Identification of Antibodies to DQß:DRα Interisotypic Heterodimers in Human Sera.

BACKGROUND: HLA class II antigens, DR, DQ, and DP, comprised an α and ß chains, which typically combine, within the same isotype, to form the major histocompatibility complex:peptide complex. Interisotypic pairing is not commonly observed. Although reports of DQß:DRα heterodimers exist, the pairing was reported to be unstable and, therefore, not studied to any extent. METHODS: DQß:DRα single antigens were produced through transfectant cell lines and used to identify and characterize positive reactive human sera by a multiplex bead-based assay. RESULTS: Stable DQß:DRα transfectants were constructed. Cell surface staining with class II-specific monoclonal antibodies revealed that some DQB1 alleles appear to be more efficient in expressing DQß:DRα heterodimers. Interestingly, alleles within the same serological group varied in their efficiency of forming dimers on the cell surface. For example, DQß0601:DRα had the highest transfection and cell membrane expression efficiency among 16 common DQB1 alleles tested. In contrast, DQß0603:DRα-positive transfectants demonstrated minimal surface expression. Assembly of DQß0601:DRα was not affected by the presence of a DQα chain. DQß0601:DRα and DQß0603:DRα single-antigen beads were used to screen human sera. Positive sera were identified that reacted to the unique epitopes of DQß0601:DRα protein on the cell surface of the transfectants. CONCLUSIONS: Our studies have demonstrated that unique DQß:DRα heterodimers can be formed and are stably expressed on the cell surface. Such antigenic combinations, presented on single-antigen beads, demonstrated that patient sera can react with such heterodimers. Investigations on the potential clinical roles of antibodies against such interisotypic heterodimers are now possible.

HLA class I peptide polymorphisms contribute to class II DQß0603:DQα0103 antibody specificity.

Antibodies reactive to human leukocyte antigens (HLA) represent a barrier for patients awaiting transplantation. Based on reactivity patterns in single-antigen bead (SAB) assays, various epitope matching algorithms have been proposed to improve transplant outcomes. However, some antibody reactivities cannot be explained by amino acid motifs, leading to uncertainty about their clinical relevance. Antibodies against the HLA class II molecule, DQß0603:DQα0103, present in some candidates, represent one such example. Here, we show that peptides derived from amino acids 119-148 of the HLA class I heavy chain are bound to DQß0603:DQα0103 proteins and contribute to antibody reactivity through an HLA-DM-dependent process. Moreover, antibody reactivity is impacted by the specific amino acid sequence presented. In summary, we demonstrate that polymorphic HLA class I peptides, bound to HLA class II proteins, can directly or indirectly be part of the antibody binding epitope. Our findings have potential important implications for the field of transplant immunology and for our understanding of adaptive immunity.

Monitoring cell-mediated immunity during immunosuppression reduction in heart transplant recipients with severe systemic infections.

BACKGROUND: Treatment for severe systemic infections in heart transplantation is reduction in immunosuppression while treating the infection. An assay that measures adenosine triphosphate production in activated lymphocytes (ImmuKnow® ) objectively monitors cellular immunity of transplant recipients. In this study, we used ImmuKnow® to adjust immunosuppression in heart transplant recipients with severe systemic infections. METHODS: Heart transplant recipients were followed with ImmuKnow® at the time of biopsy and diagnosis of systemic infection. Patients who developed an infection were monitored by ImmuKnow® assay with adjustments in immunosuppression based upon the results of the assay. Maintenance immunosuppression was reinstituted when the ImmuKnow® increased to >225 ng/mL of ATP. RESULTS: Two or more ImmuKnow® assays were performed in 80 patients. Thirteen patients developed severe systemic infections. ImmuKnow® mean value at the time of diagnosis of infection was 109 ± 49.2 ng/mL. Reduction in immunosuppression and treatment of infection resulted in normalization of ImmuKnow® level, resolution of infection, and no episodes of rebound rejection. CONCLUSION: Heart transplant recipients with severe systemic infections presented with a decreased ImmuKnow® , suggesting over immunosuppression. ImmuKnow® can be used as an objective measurement in withdrawing immunosuppression in heart transplant recipients with severe systemic infections.

Outcomes in highly sensitized pediatric heart transplant patients using current management strategies.

BACKGROUND: Previous studies have suggested that children with pre-formed anti-HLA antibodies (PRA) undergoing orthotopic heart transplantation (OHT) have increased risk for rejection, coronary artery vasculopathy (CAV) and death. In 2005, our program started utilizing aggressive desensitization (including plasmapheresis, IVIg, pulse cytoxan and rituximab) with the goal of improving outcomes for these patients. The purpose of this study was to compare outcomes with this new strategy in recipients with pre-OHT high PRA (>10%) vs low PRA ≤10%). METHODS: A retrospective study of 70 consecutive pediatric OHT patients was undertaken between January 2005 and July 2013 to identify patients with pre-OHT PRA >10% (high PRA), or PRA ≤10% (low PRA). Demographic/data information and detailed post-OHT outcomes, including rejection, 30-day and overall mortality, freedom from significant rejection, and CAV, were analyzed. RESULTS: Fourteen (20%) patients had high PRA and 56 (80%) did not. There was a significant decrease in PRA values before and after desensitization. Thirty-day and overall mortality and the proportion of patients with rejections or CAV were lower in the high PRA group, although the difference was not statistically significant. Kaplan-Meier survival analysis revealed no significant difference in survival between the two groups. There was a significant difference in survival in our sensitized patients before 2005 vs after 2005. CONCLUSIONS: We identified no significant differences in outcomes between high or low PRA patients. These preliminary findings may suggest improvement in OHT outcomes for high PRA patients as a result of aggressive desensitization. A larger study is warranted to confirm these findings.

Management of children undergoing cardiac transplantation with high Panel Reactive Antibodies.

Highly sensitised children in need of cardiac transplantation have overall poor outcomes because of increased risk for dysfunction of the cardiac allograft, acute cellular and antibody-mediated rejection, and vasculopathy of the cardiac allograft. Cardiopulmonary bypass and the frequent use of blood products in the operating room and cardiac intensive care unit, as well as the frequent use of homografts, have predisposed potential recipients of transplants to allosensitisation. The expansion in the use of ventricular assist devices and extracorporeal membrane oxygenation has also contributed to increasing rates of allosensitisation in candidates for cardiac transplantation. Antibodies to Human Leukocyte Antigen can be detected before transplantation using several different techniques, the most common being the "complement-dependent lymphocytotoxicity assays". "Solid-phase assays", particularly the "Luminex® single antigen bead method", offer improved specificity and more detailed information regarding specificities of antibodies, leading to improved matching of donors with recipients. Allosensitisation prolongs the time on the waiting list for potential recipients of transplantation and increases the risk of complications and death after transplantation. Aggressive reduction of antibodies to Human Leukocyte Antigen in these high-risk patients is therefore of vital importance for long-term survival of the patient and cardiac allograft. Strategies to decrease Panel Reactive Antibody or percent reactive antibody before transplantation include plasmapheresis, intravenous administration of immunoglobulin, and specific treatment to reduce B-cells, particularly Rituximab. These strategies have resulted in varying degrees of success. Antibody-mediated rejection and cardiac allograft vasculopathy are two of the most important complications of transplantation in patients with high Panel Reactive Antibody. The treatment of antibody-mediated rejection in recipients of cardiac transplants is largely empirical and includes the use of high-dose corticosteroids, plasmapheresis, intravenous administration of immunoglobulins, anti-thymocyte globulin, and Rituximab. Cardiac allograft vasculopathy is believed to be secondary to chronic complement-mediated endothelial injury and chronic vascular rejection. The use of proliferation signal inhibitors, such as sirolimus and everolimus, has been shown to delay the progression of cardiac allograft vasculopathy. In some non-sensitised recipients of cardiac transplants, the de novo formation of antibodies to Human Leukocyte Antigen after transplantation may increase the likelihood of adverse clinical outcomes. The use of serial testing for donor-specific antibodies after cardiac transplantation may be advisable in patients with frequent episodes of rejection and patients with history of sensitisation. Allosensitisation before transplantation can negatively influence outcomes after transplantation. A high incidence of antibody-mediated rejection and graft vasculopathy can result in graft failure and decreased survival. Current strategies to decrease allosensitisation have helped to expand the pool of donors, improve times on the waiting list, and decrease mortality. Centres of transplantation offering desensitisation are currently using plasmapheresis to remove circulating antibodies; intravenous immunoglobulin to inactivate antibodies; cyclophosphamide to suppress B-cell proliferation; and Rituximab to deplete B-lymphocytes. Similar approaches are also used to treat antibody-mediated rejection after transplantation with promising results.

Donor-specific antibody against denatured HLA-A1: clinically nonsignificant?

Pre-transplant screening of a woman with end-stage renal disease (ESRD) showed no anti-human leukocyte antigen (HLA) alloantibodies by anti-human globulin-complement-dependent cytotoxicity (AHG-CDC; class I) or enzyme-linked immunosorbent assay (class II). Following a negative AHG-CDC crossmatch, an HLA*01:01+ deceased donor (DD) kidney was transplanted in September 2005. Subsequent screening of pre-transplant serum by LABScreen Single Antigen (SA) array showed strong reactivity versus A*01:01. Despite that reactivity, at 5 years post-transplant, the patient has a serum creatinine of 1.6 mg/dl and has never experienced humoral or cellular rejection. Retrospective flow-cytometric crossmatch of pre- and post-transplant sera versus DD cells was negative. Rescreening of multiple pre- and post-transplant sera revealed anti-A1 reactivity persisting from the first through the last samples tested. The patient's anti-A1 was almost two fold more reactive with denatured A*01:01 FlowPRA SA beads after denaturation with acid treatment (pH 2.7) than with untreated beads. Parallel results were observed with pH 2.7 treated versus untreated A1+ T cells in FXM. These data highlight the difficulty in interpreting screening results obtained using bead arrays, because of antibodies that appear to recognize denatured but not native class I HLA antigens. We suggest that such bead-positive, flow cytometric crossmatch negative antibodies are not associated with humoral rejection, may not necessarily be detrimental to a graft, and deserve further evaluation before becoming a barrier to transplantation.

The acceptable reactive crossmatch (ARC), post-transplant monitoring, and their impact on kidney transplantation: a single center experience.

Although the adverse allograft outcomes associated with HLA antibodies are well documented, some controversy exists regarding the importance of low-level donor specific anti-HLA antibodies (DSA). To provide further detail on this controversy, we prospectively looked at low-level DSA in negative T- and B-cell flow cytometric crossmatch (FCXM) or acceptable reactive crossmatch (ARC) patients who each underwent protocol based post-transplant antibody monitoring. HLA Class I and II antibody screening and specificity determination was conducted via a solid phase assay (SPA) and FCXM versus donor and autologous T and B cells. Post-transplant patients were immunosuppressed with quadruple maintained immunosuppressive therapy, rabbit anti-thymocyte globulin induction, and HLA antibody monitoring. Out of 31 ARC patients transplanted, 65% had a PRA > 50% and 26% showed increased DSA at 7-14 days post-transplant. Antibody mediated rejection (AMR) was treated with pharmacological and/or plasmapheresis (PP) therapy. DSA were lowered and remained at low-levels (MFI 1000- 3000) or below FCXM cutoffs. None of the 31 patients transplanted developed de-novo antibodies. Two patients lost their allografts, one to polyoma (BK) virus, and one to antibody mediated rejection (AMR). In conclusion, our experience demonstrates that patients deemed higher risk for an immunological event due to low-level DSA should be transplanted with an ARC and followed post-transplant according to an established alloantibody monitoring protocol. With close monitoring, 5-year outcomes can be expected to approach that of low-immunologic risk transplant patients.

Assessing relative risks of infection and rejection: a meta-analysis using an immune function assay.

BACKGROUND: Long-term use of immunosuppressants is associated with significant morbidity and mortality in transplant recipients. A simple whole blood assay that has U.S. Food and Drug Administration clearance directly assesses the net state of immune function of allograft recipients for better individualization of therapy. A meta-analysis of 504 solid organ transplant recipients (heart, kidney, kidney-pancreas, liver and small bowel) from 10 U.S. centers was performed using the Cylex ImmuKnow assay. METHODS: Blood samples were taken from recipients at various times posttransplant and compared with clinical course (stable, rejection, infection). In this analysis, 39 biopsy-proven cellular rejections and 66 diagnosed infections occurred. Odds ratios of infection or rejection were calculated based on measured immune response values. RESULTS: A recipient with an immune response value of 25 ng/ml adenosine triphosphate (ATP) was 12 times (95% confidence of 4 to 36) more likely to develop an infection than a recipient with a stronger immune response. Similarly, a recipient with an immune response of 700 ng/ml ATP was 30 times (95% confidence of 8 to 112) more likely to develop a cellular rejection than a recipient with a lower immune response value. Of note is the intersection of odds ratio curves for infection and rejection in the moderate immune response zone (280 ng/ml ATP). This intersection of risk curves provides an immunological target of immune function for solid organ recipients. CONCLUSION: These data show that the Cylex ImmuKnow assay has a high negative predictive value and provides a target immunological response zone for minimizing risk and managing patients to stability.

Sensitization of renal transplant candidates by cryopreserved cadaveric venous or arterial allografts.

BACKGROUND: Cryopreserved cadaveric venous or arterial allografts are used in ESRD patients as an alternative to synthetic grafts for hemodialysis access. We evaluated the effect of these allografts on the PRA against HLA class I and II antigens in 11 ESRD patients awaiting a kidney transplant. METHODS: Flow Cytometry using purified antigen coated beads (Flow PRA Beads) was used to determine PRA against HLA class I and II antigens. RESULTS: Patients with no antibody prior to allograft implantation were all positive for HLA class I and II antibodies after implantation. Patients with anti-HLA class I and II antibodies prior to allografting, developed higher antibody titers. Of the 11 patients that received cryopreserved cadaveric allografts no deaths were reported. Two grafts were removed due to thrombosis consistent with rejection. CONCLUSION: Recipients of cadaveric venous or arterial allografts elicit an HLA antibody response attesting to the antigenicity of cryopreserved cadaveric allografts.