anti-DP Antibodies Research Articles

A personalised delisting strategy enables successful kidney transplantation in highly sensitised patients with preformed donor-specific anti HLA antibodies.

This study investigates kidney transplant outcomes in highly sensitised patients after implementing a delisting strategy aimed at enabling transplantation despite preformed donor-specific antibodies (preDSA), with the goal of reducing acute antibody-mediated rejection (aAMR) risk. Fifty-three sensitised recipients underwent kidney transplant after delisting prohibited HLA antigens, focusing initially in low MFI antibodies (<5000), except for anti-HLA-DQ. If insufficient, higher MFI antibodies were permitted, especially for those without an immunogenic eplet pattern assigned. Delisting of Complement-fixing antibodies (C1q+) was consistently avoided. Comparison cohorts included 53 sensitised recipients without DSA (SwoDSA) and 53 non-sensitised (NS). The average waiting time prior to delisting was 4.4 ± 1.8 years, with a reduction in cPRA from 99.7 ± 0.5 to 98.1 ± 0.7, followed by transplantation within 7.2 ± 8.0 months (analysed in 34 patients). Rejection rates were similar among preDSA, SwoDSA, and NS groups (16%, 8%, and 11%, respectively; p = 0.46). However, aAMR was higher in the preDSA group (12%, 4%, and 2%, respectively; p = 0.073), only presented in recipients with DSA of MFI >5000. The highest MFI DSA were against HLA-DP (Median: 10796 MFI), with 50% of preDSA aAMR cases due to anti-DP antibodies (n = 3). Graft survival rates at 1 and 5 years in preDSA group were 94%, and 67%, comparable to SwoDSA (94%, and 70%; p = 0.69), being significantly higher in the NS group (p = 0.002). The five-year recipient survival rate was 89%, comparable to SwoDSA and NS groups (p = 0.79). A delisting strategy enables safe kidney transplant in highly sensitised patients with preDSA, with a slight increase in aAMR and comparable graft and patient survivals to non-DSA cohorts.

Anticorps anti-Cw, anti-DP et décision de transplantation rénale : à propos de deux cas

AbstractDue to the harmful impact of preformed anti-HLA-A/B/DR/DQ DSA (Donor Specific Antibody) on kidney transplantation, organ allocation systems take these antibodies into account in the transplant decision. Nevertheless, more and more studies point to the deleterious role of preformed anti-Cw and anti-DP antibodies as well.Indeed, it appears from the literature that the presence of anti-Cw and antiDP DSA adversely affects the survival of the graft.These DSA are incriminated in the occurrence of graft rejection, with a correlation of their MFI (Mean Fluorescence Intensity) thresholds with their pathological potential. However, no consensus has yet been established.We present two cases of patients candidates for kidney transplantation, for whom the presence of anti-Cw and anti-DP DSA hampered access to the transplant.This strategy, which takes these DSAs into account in the renal transplant decision, requires carrying out the HLA-Cw and HLA-DP typings of the donor as soon as the potential recipient presents an anti-Cw and/or anti-DP immunization.

P119 Correlation between the strength of donor specific antibody (DSA) using single antigen bead assay (SAB) and the results of CDC- cytotoxicity and flow cytometric crossmatch

Aim In this study we correlated flow and CDC- crossmatch outcomes with results of Luminex-single antigen bead (SAB). Methods Between 2010–2016 HLA-antibody Sera were tested by screening and SAB class I & II (One lambda) according to manufacturer recommendation. MFI of ⩾1000 were considered positive. These sera were used to perform the following: 1. 80 crossmatches using isolated PB or spleen lymphocytes from healthy deceased or living donors respectively by CDC–crossmatch. Correlation was performed on sera with single antibody or multiple classes I and II antibodies. Titer of CDC was given as the inverse of the highest dilution yielding positive XM. 2.108 FCXM cross using isolated PB donor lymphocytes expressing one HLA- antigen (class I or II) and 78 crossmatches using cells expressing multiple antigens detected by the sera. Analysis performed on FACSCanto II & mean channel shift (MCS) was calculated. The cut off for T &B are 64, 100 respectively. T- Positive, B- negative results in the absence of HLA- antibody were excluded from the study. Results Our results indicate good correlation of FCXM and CDC-XM with LUMINEX -SAB assay. SAB assay has good correlation with T-FCXM (r2 = 0.7) but not with B-FCXM. Fig. 1 A cutoff MFI of 3000 for SAB assay (as single or sum of multiple weak antibodies) had a sensitivity & specificity of 100% in detecting a positive FCXM. The few number of sera with single anti HLA-Cw and DP antibodies did not give positive reaction up to 5000 and 10,000 MFI respectively but were not included in the study. Single anti HLA-A, B, DRB1of ⩾10,000 MFI was associated with positive CDC-XM in most cases and multiple low levels antibody that yield this 10,000 would also give positive reaction. We noticed presence of A2 specificity among multiple anti class I or class II antibodies increased the chance of positive reaction even when sum of MFI is ⩽10000. Conclusions SAB correlates significantly with outcome of both cytotoxic and T-FCXM. Based on the threshold established it is possible to predict correctly results of crossmatch. Download : Download high-res image (164KB) Download : Download full-size image

OR39 Potential clinical relevance of anti-HLA-DP antibodies targeting the 96K and 96R epitopes

Aim HLA-DP is conserved across the human population with the majority of polymorphism occurring at six hypervariable regions (HVR) within the β -chain. Anti-DP antibodies commonly react to specific epitopes in close proximity to AA 55, AA 84, and to a lesser extent the AA 33 region. Reactivity to these polymorphic HLA-DP epitopes can be associated with a positive flow crossmatch when single antigen bead (SAB) reactivity is high. Here we confirm another variable region of HLA-DP located at AA 96, and characterized by either a lysine (96K) or arginine (96R) residue. Methods We identified two paradoxical, robust, HLA-DP reactivity patterns by SAB, and MFI data was analyzed with HLA Matchmaker to investigate potential epitope patterns—which suggested variability at AA 96. Using patient serum samples obtained before and after the appearance of AA 96 reactivity, we performed surrogate flow crossmatch to determine the validity of antibodies targeting AA 96 epitopes. Results Using HLA Matchmaker, we have verified a variable region at AA 96, and have observed that differences at this position are capable of eliciting 96K (DP: 1, 3, 5, 6, 9, 10, 11, 13, 14, 15, 18, 19, 20) or 96R (DP: 0201, 0401, 0402, 17, 23, 28) antibody response. We identified a patient that was homozygous for 96R, received a lung from a heterozygous 96K/96R donor, and quickly developed strong anti-96K DSA determined by SAB. Flow crossmatch of this 96K reactive serum versus a surrogate donor homozygous for the 96K epitope (DP5, –) yielded a strongly positive B-cell crossmatch, while crossmatch versus a surrogate donor that was heterozygous at AA 96 (K/R: DP9, DP0201) only elicited a weakly positive B-cell crossmatch. In comparison, serum samples targeting the 96R epitope were also identified and additional surrogate testing is ongoing. Conclusions We have confirmed a HLA-DP epitope located at AA 96 with potential clinical relevance. Antibodies targeting the 96K epitope were capable of eliciting a positive B-cell crossmatch. Additional surrogate testing is ongoing to better characterize HLA-DP 96K and 96R antibodies.

Acute Antibody-Mediated Rejection by De Novo Anti-HLA-DPβ and -DPα Antibodies After Kidney Transplantation: A Case Report

The presence of isolated de novo anti-DP antibodies is uncommon, making it difficult to determine the impact of anti-DP antibodies on graft outcome. We describe a case of acute antibody-mediated rejection mediated by de novo donor-specific anti-HLA-DP antibodies. Furthermore, the generation of non–donor-specific anti-DP antibodies (NDSAs) detected in the patient's sera was investigated. An 18-year-old woman with pretransplant 0% panel-reactive antibody received kidney transplantation from a living donor. She experienced combined acute T-cell-mediated and antibody-mediated rejection at 15 months after transplantation. High resolution HLA typing of the donor and the patient revealed that they were mismatched at both DPB1 (DPB1*31:01) and DPA1 (DPA1*02:02) loci. The single antigen bead (SAB) testing of patient's sera revealed antibodies against donor's DPB1*31:01 and DPA1*02:02 alleles. Antibodies against several non–donor-specific DP antigens were also detected. No antibodies against other HLA class I and II antigens were detected. In order to explain the reactivity pattern of NDSAs, HLAMatchmaker program was used to identify immunizing eplets shared between donor alleles and reactive beads. The analysis showed 84DEAV, a DPB1 eplet, as a shared eplet found on DPB1*31:01 (mismatched donor allele) and on DPB1-reactive alleles in SAB assay. Additionally, 50RA, a DPA1 eplet, was identified as a shared eplet found on DPA1*02:02 (mismatched donor allele) and on DPA1-reactive alleles in SAB assay. This case highlights the clinical significance of HLA-DP antibodies. Furthermore, the generation of NDSA anti-DP antibodies by epitope sharing underscores the importance of HLA-DP epitope matching in kidney transplantation.

Incidence and impact of anti-HLA-DP antibodies in renal transplantation.

The role of anti-HLA-DP antibodies in renal transplantation is poorly defined. This study describes the impact of donor (donor-specific antibody [DSA]) and non-donor-specific antibodies against HLA-DP antigens in renal transplant patients. Of 195 consecutive patients transplanted between September 2009 and December 2011, 166 primary kidney recipients and their donors were typed (high-resolution) for DP antigens. Sera taken pre-transplant and at 1, 3, 6, 9, and 12months, and annually post-transplant were retrospectively tested for anti-DP antibodies using single-antigen beads. In 81 (49%) patients, anti-DP antibodies were found; 64% (n=52) of patients were positive in the pre-transplant samples and 36% (n=29) were positive exclusively post-transplant. The median time from transplantation to antibody was 20.9months. Fifty-five percent (n=16) of the de novo anti-DP antibodies were accompanied by another de novo DSA. Anti-DP antibody-positive patients had a higher rate of rejection (compared with anti-DP antibody-negative patients, P=.01). The estimated glomerular filtration rate declined more with anti-DP antibodies (-5.5% vs +26%). Antibodies against HLA-DP antigens are common. De novo anti-DP antibodies commonly appear after acute rejection and accompany DSA, which makes it difficult to determine whether anti-DP antibodies are the cause or the consequence of graft injury.

Prevalence of HLA-DP antibodies in patients awaiting renal transplantation

Aim To determine the prevalence of HLA-DP IgG antibodies in patients on Renal Transplant waitlist at Rush University Medical Center, Chicago, IL. Introduction: Role of HLA-DP antibodies in solid organ and stem cell transplantation is evolving. Isolated reports on possible involvement of only HLA-DP DSA in acute rejection episodes (cases where the Donor/Recipient pairs are matched at HLA-Class , DR and DQ)] show the importance of this issue. In the light of HLA molecular typing and antigen specific HLA antibody assays there is an opportunity to evaluate the role of HLA-DP in allograft rejection. As part of this goal we investigated the prevalence of DP antibodies in our renal transplant candidates. Patients: A total of 532 patients with multiple serum samples tested. Methods HLA-specific antibodies were determined by solid phase assay (One Lambda, Inc. ®). HLA- specific antibody was scored as strong, moderate, or weak depending on their mean fluorescent index (MFI) value of > 5000, 2000–5000, 700–2000, respectively. Results Of the 532 patients, 143 (26.9%) had antibodies against Class II HLA-DP. The number of specific HLA-DP antibodies per patient ranged from 1 to 17 antibodies (Strong, Moderate, Weak), but could also be found entirely in one category as in the case with one patient in which all 16 of the anti-DP antibodies were in the strong MFI category. Conclusions Quarter of renal transplant candidates on waiting at Rush University Medical center have detectable HLA-DP antibodies with varying MFIs. While 36.5% of those antibodies express a low MFI, 31.4% of the patients had strong MFI. Multiple case studies from various centers have shown that transplant patients perfectly matched per UNOS requirements to their donor may still have antibody mediated rejection due to a mismatched at HLA-DP. Hence donor typing for HLA-DP and monitoring of patients for HLA-DP antibodies seems to be important

LBP12: A COMPARISON OF DECEASED DONOR TYPING STRATEGIES ELICITED BY A NOVEL HLA-DPB1 ALLELE

Aim A deceased donor (DD) was identified with an HLA-DPB1 type that could not be resolved by the One Lambda sequence-specific primer (SSP) assay. We sought to resolve the typing using other methodologies. Methods The initial donor HLA-DP typing was performed by SSP (Micro SSP, One Lambda). Subsequent typing was performed by real time SSP (LinkSeq, Linkage Bioscience), and sequence based typing (SBT) (SBT Resolver, Conexio and SeCore, Life Technologies). Results The Micro SSP kit identified one allele as DPB1 ∗ 03:01:01, but the other could not be determined. The LinkSeq kit identified one allele as EDP ∗ 03 and the other as EDP ∗ 28. Exon 2 analysis by both Conexio and SeCore identified DPB1 ∗ 28:01 with either DPB1 ∗ 03:01:01, ∗ 104:01, or ∗ 124:01. Exon 3 and 4 analysis by SeCore confirmed one allele as DPB1 ∗ 03:01:01, but the other allele was unresolved. There was nearly a complete loss of heterozygosity in Exon 3 (matching DPB1 ∗ 03:01:01), except for a single nucleotide which retained the expected sequence for DPB1 ∗ 03:01:01 and DP ∗ 28:01. The consensus typing is HLA-DPB1 ∗ 03:01:01, along with a novel allele which has the Exon 2 sequence of DPB1 ∗ 28:01 and a unique Exon 3 sequence which resembles DPB1 ∗ 03:01:01 in all but one nucleotide. Conclusion DD typing must be performed by molecular methods, with serological equivalents as the minimal resolution. DP typing is performed upon request, often because a recipient has anti-DP antibodies. As the number of retransplant candidates increases, the number of patients with strong DP specificities increases. Serological equivalent typing is most useful (at the resolution of the antigens available on Luminex single antigen beads), because it aids the process of virtual crossmatching. The results of the LinkSeq typing are reported as epitypes, grouping the possible alleles according to their amino acid sequence in the six hypervariable regions of Exon 2. The Micro SSP kit is high resolution and generally gives more precise allele assignments. SBT gives the highest resolution but has limited utility for DD typing because of the associated time and cost. Despite the fact that it was unable to identify the novel DP allele, the LinkSeq result is most useful, as it identifies probable reactivity to the donor for recipients with anti-HLA-DP ∗ 28:01 antibodies within the time constraints of DD typing.

High Incidence of Rejection Caused By Donor Specific Anti-HLA-DQ Antibodies in Kidney Transplant Recipients.

Objective: Anti-HLA antibodies post transplant follow up has been suggested by several studies as a useful tool to identify patients with acute or chronic rejection risk, and therefore useful to implement the necessary therapeutic measures that would help us minimize its clinical impact. Materials and methods: We had carried out a transversal and prospective study of 342 kidney recipients. Anti-HLA antibodies were tested using Luminex technology, screening and single antigen (Genprobe). The median number of tests performed per patient was 3.3. Single antigen with MFI > 1500 and specificity against any HLA donor antigen, determined by HLA-A/B/DR typing or linkage disequilibrium when HLA-DQB1* was not available, was assigned as donor specific antibody (DSA). Results: Post transplant anti-HLA antibodies were detected in 126 patients (37%): 31% (39/126) were antibodies anti-HLA class I and II, 9.5% (12/126) were anti-HLA class I and 59.5% (75/126) were anti-HLA class II. Within the 126 positive patients, 40 (31.7%; 40/126) were de novo and most of them anti-HLA class II (39/40; 97.5%). De novo anti-HLA class II antibodies were DSA in 35 patients (75%; 35/40), anti-DQB1* was the most frequent specificity (82.8%; 29/35), anti-DR was found in five patients (17.2%; 6/35) most of them anti-DRB4* (4/6). DSA anti-DR and anti-DQB1* was only present in one patient. Anti-DP antibodies were not considered as we lacked typing data. Fifteen patients from the group with de novo anti-DQB1* DSA (51.7%; 15/29) suffered a rejection episode, causing graft failure in five recipients who failed to respond to therapy. Conclusions: Anti-HLA antibodies monitoring allowed us to discover a high frequency of de novo anti-HLA class II DSA in kidney recipients, most of them against HLA-DQB1* specifities. These patients were found to have a higher rejection incidence. HLA-DQB1* typing should be recommended in donors and patients in order to take its compatibility into account.

Do Pre or Postransplant Anti-DP Antibodies Impact Kidney Graft Survival?

Solid-phase assays on Luminex platform employing beads with purified HLA antigens assess the presence of antibodies against classical HLA antigens A, B and DR, but also C, DQ or DP. DP antibodies have been considered less immunogenic than other class II antibodies. There is not much information about the frequency and impact on graft survival of DP antibodies in kidney transplantation (KT). We have evaluated the prevalence of DP antibodies in our cohort of transplant patients and the impact of DP antibodies in patients with and without donor-specific antibodies (DSA) Methods Prospective study of 440 KT recipients (performed 1979-2012 across negative CDC XM) recruited I/2008-III/2012. Pre and postransplant serum samples were tested with Luminex Lifecodes LifeScreen y LSA Class II (Gen-probe,Stanford,CT) kits. Raw-MFI values >1000 for any single antigen bead were considered positive. Results 291 KT patients had preTR samples tested: 68 (23.4%) had HLA class II antibodies, DSA in 36 cases (52.9%). DP antibodies were present in 27/68 (39.7%) patients with pretransplant HLA antibodies and in 17/36 (47.7%) with DSA. Death-censored graft survival of pretransplant DSA-II patients (median 76.5 months) was worse than HLA non-DSA patients (p=0.005). The presence of DP antibodies did not influence graft survival (with or without DSA)[Figure 1].346 KT patients were monitored after KT (median: 54 months): 59 (17.1%) had HLA class II antibodies and 33/59 (56%) with DP antibodies. Twenty-three patients had DSA-II (39%) and 14/23 (60.9%) with anti-DP. At 40 months, death-censored graft survival was worse in patients with DSA II (p=0,022). DP antibodies did not change these results.Figure: No Caption available.Conclusions DP antibodies are detected in around half KT recipients with pretransplant DSA and 61% after transplantation. Detection of DSA class II with single antigen beads before or after transplantation associates with worse graft-survival, but the presence of DP antibodies does not modify the impact.

Late antibody-mediated rejection by de novo donor HLA-DP-specific antibody after renal transplantation: A case report

The role of donor HLA-DP-specific antibodies after renal transplantation is controversial, and only preformed HLA-DP-specific antibodies have been shown to mediate rejection. Here we present a case of late humoral rejection mediated by de novo donor HLA-DP-specific antibodies in a non-sensitized recipient. This unique case demonstrates the pathogenic role of de novo anti-DP antibodies and suggests that HLA-DP matching might be relevant for renal transplantation.

Pfit Is a Structurally Novel Crohn's Disease-Associated Superantigen

T cell responses to enteric bacteria are important in inflammatory bowel disease. I2, encoded by the pfiT gene of Pseudomonas fluorescens, is a T-cell superantigen associated with human Crohn's disease. Here we report the crystal structure of pfiT at 1.7Å resolution and provide a functional analysis of the interaction of pfiT and its homolog, PA2885, with human class II MHC. Both pfiT and PA2885 bound to mammalian cells and stimulated the proliferation of human lymphocytes. This binding was greatly inhibited by anti-class II MHC HLA-DR antibodies, and to a lesser extent, by anti HLA-DQ and DP antibodies, indicating that the binding was class II MHC-specific. GST-pfiT efficiently precipitated both endogenous and in vitro purified recombinant HLA-DR1 molecules, indicating that pfiT directly interacted with HLA-DR1. Competition studies revealed that pfiT and the superantigen Mycoplasma arthritidis mitogen (MAM) competed for binding to HLA-DR, indicating that their binding sites overlap. Structural analyses established that pfiT belongs to the TetR-family of DNA-binding transcription regulators. The distinct structure of pfiT indicates that it represents a new family of T cell superantigens.

48-P

Virtual crossmatches (VXM) are used by transplant programs to evaluate compatibility with potential donors using a recipient’s HLA antibody profile and donor HLA typing. The aim of this study was to determine if the MFIs of single antigen donor specific antibodies (DSA) could be used to predict flow cytometric crossmatches (XM). Patient samples were screened for antibodies to mismatched donor antigens using Luminex single antigen (LSA) beads. The LSA bead MFI values were then correlated with the XM value. Only single antigen DSA was used for this study. Patients with anti-Cw or anti-DP antibodies and MFI values on beads known to give false positive results were also removed. The mean LSA bead MFI values of the recipients correlated with their XM values ( r 2 = 0.81 for class I DSA; r 2 = 0.75 for class II DSA). A significant difference between the DSA MFI values in patients whose XM was weak versus strong was found for both class I and class II antibodies. A cutoff MFI value of ⩾2000 predicted 100% of the moderate and strong XMs for both class I and class II antibodies. A prospective XM would be recommended for LSA bead MFI values from 1000-1999 (grey zone). [Fig. 1] A patient’s DSA profile can be used for a VXM and its predictive power is better when the LSA MFI is higher. Single antigen DSA MFI values can be used to predict moderate and strong XM. Further analysis of multiple DSA with VXM needs to be performed.

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The introduction of Luminex-Single Antigen bead (LSA) assay significantly improved identification of HLA antibodies in sera from highly sensitized transplant candidates. Variants of this assay allowed to detect IgM antibodies and complement-fixing antibodies. Recently, the LSA assay was shown to be prone to the complement-mediated prozone effect leading to false-negative results. In this study we performed a comprehensive analysis of the HLA class I and class II antibodies present in sera from 16 highly sensitized kidney transplant candidates (%FlowPRA class I and II: 96 ± 4 and 95 ± 5 respectively) using four variants of the LSA technique: IgG-LSA, IgM-LSA, C1q-LSA and also EDTA-IgG-LSA to overcome the complement-mediated prozone effect. In 61% of EDTA-IgG-LSA tests we evidenced a strong increment of several antibody row-value (13,709 ± 5,863) respect to IgG-LSA results. This method was also able to reveal additional HLA class I and II antibodies (A2, A25, A68, A69, B42, B57, B59, B67, B77,DR17, DQ7, DQA1∗05:01) in 33% of the performed tests. Correlating EDTA-IgG-LSA and C1q-LSA results we found that all but one sera contained both complement-fixing and non complement-fixing antibodies; in the remaining serum we only detected anti-DP antibodies unable to fix complement. In one case, EDTA-IgG-LSA assay revealed the presence of a wide antibody pattern (anti-DQ2, -DQ4, -DQ7, -DQ8, -DQ9) specific for 84-87QLEL/89-90TT epitopes while C1q-LSA assay identified a similar but smaller antibody pattern (anti-DQ7, -DQ8, -DQ9) due to the recognition of 55P epitope. In 5 sera, IgM-LSA assay was able to detect HLA antibodies not revealed by IgG-LSA technique; unexpectedly several of these antibodies were HLA-Cw specific. In conclusion our findings evidenced the importance to make use of all LSA potentiality for characterization of HLA pre-sensitization status of transplant candidates in order to assess the real immunological risk of a potential kidney transplant.

1024-LB

Aim In 2008, we defined 18 HLA DQ epitopes using mouse monoclonal antibodies and alloantibodies adsorbed and eluted (Ads/Elut) from HLA DQ homozygous cell lines. Here, we confirm 11 of the 18 epitopes and define 10 NEW epitopes of alloantibodies purified by Ads/Elut using recombinant single antigen cell lines. Methods Alloantibodies were purified from the sera of 36 kidney transplant patient by Ads/Elut using HLA DQ recombinant single antigen cell lines (rHLA-DQ). Each serum was Ads/Elut using several different cell lines to insure monoclonality. Eluted antibody specificities were determined by the HLA Class II Single Antigen (SA) beads. Epitope search software was used to identify amino acids exclusively shared among all positive DQ heterodimers (DQA or DQB) on the SA beads. These amino acids define the epitope. Results Ten new HLA DQ epitopes, 2 Alpha & 10 Beta chain, are defined (partial list in table below) and eleven previously defined DQ epitopes were confirmed. Donor Specific Antibodies (DSA) were found in almost all sera. And in most sera, we found more than one antibody positive with the same mismatched antigen, but each targets a different epitope. When anti DR or DP antibodies were present in the sera, the eluted antibodies were negative with the DR and DP beads indicating only DQ-specificities. Partial list of new DQA,B epitopes. Serum # mismatched DQ (A or B chain) Ads/Elut Cell # Cell heterodimer DQA.DQB Antigens positive with eluted antibody Epitope location (chain) Position/aa define epitope #02 DQA1*05:01 nn NA DQA4,5,6 A 40G #10 DQB1*05:02 RC893 DQA102.DQB502 DQB2,5,6 B 140A #10 DQB1*05:02 RC753 DQA301.DQB201 DQB2,5 B 77R #14 DQB1*03:01 RC883 DQA301.DQB303 DQB6,7,8,9 B 75L #22 DQB1*06:02 RC841 DQA102.DQB602 DQB6 B 87F #31 DQB1*05:01 nn na DQB1*05:01 B 125S+126Q nk = not known, nn = not needed, na = not applicable, xx = exact allele not known Conclusions Using rHLA-DQ single antigen cell lines to purify anti HLA-DQ antibodies and testing the antibodies with the HLA-DQ single antigen beads proved very effective in defining DQA and DQB epitopes.

HLA-DPA1 and -DPB1 Antibodies in Kidney Transplantation

Introduction: All preformed HLA antibodies have a deleterious impact on kidney graft outcome. Moreover, HLA sensitization often shows in broad antibody patterns since antibodies bind to epitope/s of HLA molecules and these target epitopes can be shared between several molecules intra- and inter-HLA loci. Current HLA class II matching strategies for kidney transplantation only consider the DR antigens; DQ matching is sometimes considered for organ sharing program. But both α and β chains of DP molecules are polymorphic and can elicit a humoral immune response. DP molecules have generally low expression but are upregulated upon inflammation that may occur during transplantation. Detection of anti-DP antibodies have been difficult using complement-dependent citotoxicity but newer assays for antibody analysis, such as Luminex-Single Antigen beads (One Lambda, CA), greatly enhanced detection of anti-DP antibodies and allowed to distinguish between anti-DPB1 and -DPA1. Methods: We investigated the incidence of anti-DP alloantibodies in 248 renal transplant candidates showing production of HLA class II antibodies. Antibody characterization was performed using Luminex Single Antigen beads that contain 26 microbeads coated with DPA1/DPB1 heterodimers (4 DPA1 alleles and 19 DPB1 alleles variously combined each other). Using an online database (www.ebi.ac.uk/imgt/hla), we also analyzed the amino acid sequences of DPA1/DPB1 molecules corresponding to the detected antibodies to identify the putative epitopes for antibody formation. All the anti-DP positive patients had been typed for DPB1/DPA1 alleles by SSP methods. Results: Ninety-two (37%) of the HLA class II positive patients showed production of anti-DP antibodies; 71% of these patients had anti-DPB1, 2% had anti-DPA1 and the remaining 27% of patients had both anti-DPA1 and-DPB1.As for sensitizing events, 66 (72%) of the anti-DP positive patients had had a previous transplant, 21 (23%) had had pregnancies and 5 (5%) received transfusions only. Noteworthy, anti-DP antibodies were exclusively present in sera from 8 of the 66 (12%) patients sensitized by a previous transplant; 2 of these re-transplant patients developed anti-DPA1 only. Analyzing epitopes whose recognition determined antibody formation, we evidenced that 42 (64%) of the retransplant patients developed a broad antibody pattern (anti-DPB1*01, *03, *05, *06, *09, *10, *11, *13, *14, *17, *19, *20) due to the recognition of 84-87DEAV epitope of the previous graft. Conclusion: This study confirms that both α and β chain epitopes of HLA-DP molecules are immunogenic. The most important stimulus for anti-DP antibody production was a previous transplant, but also pregnancies and transfusions may determine the development of anti-DP α/β chain specific antibodies. The anti-DPB1 humoral response often resulted in broad antibody patterns because of the sharing of the same epitope by many DPB1 molecules. Consequently, in renal transplantation, HLA matching strategy should consider all the HLA class II molecules especially for re-transplant patients.

Impact of Pretransplant Human Leukocyte Antigen-C and -DP Antibodies on Kidney Graft Outcome

The aim of our study was to determine whether the presence of specific human leukocyte antigen (HLA)-C and -DP antibodies before transplantation influenced graft outcomes in immunized recipients. Two groups of pretransplant immunized recipients were studied: patients with only classical HLA-A, -B, -DR, -DQ antibodies ( n = 176) and those with classical plus HLA-C and/or -DP antibodies ( n = 27). Acute antibody-mediated rejection was preferentially associated with the presence of pretransplant anti-HLA-C and -DP antibodies (5/6 cases). In four cases, acute rejection episodes were followed by graft loss within 15 months after transplantation. There was a significant increase in the number of acute rejection episodes especially antibody-mediated acute rejections ( P = .036) and in the number of graft losses for immunologic reasons ( P < .001) among the group with pretransplant anti-C and -DP antibodies. Pretransplant anti-DP antibodies seemed to be involved more frequently in poor graft outcomes as shown in several recent published cases. We need to investigate their specific role among a larger cohort, taking into account an epitope analysis.

Algorithm to Manage Highly Sensitized Kidney Transplant Recipients in Poland

Objective Programs for immunized transplant recipients are essential to achieve graft survivals comparable to those of non-immunized recipients. The threshold in Poland is a PRA by the complement-dependent cytotoxicity (CDC) method greater than 50%, which includes approximately 3.8% of the patients. At the same time the United Network for Organ Sharing there recipients represent approximately 16% of the waiting list in the United Network for Organ Sharing (UNOS). The underestimation of the immunized group in Poland may be due to differences in laboratory techniques to assess alloantibodies. Materials and methods This study investigated 55 potential recipients with a PRA by CDC > 50%. We used the following algorithm to assess their immunization: Luminex screening test for an HLA antibody; specificity assessed with Luminex Single Antigen, vPRA (evaluation of immunization of the patient); and analysis of acceptable HLA incompatibilities (HLAMatchmaker). Results All recipients were positive class I anti-HLA antibodies and 94.5% were positive for class II. For the groups of subjects with PRA-CDC from 50% to 79% versus those greater than 80%, the average values of PRA-CDC were 62.2% and 89.5%, respectively. The virtual PRA results for these groups were 95.7% and 97.2%, respectively. In addition, anti-HLA-Cw, anti-DQ and anti-DP antibodies were detected in 77%, 84%, and 51% of recipients, respectively. Immunized recipients reported to the next transplant were characterized by the antibodies against mismatch only in 68%. For all potential recipients, additional acceptable non-compliance was determined with HLAMatchmaker: 152 specificity for locus A and 252 for locus B. Conclusions Evaluation of immunization status of recipient candidates should be routinely performed using tests to assess class and specificity as well as level of alloantibodies to enable determination of a safe potential donor. As a routine test, PRA-CDC underestimates the number of highly immunized patients. Exclusion from the list of patients with repeated non-compliance is a simplification, which reduces their chance for transplantation.

Anti-HLA Antibodies and Graft Rejection In Hematopoietic Stem Cell Transplantation

AbstractAbstract 519We have previously identified a high risk of primary graft failure (PGF) in patients with DSA in T-cell depleted haploidentical transplantation (HaploSCT); 3/4 patients with DSA had PGF compared with 1/20 patients without DSA. All patients with DSA against anti-HLA A, B and DRB1 developed PGF, while 1 patient with anti-DP DSA did not. We now hypothesize that anti-DP antibodies may have a significant but less deleterious impact on engraftment. Methods:We evaluated the occurrence of PGF in 592 patients who received matched unrelated donor transplants at our institution after 1/2005. 88 % of the transplants were matched in 8/8 alleles of HLA-A,B,C,DRB1 in the HvG vector; approximately 75% of the transplants were mismatched in either DRB 3/4/5, DQB1 or DPB1. The presence of DSA was determined by testing the patients' sera with a panel of fluorescent beads coated with single HLA antigen preparations using a Luminex™ platform; results were interpreted as fluorescence intensity (FI) against DSA mismatch. All HLA loci were typed by high resolution methods. Results:19/592 patients (3%) had either PGF (N=9) or died early without engraftment (ED) (N=10). The only DSA identified were against the DP molecule in 8 patients matched in 8/8 HLA alleles with no apparent specificity. 3/8 (37.5%) patients had PGF/ED compared with 16/584 (2.7%) who did not have DSA (p=0.001, RR=23.3). One patient with PGF in the presence of anti-DP DSA had a second transplant in the absence of antibodies and engrafted cells from the same donor. In spite of common HLA sensitization (116/592, 19.6%) only 8 patients (1.4%) presented DSA in their pre-transplant specimens, mostly females (N=7) with a median age 49 years. DSA ranged from 1558 to 9845 FI, similar in patients with and without engraftment. Overall there was a 3% risk of PGF/ED without HLA antibodies, 6% risk in the presence of antibodies but not DSA, and 37.5% risk in the presence of DSA. Multivariate analysis revealed that DSA (p=0.0001) and ABO mismatch (p=0.04) were the only variables associated with graft failure. There was a significant association between female gender and allosensitization, 30.8% of females had anti HLA antibodies vs. 12.1% males (p<0.0001). While no difference in the incidence of HLA antibodies was observed between females with no prior pregnancies and males (p=0.24), this became apparent when allosensitization was evaluated in males vs. females with one pregnancy (p=0.008) and females with 2 or more pregnancies (p=0.0003). Conclusions:These results, combined with our previous findings in HaploSCT, suggest that DSA are associated with graft rejection in hematopoietic stem cell transplantation. Graft failure occurs less often with anti-DP DSA (3/9) compared with DSA against high expression HLA molecules (3/3, HLA-A, B or DRB1) and may confer a lower risk for graft rejection. The differences may reside in the lower levels of DP molecules expressed on cell surface. DSA screening is warranted when considering donors with HLA mismatches as strategies for donor selection and/or antibody level reduction may be needed to decrease the risk of PGF in allogeneic HSCT from partially HLA-matched donors. Disclosures:No relevant conflicts of interest to declare.

110-P: Donor-Specific (DSA) Anti-DP Antibodies Associate With Graft Failure (PGF) in Unrelated Donor Hematopoietic Stem Cell Transplantation (MUDHSCT)

110-P: Donor-Specific (DSA) Anti-DP Antibodies Associate With Graft Failure (PGF) in Unrelated Donor Hematopoietic Stem Cell Transplantation (MUDHSCT)