Cross-reactive Group Research Articles

Analysis of transfusion effect of different platelet matching schemes in patients with platelet transfusion refractoriness

Objective: To analyze the transfusion effect of different platelet matching schemes in patients with platelet transfusion refractoriness (PTR). Methods: A total of 94 patients with PTR received by Taiyuan Blood Center from January to December 2021 were retrospectively analyzed, including 26 males and 68 females, aged 53(34,66) years. Platelet antibody screening was performed by enzyme-linked immunosorbent assay (ELISA). For patients with positive human leukocyte antigen (HLA) class Ⅰ antibodies, Luminex platform liquid chip assay was used to identify the specificity of antibodies, and platelets with missing allelic expression antigen corresponding to their specific antibodies were found in the platelet donor gene database established in our laboratory. For patients with negative class HLA-Ⅰ antibody screening, medium and high-resolution HLA-A and B alleles were genotyped by polymerase chain reaction restriction sequence specific oligonucleotide (PCR-SSO), and the compatible platelets were searched from the platelet donor gene database by HLA cross-reactive group genotype matching scheme or directly selected by serological cross-matching. The PCI compliance rate and total transfusion effective rate of different mismatch site groups and different matching scheme groups were statistically analyzed. Results: Platelet antibody was detected in 39 of 94 PTR patients with a positive rate of 41.5%, and all of them were HLA-Ⅰ antibodies, and 1 case was accompanied by human platelet antigen (HPA) antibody. A total of 134 times of compatible platelets were supplied to 39 patients with HLA-Ⅰ antibody positive by using antibody avoidance matching method. And the total effective rate of transfusion was 97.8% (131/134); The PCI compliance rates of HLA-A antigen mismatch, HLA-B antigen mismatch and HLA-A and B antigen mismatch groups were 81.6% (31/38), 86.5% (32/37) and 78.6% (22/28), respectively. The total effective rate of transfusion was 97.4% (37/38), 94.6% (35/37) and 100% (28/28), respectively, with no statistical significance (all P>0.05). A total of 118 times of compatible platelets were provided by HLA antigen cross-reaction group genotype matching and serological cross-matching, 90 transfusion effects were collected during follow-up, and the total effective rate was 76.7% (69/90). Conclusion: The combination of different platelet matching schemes can improve the PCI compliance rate and the total effective rate of transfusion in PTR patients.

Anti-HLA-B7/HLA-B44 strong cross immunoreactivity observed in flow cytometry HLA-B27 immunotyping.

Cross reactivities are known for human leukocyte antigen inside HLA-B7 related Cross-Reactive Group (B7CREG). Some CE-IVD flow-cytometry kits use double monoclonal antibodies (mAb) to distinguish HLA-B27 and HLA-B7 but practice reveals more complexes results. This study explores the performances of this test. Analysis of 466 consecutive cases using HLA-B27 IOTest™ kit on a Navios™ cytometer from Beckman-Coulter, partially compared to their genotypes. Expected haplotypes HLA-B27-/HLA-B7- (undoubtedly HLA-B27 negative) and HLA-B27+/HLA-B7- (undoubtedly HLA-B27+) were clearly identified according to the manufacturer's instructions. On the opposite, patients strongly labeled with anti-HLA-B7 showed three different phenotypes regarding anti-HLA-B27 labeling: (1) most of the cases were poorly labeled in accordance with cross reactivity inside B7CREG (HLA-B27-/HLA-B7+ haplotype); (2) rare cases had strong B7 and B27 labeling corresponding to HLA-B27+/HLA-B7+ haplotype; (3) even less cases had strong labeling by anti-HLA-B7 but non for anti-HLA-B27, all expressing HLA-B44 and no B7CREG molecules. Surprisingly, more cases were not labeled with anti-HLA-B7 antibody but partially labeled with anti-HLA-B27 suggesting another cross reactivity out of B7CREG. mAb HLA typing suggests new, cross reactivities of anti-HLA-B27 antibody to more molecules out of B7CREG and of anti-HLA-B7 antibody but not anti-HLA-B27 to HLA-B44 molecule also out of B7CREG. HLA-B27 could surely be excluded in most samples labeled with HLA-B27, below a "grey zone" on intermediate intensity. More comparison is needed in future studies.

Structural principles of peptide-centric chimeric antigen receptor recognition guide therapeutic expansion.

Peptide-centric chimeric antigen receptors (PC-CARs) recognize oncoprotein epitopes displayed by cell-surface human leukocyte antigens (HLAs) and offer a promising strategy for targeted cancer therapy. We have previously developed a PC-CAR targeting a neuroblastoma-associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes. Here, we determine the 2.1-angstrom crystal structure of the PC-CAR-PHOX2B-HLA-A*24:02-β2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). This PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactive group, covering a combined global population frequency of up to 46.7%. Biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation, and CAR T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.

Living Donor Liver Transplantation in Patients with Preformed Donor-Specific Anti-Human Leukocyte Antigen Antibodies Using Preoperative Desensitization Therapy According to Intensity of Donor-Specific Antibodies: A Single-Center Study.

BACKGROUND In liver transplantation (LT), preoperative desensitization therapy is considered necessary in patients positive for donor-specific anti-human leukocyte antigen antibodies (DSAs). However, the relationship between DSA intensity and the necessary desensitization therapy is unclear. MATERIAL AND METHODS A total of 37 adult living donor (LD) LTs performed between January 2016 and March 2022 were examined. Mycophenolate mofetil (MMF) was administered preoperatively in DSA-positive cases with positive lymphocyte cross-matching who underwent LDLT. In those with strongly positive DSA (mean fluorescence intensity 10 000), rituximab was administered 2 weeks before LDLT in addition to MMF. Cross-reactive epitope group antigen (CREG)-alone-positive cases were also treated with preoperative MMF when lymphocyte cross-matching was positive. RESULTS Of the 37 patients, 9 were DSA-positive, 7 were CREG-alone-positive, and the others were double-negative. Of 9 DSA-positive cases, desensitization therapy was performed in 7, among which rituximab administration was performed in 3 strongly DSA-positive cases. Of 7 CREG-alone-positive cases, 2 were lymphocyte cross-match-positive and underwent desensitization therapy. The 1-year survival rate was 100% in both DSA- and CREG-alone-positive cases. The frequency of T-cell mediated rejection in DSA-positive, CREG-alone-positive, and double-negative cases was 22%, 43%, and 29%, respectively, with no significant difference. Antibody-mediated rejection occurred in only 1 patient, who was strongly DSA-positive and blood-group incompatible. There was also no significant difference among the 3 groups in terms of the frequency of biliary complications or 90-day mortality. CONCLUSIONS Satisfactory LDLT results were achieved in DSA- and CREG-alone-positive cases following desensitization therapy.

Structure-guided design of a broadly cross-reactive multivalent group a streptococcal vaccine

The M protein of group A streptococci (Strep A) is a major virulence determinant and protective antigen. The N-terminal region of the M protein is variable in sequence, defines the M/emm type, and contains epitopes that elicit opsonic antibodies that protect animals from challenge infections. Although there are >200 M types of Strep A, there is now evidence that structurally related M proteins can be grouped into clusters and that immunity may be cluster-specific in addition to M type-specific. This observation has led to recent studies of structure-based design of multivalent M peptide vaccines to select peptides predicted to cross-react with heterologous M types to improve vaccine coverage. In the current study, we have applied a refined series of peptide structural algorithms to predict immunological cross-reactivity among 117 N-terminal M peptides representing the most prevalent M types of Strep A. Based on the results of the structural analyses, in combination with global M type prevalence data, we constructed a 32-valent vaccine containing 19 cross-reactive vaccine candidates predicted to cross-react with 37 heterologous M peptides to which were added 13 type-specific M peptides. The 4-protein recombinant vaccine was immunogenic in rabbits and elicited significant levels of antibodies against 31/32 (97%) vaccine peptides and 28/37 (76%) peptides predicted to cross-react. The vaccine antisera also promoted opsonophagocytic killing of vaccine and cross-reactive M types of Strep A. Based on a recent analysis of M type prevalence of Strep A, the potential global coverage of the 32-valent vaccine is ∼90%, ranging from 68% in Africa to 95% in North America. Our results indicate the utility of structure-based design that may be applied to future studies of broadly protective M peptide vaccines.

Development and Validation of a Protein Array for Detection of Antibodies against the Tick-Borne Pathogen Borrelia miyamotoi.

Current serological tests for the emerging tick-borne pathogen Borrelia miyamotoi lack diagnostic accuracy. To improve serodiagnosis, we investigated a protein array simultaneously screening for IgM and IgG reactivity against multiple recombinant B. miyamotoi antigens. The array included six B. miyamotoi antigens: glycerophosphodiester phosphodiesterase (GlpQ), multiple variable major proteins (Vmps), and flagellin. Sera included samples from cases of PCR-proven Borrelia miyamotoi disease (BMD), multiple potentially cross-reactive control groups (including patients with culture-proven Lyme borreliosis, confirmed Epstein-Barr virus, cytomegalovirus, or other spirochetal infections), and several healthy control groups from regions where Ixodes is endemic and regions where it is nonendemic. Based on receiver operating characteristic (ROC) analyses, the cutoff for reactivity per antigen was set at 5 μg/mL for IgM and IgG. The individual antigens demonstrated high sensitivity but relatively low specificity for both IgM and IgG. The best-performing single antigen (GlpQ) showed a sensitivity of 88.0% (95% confidence interval [CI], 78.9 to 93.5) and a specificity of 94.2% (95% CI, 92.7 to 95.6) for IgM/IgG. Applying the previous published diagnostic algorithm-defining seroreactivity as reactivity against GlpQ and any Vmp-revealed a significantly higher specificity of 98.5% (95% CI, 97.6 to 99.2) but a significantly lower sensitivity of 79.5% (95% CI, 69.3 to 87.0) for IgM/IgG compared to GlpQ alone. Therefore, we propose to define seroreactivity as reactivity against GlpQ and any Vmp or flagellin which resulted in a comparable sensitivity of 84.3% (95% CI, 74.7 to 90.8) and a significantly higher specificity of 97.9% (95% CI, 96.9 to 98.7) for IgM/IgG compared to GlpQ alone. In conclusion, we have developed and validated a novel serological tool to diagnose BMD that could be implemented in clinical practice and epidemiological studies. IMPORTANCE This paper describes the protein array as a novel serological test for the diagnosis of Borrelia miyamotoi disease (BMD), by reporting the methodology, the development of a diagnostic algorithm, and its extensive validation. With rising numbers of ticks and tick bites, tick-borne diseases, such as BMD, urgently deserve further societal and medical attention. B. miyamotoi is prevalent in Ixodes ticks across the northern hemisphere. Humans are exposed to, and infected by, B. miyamotoi and develop BMD in Asia, in North America, and to a lesser extent in Europe. However, the burden of infection and disease remains largely unknown, due to the noncharacteristic clinical presentation, together with the lack of awareness and availability of diagnostic tools. With this paper, we offer a novel diagnostic tool which will assist in assessing the burden of disease and could be implemented in clinical care.

Relevance of Anti-HLA Antibody Strength Underestimation in Single Antigen Bead Assay for Shared Eplets.

HLAs contain combinations of multiple eplets, sometimes shared between numerous HLA alleles. Some authors suggested that single antigen bead (SAB) assays may underestimate the signal of anti-HLA antibodies (Ab) when several beads share the targeted eplet. However, this assumption has not yet been validated experimentally. We selected 5 eplets shared by 1-24 beads of the routine SAB kits: the eplet 163LS/G; the 3 eplets 127K, 62GE, and 62GRN thereafter called cross-reactive group 2C; the 82LR eplet, well-known as Bw4; the locally called QB2A5 eplet associated with the DQA1*05:01/DQB1*02:01 combination; and the 40GR DQ eplet. We selected a dozen of sera for each eplet with Ab mean fluorescence intensity (MFI) between 1000 and 15 000 for the beads carrying the targeted eplet. We tested them with the classical SAB panel (SABp), with an isolated bead carrying the eplet (isolated SAB [SABi]) and with a mixture of both (SABp+i). No significant difference in MFI was detected among SABi, SABp, and SABp+i conditions for all the eplets. We noticed only a nonsignificant difference in the Ab MFI signal due to eplet sharing on the SAB assay. We, therefore, conclude that this phenomenon should no longer be considered as a significant risk factor during patient follow-up pre- or posttransplantation.

Hidden Patterns of Anti-HLA Class I Alloreactivity Revealed Through Machine Learning.

Detection of alloreactive anti-HLA antibodies is a frequent and mandatory test before and after organ transplantation to determine the antigenic targets of the antibodies. Nowadays, this test involves the measurement of fluorescent signals generated through antibody–antigen reactions on multi-beads flow cytometers. In this study, in a cohort of 1,066 patients from one country, anti-HLA class I responses were analyzed on a panel of 98 different antigens. Knowing that the immune system responds typically to “shared” antigenic targets, we studied the clustering patterns of antibody responses against HLA class I antigens without any a priori hypothesis, applying two unsupervised machine learning approaches. At first, the principal component analysis (PCA) projections of intra-locus specific responses showed that anti-HLA-A and anti-HLA-C were the most distantly projected responses in the population with the anti-HLA-B responses to be projected between them. When PCA was applied on the responses against antigens belonging to a single locus, some already known groupings were confirmed while several new cross-reactive patterns of alloreactivity were detected. Anti-HLA-A responses projected through PCA suggested that three cross-reactive groups accounted for about 70% of the variance observed in the population, while anti-HLA-B responses were mainly characterized by a distinction between previously described Bw4 and Bw6 cross-reactive groups followed by several yet undocumented or poorly described ones. Furthermore, anti-HLA-C responses could be explained by two major cross-reactive groups completely overlapping with previously described C1 and C2 allelic groups. A second feature-based analysis of all antigenic specificities, projected as a dendrogram, generated a robust measure of allelic antigenic distances depicting bead-array defined cross reactive groups. Finally, amino acid combinations explaining major population specific cross-reactive groups were described. The interpretation of the results was based on the current knowledge of the antigenic targets of the antibodies as they have been characterized either experimentally or computationally and appear at the HLA epitope registry.

Comparison of Single Antigen Bead–Based, Cell-Based, and Solid Phase–Based Crossmatch Methods

AimAntibody assessment during pretransplantation term is important to detect donor specific antibodies. These donor-specific antibodies are determined by various crossmatch methods (flow cytometric [FCXM], complement-dependent cytotoxic [CDCXM], and Luminex [LMXM]). Recently, single antigen bead (SAB) assays have been used for the assessment of hypersensitized patients. The aim of this study was to compare sensitivity and specificity of the 3 crossmatch methods in reference to the SAB method. MethodIn this study, 69 hypersensitized patients with high class I and/or class II panel reactive antibodies were tested using the flow cytometric SAB method. Serum samples were cross-matched by 3 crossmatch methods with the cells of a volunteer healthy individual, and the results were evaluated according to HLA and cross-reactive epitope groups (CREGs). ResultsSensitivity was found to be better with T FCXM (0.91) and class I LMXM (0.87). Specificity of peripheral blood lymphocyte CDCXM method (1.0) was found to be better than the other 2 methods (0.33 and 0.57, respectively). Sensitivity of class II LMXM (0.88) was found to be better than the others (0.42 for B CDCXM and 0.82 for B FCXM, respectively). The specificity of the B CDCXM, B FCXM, and class II LMXM was similar (0.44, 0.44, and 0.33, respectively). CREGs results were similar to HLA results. ConclusionAlthough CDCXM has high specificity for the detection of anti-HLA antibodies, it has low sensitivity. To increase sensitivity, FCXM or LMXM methods may be used with the CDCXM test. These results will be beneficial for laboratories and clinicians during graft survival and patient health assessment.

Anti-HLA Antibody: The Role of Epitopes in Organ Transplantation.

Recent developments have been achieved for better matching in solid-organ transplantation by epitope matching. HLA matching remains a standard immunologic strategy to determine organ compatibility for recipients. Advancements in tissue typing have introduced HLA matching at the epitope level. For this, B cells are able to recognize polymorphic amino acid configurations, which are named epitopes. However, antibody production against these epitopes may be a leading cause of rejection. Although data to support the added clinical benefit of epitope matching over traditional antigen matching are still lacking, there are at least 2 theoretical reasons for epitope matching: (1) to avoid future allosensitization with the development of anti-HLA antibodies and (2) to allow selection of a suitable allograft for highly sensitized patients through virtual crossmatch. Sensitive antibody tests with a comprehensive panel of single alleles have yielded informative, donor-specific antibody reactivity patterns that can be analyzed with a computer algorithm. This program (HLAMatchmaker) uses structurally defined eplets to describe epitopes that can react with specific antibodies. Although low mean fluorescence intensity levels by Luminex (Austin, TX, USA) assay are usually considered low risk for solid organ transplant, it could be important in posttransplant humoral rejection. Thus, specific shared eplets should always be investigated with respect to previous transplant mismatches. Epitopes are present on a single HLA (private epitope) or shared by multiple antigens (public epitope). The phenomenon of crossreactivity in HLA testing, often explained as crossreactive groups of antigens with antibody, can be clearly explained now by public epitopes, an antibody targeting an epitope that shows positive reaction with all antigens sharing the epitope. A better understanding of the immunogenicity and structural characteristics of HLA epitopes will guide us to consider epitope matching as an important parameter for donor selection in kidney transplant in the near future.

Efficacy of HLA-Matched PLT Transfusions for Platelet Transfusion Refractoriness in HSCT Patients

Introduction: Cross-matched platelet (cross-matched PLT) transfusions are effective for immune-mediated platelet transfusion refractoriness (PTR). However, cross-matched PLT is more costly and takes more effort to cross match than a standard PLT unit. Recently, the utility of virtual HLA cross-matched PLT (HLA-matched PLT) has been reported. HLA-matched PLT is defined as HLA-A/B matched or cross-reactive groups excluded. Cross-matched PLTs are collected from donors selected based on patient's HLA class I antibody and HLA typing of the donor. The products are supplied after confirmation of cross-matched negative. Hematopoietic stem cell transplantation (HSCT) patients frequently receive many transfusions before HSCT. Therefore, HLA antibody is often detected, which causes PTR. After HSCT, many complications, such as infection, bleeding, and thrombotic microangiopathy, may occur, which can cause PTR. Inadequate post-transfusion PLT increment occurs in more than 50% of PLT transfusions in patients undergoing HSCT. Here, we evaluated the effect of HLA-matched PLT for PTR in post-HSCT recipients.Methods: The records of all patients who underwent HSCT at Okayama University Hospital between 2010 and 2017 were reviewed to identify patients who received either cross-matched or HLA-matched PLT products after HSCT for hematologic malignancy. All patients had HLA Class I antibody and immune-mediated PLT transfusion refractoriness (PTR). An analysis of the initial episode of the PLT transfusion refractoriness was conducted for each patient. The following data were recorded for each patient: disease, age, sex, body surface area, HSCT donor source, HLA typing (Class I), HLA Class I antibody screen, WHO Grade 3 to 4 bleeding episodes, complications, death, and platelet-corrected count increment (CCI). Non-immune mediated causes were defined as an episode of fever (≥38 °C), bleeding (Grade 3 to 4), DIC, infection, thrombotic microangiopathy, sinusoidal obstructive syndrome, and splenomegaly. PLT products (cross-matched and HLA-matched units) were supplied by the Japanese Red Cross Society. The exact PLT content of each unit was obtained from the Japanese Red Cross Society. The 24-hour corrected count increments (CCI-24) were calculated to evaluate the effect of PLT transfusions. A CCI-24 more than 4500/μL was considered a successful transfusion.Results: Sixteen patients received a total of 241 PLT transfusions; 139 PLT transfusions were cross-matched, and 102 PLT transfusions were HLA-matched. The median CCI-24 for cross-matched PLT transfusions and HLA-matched transfusions was 2626/μL and 6137/μL, respectively, (p<0.001). The percentage of successful transfusions was 38.1% for cross-matched PLT and 57.8% for HLA-matched PLT, respectively, (p<0.001). Eighty-four PLT transfusions were used under situations without cause for non-immune mediated PTR. The percentage of successful transfusions (median CCI-24) was 62.8% (6684/μL) for cross-matched PLT and 65.1% (7108/μL) for HLA-matched PLT(p=0.825). Of the 241 transfusions, 157 (65.1%) transfusions were used under situations with cause for non-immune mediated PTR. When patients had non-immune mediated PTR, the percentage of successful transfusions was only 28.1% for cross-matched PLT and 51.1% for HLA-matched PLT (p=0.001). The median CCI-24 was 1856/μL for cross-matched PLT and 5824/μL for HLA-matched PLT (p<0.001). HLA-matched PLT resulted in a higher response, and episodes of bleeding and splenomegaly were lower by multiple linear regression analysis (Table 1).Conclusions: HLA-matched PLT was not inferior to cross-matched PLT for post-HSCT PTR. Both cross-matched PLT and HLA-matched PLT resulted in a similar response for immune-mediated PTR, but HLA-matched PLT was superior for non-immune mediated PTR. The difference of selection method between cross-matched PLT and HLA-matched PLT may influence the effectiveness of the transfusion. The reasons of superior CCI-24 result in HLA-matched PLT are currently under investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

P112 Clearance of pre-formed donor specific antibodies after liver transplantation is specific for DSA and cross-reactive antigens

Aim Liver transplant recipients with pre-formed HLA DSA are reported to “clear” antibodies post-transplant. We sought to determine if the reduction in HLA antibodies was specific for donor antigens or represented a global decline in alloantibodies. Methods We retrospectively analyzed the post-transplant DSA course for 27 liver allograft recipients transplanted with pre-formed HLA DSA, measured by single antigen assay. Results In 16 patients, DSA was persistently seen at last follow-up (although weaker than pre-transplant in all patients); another 2 patients also developed de novo DSA to other donor HLA. In the remaining 9 patients, DSA declined dramatically post-transplant and was not detected at last follow-up. We asked whether third party antibodies also declined (i.e. global immunosuppression) or whether the patterns were specific for DSA and cross-reactive antigens. In 6 of 9 patients whose DSA declined, some non-DSA remained strong while the third party antibodies that reduced were clearly in cross-reactive groups with the donor specific antibodies. In one of the remaining patients, all of their antibodies disappeared post-transplant, in another the DSA did not fit into a CREG, and in the last patient DSA was to HLA-C locus. One example is given in the figure below, where strong antibodies persisted to third party A1 CREG; but DSA to A32 and antibodies to other A10/A19 CREG antigens declined post-transplant. In the same patient DSA to DQ6 disappeared during follow-up, as did non-DSA DQ5 antibodies; but antibodies to DQ2 persisted. Conclusions Overall, liver allograft recipients experienced a global reduction in HLA antibodies, but the most profound effect was on DSA and CREG-related antigens. These cases suggest that liver clearance of HLA antibodies is specific for donor reactive antibodies and cross-reactive clones.

OR50 Donor specific HLA antibody formation following distal tibial allograft and subsequent graft resorption: A case report

Background The association between donor specific Human Leukocyte Antigen (HLA) antibody formation and small bone allograft resorption has not been studied. We present the case of a patient treated for glenoid bone loss with a distal tibial allograft who formed donor specific HLA antibodies against the allograft and had subsequent graft resorption. Methods The patient received a right glenoid reconstruction using distal tibia allograft with Bankart repair in June 2015. X-ray and CT-Scans were performed pre-and post-surgery at standard checkpoints. Patient blood and serum samples were collected pre-and-post-surgery for HLA typing and HLA antibody testing. Results Pre-operative CT scans revealed large Hill-Sachs and bony Bankart lesions, and pre-operative HLA antibody testing revealed no HLA antibodies. The 2-week post-operative radiograph revealed proper graft fixation, and the patient had good range of motion 6-weeks post-surgery. However, 6-week post-operative HLA antibody testing revealed development of donor specific HLA antibodies directed at HLA-A2 antigen as well as several antigens belonging to the A2 crossreactive group (A24, A68, A69, B57 and B58), cPRA of 69%. Eplet analysis showed reactivity to 62GE and 151AHV eplets. At 3-months post-surgery, the patient was weak in internal rotation, and the HLA-A2 antibodies continued to be detected at 5-months post-surgery. Shortly after the antibody peak, the 6-month post-operative CT arthrogram revealed significant graft resorption. Interestingly, the donor specific HLA-A2 antibodies were undetectable 6 months after resorption was complete. Discussion This case report shows that small bone allografts can lead to HLA sensitization. The temporal correlation between donor specific HLA antibody formation and clinical findings strongly suggest an association between HLA antibody formation and graft resorption. It is unclear what impact HLA antibody formation may have on future regrafting in this patient. This case shows a clear association between the formation of donor specific HLA antibodies and graft resorption, which suggests a link between the two events. Our future studies will address the frequency of HLA antibody formation, and investigate whether tissue processing techniques could be improved to reduce bone allograft antigenicity.

Separation of Multiple HLA Antibody Specificities from Some Cross-Reactive Group (CREG).

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Does human leukocyte antigens sensitization matter for xenotransplantation?

The major histocompatibility complex class I and class II human leukocyte antigens (HLA) play a central role in adaptive immunity but are also the dominant polymorphic proteins targeted in allograft rejection. Sensitized patients with high levels of panel-reactive anti-HLA antibody (PRA) are at risk of early allograft injury, rejection, reduced allograft survival and often experience prolonged waiting times prior to transplantation. Xenotransplantation, using genetically modified porcine organs, offers a unique source of donor organs for these highly sensitized patients if the anti-HLA antibody, which places the allograft at risk, does not also enhance anti-pig antibody reactivity responsible for xenograft rejection. Recent improvements in xenotransplantation efficacy have occurred due to improved immune suppression, identification of additional xenogeneic glycans, and continued improvements in donor pig genetic modification. Genetically engineered pig cells, devoid of the known xenogeneic glycans, minimize human antibody reactivity in 90% of human serum samples. For waitlisted patients, early comparisons of patient PRA and anti-pig antibody reactivity found no correlation suggesting that patients with high PRA levels were not at increased risk of xenograft rejection. Subsequent studies have found that some, but not all, highly sensitized patients express anti-HLA class I antibody which cross-reacts with swine leukocyte antigen (SLA) class I proteins. Recent detailed antigen-specific analysis suggests that porcine-specific anti-SLA antibody from sensitized patients binds cross-reactive groups present in a limited subset of HLA antigens. This suggests that using modern genetic methods, a program to eliminate specific SLA alleles through donor genetic engineering or stringent donor selection is possible to minimize recipient antibody reactivity even for highly sensitized individuals.

Epitopes and motifs of the HLA-B*14 allele family products and related HLA-B14 cross-reactive specificities.

The split specificities of HLA-B14 (B64, B65) are assigned to the B*14:01 (B64) and B*14:02 (B65) products only. Of the further 50 B*14 expressed products, only B*14:03 and B*14:06 are officially designated as HLA-B14. The B*14:08 product differs from B64 by a single amino acid substitution of W97R, while the B*14:53 specificity (which is a "short" B14 and neither B64 nor B65) differs from B64 by three residues (W97S, Y113H and F116Y). Comprehensive testing of B*14:08:01 cells (using 49 alloantisera with B64 or B64, B65 specificities, and five monoclonal antibodies with B65 or B64, B65 activity) showed that the B*14:08 specificity is, like the B*14:53 product, neither B64 nor B65 and appears as a "short" B14 specificity. To help understand the serological reactivity of the B*14:08 and B*14:53 products, and B64 and B65, we identified seven published epitopes (11AV, 97W, 61ICT, 116F, 131S+163T, 170RH and 420) and, by inspection, 29 motifs, that encompass one or more of B64, B65 and various HLA-B14 cross-reactive group specificities. We then considered the possession of these epitopes and motifs by the products of B*14:01 to B*14:06, B*14:08 and B*14:53. Seventeen of the 29 motifs fully complied with the one-/two-patch functional epitope concept for amino acid proximity, as determined by Cn3D software, the remainder partially complied. The nature and patterns of epitopes and motifs possessed by both B*14:08 and B*14:53 specificities supported their designation as HLA-B14 but non-B64/B65. Also that epitope 97W, with 11S or 11A, is critical for serological B64 and B65 reactivity. And conversely, that epitope 116F, and several identified motifs, are probably unimportant for HLA-B14 antibody reactivity. The previous submission that the B*14:03 specificity is HLA-B65 was compatible with its epitope/motif pattern. B*14:04 cells would also be expected to react as B65, based on its epitope/motif pattern, and not as B64 as previously implied. Also, from their epitope/motif patterns, and external serological information, it is probable that the B*14:05 and B*14:06 specificities will both appear as "short" HLA-B14, non-B64/B65. Several epitopes and motifs encompassed a range of HLA-B specificities included in the serological HLA-B14 cross-reactive group, thus supporting these original serological findings.

Cucurbit[6]uril-Promoted Click Chemistry for Protein Modification.

Azide-alkyne cycloaddition is a powerful reaction for the formation of bioconjugates. When catalyzed by Cu(I) or strain promotion, this cycloaddition is considered to be a "click" reaction with many applications in chemical biology and materials science. We report a new type of azide-alkyne click chemistry for the synthesis of protein conjugates using cucurbit[6]uril (CB6) supramolecular chemistry. CB6-promoted azide-alkyne cycloaddition has been previously used for the synthesis of rotaxanes but has not been applied to the development of complex bioconjugates. By developing new substrates for CB6 click that do not contain any cross-reactive functional groups and by optimizing reaction conditions, we converted CB6 click chemistry from a rotaxane synthesis tool into a useful bioconjugation technique. Using these new parameters, we synthesized a series of protein conjugates including protein-peptide, protein-DNA, protein-polymer, and protein-drug conjugates. We further demonstrated that CB6 click can be used in conjunction with strain-promoted azide-alkyne cycloaddition to generate distinct bioconjugates in protein mixtures. CB6 click is a promising new reaction for the development of protein conjugates and can be applied toward the synthesis of complex biomaterials for a wide range of applications.

A B Cell Epitope Peptide Derived from the Major Grass Pollen Allergen Phl p 1 Boosts Allergen-Specific Secondary Antibody Responses without Allergen-Specific T Cell Help.

More than 40% of allergic patients suffer from grass pollen allergy. Phl p 1, the major timothy grass pollen allergen, belongs to the cross-reactive group 1 grass pollen allergens that are thought to initiate allergic sensitization to grass pollen. Repeated allergen encounter boosts allergen-specific IgE production and enhances clinical sensitivity in patients. To investigate immunological mechanisms underlying the boosting of allergen-specific secondary IgE Ab responses and the allergen epitopes involved, a murine model for Phl p 1 was established. A B cell epitope–derived peptide of Phl p 1 devoid of allergen-specific T cell epitopes, as recognized by BALB/c mice, was fused to an allergen-unrelated carrier in the form of a recombinant fusion protein and used for sensitization. This fusion protein allowed the induction of allergen-specific IgE Ab responses without allergen-specific T cell help. Allergen-specific Ab responses were subsequently boosted with molecules containing the B cell epitope–derived peptide without carrier or linked to other allergen-unrelated carriers. Oligomeric peptide bound to a carrier different from that which had been used for sensitization boosted allergen-specific secondary IgE responses without a detectable allergen-specific T cell response. Our results indicate that allergen-specific secondary IgE Ab responses can be boosted by repetitive B cell epitopes without allergen-specific T cell help by cross-linking of the B cell epitope receptor. This finding has important implications for the design of new allergy vaccines.

HLA Epitopes: The Targets of Monoclonal and Alloantibodies Defined.

Sensitization to human leukocyte antigens (HLA) in organ transplant patients causes graft rejection, according to the humoral theory of transplantation. Sensitization is almost ubiquitous as anti-HLA antibodies are found in almost all sera of transplant recipients. Advances in testing assays and amino acid sequencing of HLA along with computer software contributed further to the understanding of antibody-antigen reactivity. It is commonly understood that antibodies bind to HLA antigens. With current knowledge of epitopes, it is more accurate to describe that antibodies bind to their target epitopes on the surface of HLA molecular chains. Epitopes are present on a single HLA (private epitope) or shared by multiple antigens (public epitope). The phenomenon of cross-reactivity in HLA testing, often explained as cross-reactive groups (CREGs) of antigens with antibody, can be clearly explained now by public epitopes. Since 2006, we defined and reported 194 HLA class I unique epitopes, including 56 cryptic epitopes on dissociated HLA class I heavy chains, 83 HLA class II epitopes, 60 epitopes on HLA-DRB1, 15 epitopes on HLA-DQB1, 3 epitopes on HLA-DQA1, 5 epitopes on HLA-DPB1, and 7 MICA epitopes. In this paper, we provide a summary of our findings.

P010 Complex constellation of HLA antibodies contributes to decreasing trend of kidney transplant rate in candidates with 100% CPRA

The key component of the new kidney allocation system is increased priority for sensitized candidates. While transplants (tx) have increased 6 fold for recipients with a CPRA of ⩾99%, the trend in tx for these patients began to decrease over the 1st year. To determine if HLA antibody composition contributes to the decreasing tx rate, we compared the distribution of Cross-REactive Groups (CREG) in our waitlist candidates with different CPRA ranges. Kidney waitlist candidates (n = 5281) were tested for HLA antibodies using One Lambda single antigen beads on DTT-treated sera. CPRA values were determined based on unacceptable antigens (UA), and were clustered into 100%, 99%, 98%, 80–97% and 20–79% CPRA groups. The number of UA differs significantly between CPRA groups (Table). The 100%-CPRA group display 18–107 UA (98% of this group display >25 UA and 72% display ⩾50 UA). The 99%-CPRA group display 13–61 UA (78% display ⩾25 UA) while the 98%-CPRA group display 8–60 UA (66% display ⩾25 UA). The incidence of CREG HLA antibodies differs significantly among CPRA groups. Most candidates with 100% CPRA display 2–3 fold increase in the number of CREGs compared to candidates with 99% or 98% CPRA. More than 3 CREGs were observed in 63.8% of the 100%-CPRA group, but only in 21.3% of the 99%-CPRA group, in 2% of the 98%-CPRA group and in 1.4% of the 80–97%-CPRA group. Candidates with CPRA <80% did not display more than 4 CREGs. Over 80% of the candidates in the 100%-CPRA group are homozygotes at multiple HLA loci, and display antibodies to all antigens of one or more HLA loci except to self. Moreover, the probability of finding HLA-A/B/DR antigen matched kidney is zero for 43.6% of the candidates with 100% CPRA. These results suggest that the majority of the candidates in the100%-CPRA group are very broadly sensitized and are hard to find a compatible kidney, and thus need an alternative approach such as desensitization and living-donor kidney exchange. These data support the importance of HLA matching at least at the level of CREG.