA1∗05 were strongly B-cell positive (210–420 MCS), suggesting the DSA was directed against native DQA chain. Functionally, C1q testing demonstrated the DQA DSA’s considerable ability to fix complement (5300–9500 MFI), while CDC XM with DQA1∗05 + surrogate donors were all negative. Despite the prolonged presence of this DQA DSA, other than an episode of acute cellular rejection (Banff II C4d-) diagnosed shortly after transplant which was promptly resolved, the patient’s 10 + years of post-TX course has been unremarkable with a stable kidney function (creatinine 1.5 ± 0.2 mg/dL; BUN 13.4 ± 2.7 mg/dL; GFR 55.0 ± 6.8 mL/min). This case highlights the fact that not all HLA DSA are created equal in terms of their clinical significance, and may also suggest a protective effect of a subset of DSA when directed against only to the DQ alpha chain. Download : Download high-res image (313KB) Download : Download full-size image

HLA-DQ Specific Alloantibodies Modify Allogenic Endothelial Cell Immunogenicity and Impair the Expansion of Anti-Inflammatory FoxP3 High Regulatory T Lymphocytes

Recent clinical studies have highlighted the prevalence of de novo circulating Donor-Specific Antibodies (DSA) against HLA-DQ following solid organ transplantation. The emergence of HLA-DQ DSA has been associated with poor graft outcome. The allograft microvascular endothelium is known to upregulate HLA-DQ expression during rejection. The allograft endothelium is directly exposed to circulating anti-donor antibodies and as a result, becomes a principal site of DSA binding and thereby the target of antibody-mediated damage. Endothelial activation is a feature of antibody-mediated rejection and is reported to arise from inflammation, DSA binding and complement activation. Activated endothelial cells modulate the recipient’s immune response as a result of their interactions with CD4+ T lymphocytes. Plasmapheresis samples were obtained from alloimmunized patients with high levels of circulating DSA. Antibodies were isolated using protein G beads, and their HLA specificity was assessed by Luminex. Antibodies were evaluated for binding to microvascular endothelial cells, and a time course of binding was established by flow cytometry. Patient antibodies specific for relevant HLA-DQ alleles were used to assess potential signal transduction from antibody-binding to endothelial cells. HLA-DQ DSA induced transient phosphorylation of Akt and S6 Kinase. This was confirmed using a monoclonal anti-HLA DQ antibody. Alloantibodies against HLA-DQ alleles not expressed by our cell model did not induce specific signalling events. Previously, we found that HLA-DR+ endothelial cells are capable of inducing alloproliferation of CD4+ T lymphocytes and selectively expanding Th17 and FoxP3high T regulatory cell (Treg) subsets (Taflin PNAS 2011, Lion Am J Transplant 2016). The presence of intragraft Th17 correlates with shorter graft survival, whilst the proportion of Treg cells has been implicated in tolerance. To assess the functional consequences of DSA binding, endothelial cells were stimulated with HLA-DQ DSA for one hour preceding co-culture with allogenic peripheral blood mononuclear cells. After seven days, CD4+ T cell subsets were analysed by intracellular cytokine staining. HLA-DQ DSA modified endothelial cell immunogenicity, resulting in a reduction in alloproliferation and in Treg expansion. Alloantibodies against HLA-DQ alleles not expressed by our cell model did not alter alloproliferation or Treg expansion. The impaired amplification of anti-inflammatory Treg cells, in combination with an unaltered expansion of the Th17 subset, leads to an overall pro-inflammatory orientation of alloreactive CD4+ T cells. This altered composition of CD4+ T cells, due to HLA-DQ binding to endothelial cells, may exacerbate allograft damage and reduce allograft tolerance. AC was supported by the Société Francophone de Transplantation (SFT).

Donor-specific HLA-DQ antibodies may contribute to poor graft outcome after heart transplantation.

BACKGROUNDHLA-DQ donor-specific antibodies (DSA) are implicated in allograft dysfunction after renal and lung transplantation. Limited data exists on the impact of HLA-DQ antibodies on heart transplant patients.OBJECTIVETo investigate the impact of DSA formation on allograft function and outcomes in heart transplant patients.DESIGNRetrospective cohort study.SETTINGCollating post-transplantation patient data from computerized database in a tertiary hospital in Riyadh, Saudi Arabia from January 2006 to October 2014.PATIENTS AND METHODSWe excluded recipients with positive preoperative complement-dependent-cytotoxicity crossmatch grafts and those with preformed DSA. Anti-HLA antibodies were identified using Luminex-based assay in sera collected before transplantation with a routine endomyocardial biopsy the first year and then annually.MAIN OUTCOME MEASURESPrimary outcome measures were all-cause mortality, development of antibody mediated rejection, treated acute cellular rejection (ACR) and cardiac allograft vasculopathy (CAV).SAMPLE SIZE127 patients.RESULTSDSA formation occurred in 43/127 (34%), with 33/43 (77%) targeting HLA-DQ antigens alone (n=7) or in combination with -DR, -A or B antibodies (n=26). Most (76%) were male and the mean (SD) age was 36 (14) years. Ten patients developed -A, -B or -DR antibodies without -DQ antibodies also present. Treated ACR (P=.011), reduced left ventricular ejection fraction (P<.001), CAV development (P=.003), and all-cause mortality (P=.01) were all significantly more prevalent in the DSA-positive cohort.CONCLUSIONHLA-DQ donor-specific antibodies were the most common type detected and may play a significant role in poor outcomes post-cardiac transplantation. This emphasizes the importance of HLA-DQ matching and monitoring for DSA formation in order to minimize post-transplantation immunological risk.LIMITATIONSRetrospective design comes with inherent biases, results from single institute, with a particularly young cohort.

P215 Donor specific antibodies after transplantation: Importance of HLA-DQ

Aim Development of de novo donor-specific HLA antibodies (DSA) after kidney and heart transplantation limits the long term success of this therapy and may lead to new transplantation. Luminex-supported single antigen bead assay (SAB) represents a major advantage in post-transplant monitoring by detecting different stages of antibody-mediated rejection (AMR). We aimed to explore the pattern of DSA generated after kidney and heart transplantation. Methods A total of 151 kidney and heart transplant recipients were tested from January in 2015. The sera collected at different time points after transplantation were screened for HLA-A, B, C, DR, DQ and DP specific antibodies by SAB (One Lambda). DSA positive patients and corresponding donors were additionally typed at high to intermediate level of resolution for antibody relevant loci. Screening results were analysed with HLA Fusion Software v3.4 at the allele and epitope level. Results DSA were detected in 32% of patients. Out of them 43% had class I and 82% class II DSA, respectively. The frequency of anti-DQ DSA (67%) were the highest of locus specific antibodies, moreover 39% of patients had anti-DQ DSA alone. Some of anti-DQ DSA were directed specifically at DQ-alpha, some at DQ-beta, and some at epitopes formed by amino acids from both chains. Our results confirmed that anti-DQ DSA recognize epitopes shared between different DQ molecules, mostly within broad serological specificities. For instance, in patients not carrying DQ3 and mismatched for DQ7, immune response was spread to DQ8 and DQ9 due to the epitope 55P on beta-chains coded from DQB1∗03:01, DQB1∗03:02 and DQB1∗03:03. Consequently, in the present allocation system all DQ3 molecules should be considered unacceptable for next transplantation. Conclusions Since epitope matching has not been implemented yet and anti-DQ specific antibodies are the predominant DSA after heart and kidney transplantation, full high to intermediate level of resolution DQ-alpha and -beta matching might be considered.

De novo DQ donor-specific antibodies are associated with worse outcomes compared to non-DQ de novo donor-specific antibodies following heart transplantation.

Antibody-mediated rejection (AMR) resulting from de novo donor-specific antibodies (dnDSA) leads to adverse outcomes following heart transplantation (HTx). It remains unclear what role dnDSA to specific HLA antigens play in adverse outcomes. This study compares outcomes in patients developing dnDSA to DQ antigens with those developing non-DQ dnDSA and those free from dnDSA. The present study was a single-center, retrospective analysis of 122 consecutive HTx recipients. The primary outcome was a composite of death or graft dysfunction. After 3.3years of follow-up, 31 (28%) patients developed dnDSA. Mean time to dnDSA was 539days. Of 31 patients, 19 developed DQ antibodies and 12 developed non-DQ antibodies. Compared to non-DQ dnDSA, DQ antibodies presented with higher MFI values (P=.001) were more likely persistent (P=.001) and appeared later post-HTx (654 vs 359days, P=.035). In a multivariable analysis, DQ dnDSA was associated with increased risk of the primary endpoint (HR 6.15, 95% CI 2.57-14.75, P=.001), whereas no increased risk was seen with non-DQ dnDSA (P=.749). dnDSA to DQ antigens following HTx are associated with increased risk of death and graft dysfunction.

P120 Correlation between FlowPRA® and LABScreen Class II single antigen beads for the detection of HLA DQB antibodies: Not all assays are created equal

Aim Several publications have shown the clinical importance of donor-specific DQ antibodies, post-transplant, in kidney, heart and lung. We wanted to determine if FlowPRA®(F-PRA) screening beads would be a cost-effective way to assess the presence of DQB antibodies. Methods Patients with monospecific and well defined DQB specificities were identified for testing. Both F-PRA screen and LABScreen Class II single antigen bead (SAB) (One Lambda, Inc) assays were performed using serial dilutions of these sera to determine the detection “end-point” for each method. Results Our data showed that for DQB1 specificities, ∗02, ∗04, ∗05/∗06, and ∗03:01/02/03, when MFI values were high; eg: >10,000, the F-PRA displayed distinct positive staining architecture. However, when diluted, SABs could identify the presence of DQB antibody at dilutions that were negative by F-PRA (Fig. 1). The corresponding SAB MFI values ranged from ∼ 750 – 5300 (Table 1). Conclusions Our data show that the F-PRA will fail to detect HLA DQB antibody at concentrations that yield moderate MFI values by LABScreen Class II SABs. Hence, F-PRA cannot be used alone to rule out the presence of HLA DQB antibodies. The reason for this discrepancy may be differences in antigen density between the two products as each one uses a different type of cell line for antigen purification. We are in the process of performing similar studies, including crossmatches, for other HLA loci. Download : Download high-res image (220KB) Download : Download full-size image Download : Download high-res image (66KB) Download : Download full-size image

Pitfall in the interpretation of DQ HLA-antibodies

HLA-DQ consist of two polymorphic chains, both contribute to immunization. SAB testing which ignores the DQ α , can lead to erroneous DQ-antibody assignments. Pitfall in the interpretation of DQ antibodies in the cases presented here had declined immunologically compatible transplant and exposed the second one to unnecessary immunesuppression. The first patient was a young male referred for kidney transplantation. HLA-antibodies revealed positive DQ7, DQ8, and DQ9 antibodies. B-Flow cross matches with the three evaluated potential living related donors was positive with donor1, 2 (sister and cousin1) but negative with the (cousin2) donor3. All donor typed DQ8. The negative result with donor3 was unexpected. DQA1∗component was thought to be responsible however, bead reactivity analysis could not rule out contribution of B∗03:02. The possibility of unexpressed DQ8 antigen was not supported by serological and high resolution typing which confirmed DQA1∗ 03:02/DQB1∗03:02 in the first two donors &DQA1∗03:01/DQB1∗03:02 in donor3. As the difference between DQA1∗03:02 and DQA1∗03:01 alleles is outside the antigen recognition site the individual DQA1∗03:01 should not account for different immune response. Furthermore, DQA1∗03:01, DQB1∗03:02 coated beads was positive. Flow PRA class II bead on BD showed positive response to homozygous DQA1∗03:02 beads. The inconclusive results in assessing the immunological risk with the donor3, denied patient transplantation for long time before he underwent uneventful transplant. Patient2 was a lady who had failed first graft from her mother and came for retransplant from her full brother. She had weak DQ2 DSA against her mother who shared this antigen with the brother. The first post retransplant SAB showed elevated DQ2, she was put under close observation despite normal creatinine level. A kidney biopsy was unremarkable in the first time but was C4d focally positive in the second one. She received anti-rejection therapy. HR typing confirmed DQB1∗02:02/DQA1∗02:01 for both donors. Reanalysis of bead reaction revealed the antibody we report was against DQB1∗02:01/DQA∗05:01 allele. The patient is now14 months post transplant with good renal function. In conclusion the results of SAB were not conclusive and misleading in the 2 patients respectively.