Hiding in plain sight: Misinterpretation of immunogenic DPB epitopes within G/P groups. Short running title: The impact of HLA G and P codes on DPB.

The clinical impact of HLA DP antibodies is poorly understood, resulting in variable clinical strategies for transplant candidates and recipients with donor-directed HLA-DP antibodies. Complicating matters further, the DPB naming convention is not based on allelic homology and requires sequence alignments to identify potential immunogenic epitopes. Historically, G and P codes, which consolidated alleles that were identical over Exon 2, were used to simplify the reporting of HLA Class II typing as differences outside of Exon 2 have not been considered immunogenic (i.e., able to induce an antibody response). Herein, we present four cases demonstrating that polymorphisms at codons 96R/K and 170I/T, in Exon 3 of DPB, are targets for alloantibody recognition. These regions "hide in plain sight" due to the current use of G/P code-level typing, potentially leading to incorrect compatibility assessments (i.e., virtual crossmatches) and misinterpreted antibody responses. The unintentional crossing of an HLA-DPB donor-specific antibody (DSA) in a solid organ or hematopoietic stem cell transplant may lead to unforeseen deleterious clinical outcomes. Our data underscore the complexities of DPB histocompatibility assessments and highlight the need for adaptable systems that align with evolving research and clinical outcomes.

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.

Loss of heterozygosity leading to incorrect HLA typing for platelet-transfusion refractory patient.

BACKGROUND: Management of platelet-transfusion refractory (PR) patients due to anti-HLA antibodies includes the provision of HLA-matched (HLAm) platelets (PLT) or PLTs that are negative for HLA antigens corresponding to the recipient antibodies. Obtaining HLAm PLTs is predicated on accurate HLA antigen typing of the recipient and donor. Here, we present the clinical implications of a case involving loss of heterozygosity (LOH) in a patient presented for PR workup. STUDY DESIGN AND METHODS: HLA typing was performed by three methods: (1) Real-time PCR; (2) Sequence-specific oligonucleotide (SSO) typing test; and (3) Next-Generation Sequencing (NGS). Cytogenomic SNP microarray was utilized to assess LOH. RESULTS: A 30-year-old female with newly diagnosed acute myelogenous leukemia was found to be PR secondary to HLA sensitization. A peripheral blood (PB) sample, containing 93% myeloid blast cells, was sent for HLA typing for the provision of HLAm PLTs. HLA typing revealed homozygosity at the HLA-A locus but was heterozygous at the -B and -C loci. After chemotherapy, HLA typing on a new PB sample, devoid of blast cells, identified HLA-A locus heterozygosity, which was subsequently confirmed by real-time PCR and NGS. Cytogenomic SNP microarray analysis demonstrated LOH of the HLA-A locus on chromosome 6p in the pretreatment sample but not in the posttreatment sample. CONCLUSION: In hematologic patients with high tumor burden, HLA homozygosity should be viewed with suspicion for potential LOH. Therefore, HLA testing should be repeated, preferably with a non-hematological source (e.g., buccal swab) or following successful reduction of the tumor burden.

Evidence of Chronic Allograft Injury in Liver Biopsies From Long-term Pediatric Recipients of Liver Transplants.

BACKGROUND & AIMS: A substantial proportion of pediatric liver transplant recipients develop subclinical chronic allograft injury. We studied whether there are distinct patterns of injury based on histopathologic features and identified associated immunologic profiles. METHODS: We conducted a cross-sectional study of 157 stable, long-term pediatric recipients of transplanted livers (70 boys; > 6 years old at time of transplantation; mean, 8.9 ± 3.46 years after liver transplantation) who underwent liver biopsy analysis from August 13, 2012, through May 1, 2014. Participants had received livers from a living or deceased donor and had consistently normal results from liver tests. Liver biopsy specimens were scored by a central pathologist; an unsupervised hierarchical cluster analysis of histologic features was used to sort biopsy samples into 3 clusters. We conducted transcriptional and cytometric analyses of liver tissue samples and performed a systems biology analysis that incorporated clinical, serologic, histologic, and transcriptional data. RESULTS: The mean level of alanine aminotransferase in participants was 27.6 ± 14.57 U/L, and the mean level of γ-glutamyl transferase was 17.4 ± 7.93 U/L. Cluster 1 was characterized by interface activity (n = 34), cluster 2 was characterized by periportal or perivenular fibrosis without interface activity (n = 45), and cluster 3 had neither feature (n = 78). We identified a module of genes whose expression correlated with levels of alanine aminotransferase, class II donor-specific antibody, portal inflammation, interface activity, perivenular inflammation, portal and perivenular fibrosis, and cluster assignment. The module was enriched in genes that regulate T-cell-mediated rejection (TCMR) of liver and other transplanted organs. Functional pathway analysis showed overrepresentation of TCMR gene sets for cluster 1 but not clusters 2 or 3. CONCLUSION: In an analysis of biopsies from an apparently homogeneous group of stable, long-term pediatric liver transplant recipients with consistently normal liver test results, we found evidence of chronic graft injury (inflammation and/or fibrosis). Biopsy samples with interface activity had a gene expression pattern associated with TCMR.

Genotyping Applications for Transplantation and Transfusion Management: The Emory Experience.

Current genotyping methodologies for transplantation and transfusion management employ multiplex systems that allow for simultaneous detection of multiple HLA antigens, human platelet antigens, and red blood cell (RBC) antigens. The development of high-resolution, molecular HLA typing has led to improved outcomes in unrelated hematopoietic stem cell transplants by better identifying compatible alleles of the HLA-A, B, C, DRB1, and DQB1 antigens. In solid organ transplantation, the combination of high-resolution HLA typing with solid-phase antibody identification has proven of value for highly sensitized patients and has significantly reduced incompatible crossmatches at the time of organ allocation. This database-driven, combined HLA antigen/antibody testing has enabled routine implementation of "virtual crossmatching" and may even obviate the need for physical crossmatching. In addition, DNA-based testing for RBC antigens provides an alternative typing method that mitigates many of the limitations of hemagglutination-based phenotyping. Although RBC genotyping has utility in various transfusion settings, it has arguably been most useful for minimizing alloimmunization in the management of transfusion-dependent patients with sickle cell disease or thalassemia. The availability of high-throughput RBC genotyping for both individuals and large populations of donors, along with coordinated informatics systems to compare patients' antigen profiles with available antigen-negative and/or rare blood-typed donors, holds promise for improving the efficiency, reliability, and extent of RBC matching for this population.

Class I and II HLA antibodies in pediatric patients with thalassemia major.

BACKGROUND: HLA alloimmunization is a potential complication of red blood cell (RBC) transfusion with detrimental consequences for future organ or hematopoietic stem cell transplantation. STUDY DESIGN AND METHODS: The study aimed to determine the prevalence and specificity of HLA antibodies among pediatric patients with thalassemia major (TM) and antibody changes over time while on leukoreduced chronic transfusion therapy. HLA antibodies were measured at two or more time points in children and young adults ages 3 to 21 years with TM. HLA Class I and II antibodies were measured by FlowPRA screening. Positive screening assays were confirmed with LabScreen single-antigen bead assays for antibody specificity. RESULTS: HLA antibodies were detected in 10 of 19 (53%) subjects: seven of 19 (37%) with HLA Class I and II antibodies, two of 19 (11%) with only HLA Class I antibodies, and one of 19 (5%) with only HLA Class II antibodies. Subjects with HLA antibodies were older (14.6 years vs. 7.1 years, p = 0.05), predominantly male (80%), and more likely to have a remote history of nonleukoreduced transfusions (p = 0.057). Median time between testing was 3.7 years. De novo HLA antibodies were detected in two of 11 patients who initially had negative panel-reactive antibody screens, while one subject lost detection of Class II antibody. Two of seven patients with HLA antibodies had antibodies to self-HLA. CONCLUSION: HLA antibodies have a high prevalence in TM patients and may be associated with nonleukoreduced transfusions and older age. For such patients, antibody identification will be useful if subsequent organ or stem cell transplantation is needed.

Consensus guidelines on the testing and clinical management issues associated with HLA and non-HLA antibodies in transplantation.

BACKGROUND: The introduction of solid-phase immunoassay (SPI) technology for the detection and characterization of human leukocyte antigen (HLA) antibodies in transplantation while providing greater sensitivity than was obtainable by complement-dependent lymphocytotoxicity (CDC) assays has resulted in a new paradigm with respect to the interpretation of donor-specific antibodies (DSA). Although the SPI assay performed on the Luminex instrument (hereafter referred to as the Luminex assay), in particular, has permitted the detection of antibodies not detectable by CDC, the clinical significance of these antibodies is incompletely understood. Nevertheless, the detection of these antibodies has led to changes in the clinical management of sensitized patients. In addition, SPI testing raises technical issues that require resolution and careful consideration when interpreting antibody results. METHODS: With this background, The Transplantation Society convened a group of laboratory and clinical experts in the field of transplantation to prepare a consensus report and make recommendations on the use of this new technology based on both published evidence and expert opinion. Three working groups were formed to address (a) the technical issues with respect to the use of this technology, (b) the interpretation of pretransplantation antibody testing in the context of various clinical settings and organ transplant types (kidney, heart, lung, liver, pancreas, intestinal, and islet cells), and (c) the application of antibody testing in the posttransplantation setting. The three groups were established in November 2011 and convened for a "Consensus Conference on Antibodies in Transplantation" in Rome, Italy, in May 2012. The deliberations of the three groups meeting independently and then together are the bases for this report. RESULTS: A comprehensive list of recommendations was prepared by each group. A summary of the key recommendations follows. Technical Group: (a) SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch (XM). (c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation Group: (a) Risk categories should be established based on the antibody and the XM results obtained. (b) DSA detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c) A renal transplantation can be performed in the absence of a prospective XM if single-antigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be taken in agreement with local clinical programs and the relevant regulatory bodies. (d) The presence of DSA HLA antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and islet cell transplantation. Posttransplantation Group: (a) High-risk patients (i.e., desensitized or DSA positive/XM negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b) Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. (c) Low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results. CONCLUSIONS: A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.

Translational applications of flow cytometry in clinical practice.

Flow cytometry has evolved over the past 30 y from a niche laboratory technique to a routine tool used by clinical pathologists and immunologists for diagnosis and monitoring of patients with cancer and immune deficiencies. Identification of novel patterns of expressed Ags has led to the recognition of cancers with unique pathophysiologies and treatment strategies. FACS had permitted the isolation of tumor-free populations of hematopoietic stem cells for cancer patients undergoing stem cell transplantation. Adaptation of flow cytometry to the analysis of multiplex arrays of fluorescent beads that selectively capture proteins and specific DNA sequences has produced highly sensitive and rapid methods for high through-put analysis of cytokines, Abs, and HLA genotypes. Automated data analysis has contributed to the development of a "cytomics" field that integrates cellular physiology, genomics, and proteomics. In this article, we review the impact of the flow cytometer in these areas of medical practice.

Clinical cytometry and progress in HLA antibody detection.

For most solid organ and selected stem cell transplants, antibodies against mismatched HLA antigens can lead to early and late graft failure. In recognition of the clinical significance of these antibodies, HLA antibody identification is one of the most critical functions of histocompatibility laboratories. Early methods employed cumbersome and insensitive complement-dependent cytotoxicity assays with a visual read-out. A little over 20 years ago flow cytometry entered the realm of antibody detection with the introduction of the flow cytometric crossmatch. Cytometry's increased sensitivity and objectivity quickly earned it popularity as a preferred crossmatch method especially for sensitized recipients. Although a sensitive method, the flow crossmatch was criticized as being "too sensitive" as false positive reactions were a know drawback. In part, the shortcomings of the flow crossmatch were due to the lack of corresponding sensitive and specific HLA antibody screening assays. However, in the mid 1990s, solid phase assays, capable of utilizing standard flow cytometers, were developed. These assays used microparticles coated with purified HLA molecules. Hence, the era of solid-phase, microparticle technology for HLA antibody detection was born permitting the sensitive and specific detection of HLA antibody. It was now possible to provide better correlation between HLA antibody detection and the flow cytometric crossmatch. This flow-based technology was soon followed by adaptation to the Luminex platform permitting a mutltiplexed approach for the identification and characterization of HLA antibodies. It is hoped that these technologies will ultimately lead to the identification of parameters that best correlate with and/or predict transplant outcomes.

HLA alloimmunization is associated with RBC antibodies in multiply transfused patients with sickle cell disease.

BACKGROUND: Alloimmunization to minor red blood cell (RBC) antigens occurs commonly in sickle cell disease (SCD). Patients with alloimmunization demonstrate increased risk for new alloantibody formation with subsequent transfusion. Alloimmunization to human leukocyte antigens (HLA) can occur with RBC transfusion and may result in graft rejection during stem cell or organ transplantation. The prevalence and risk factors for HLA alloimmunization in multiply transfused pediatric SCD patients are unknown. PROCEDURE: A cross-sectional study of HLA alloimmunization in SCD patients aged 3-21 years with a history of >or=3 RBC transfusions was performed to test the hypothesis that HLA alloimmunization is associated with RBC alloimmunization. Antibodies to class I and class II HLA were measured by Flow Panel Reactive Antibody (FlowPRA). RESULTS: Seventy-three SCD patients (30 with RBC antibodies) were tested. HLA antibodies were detected in 25/73 (34%) patients; class I HLA antibodies occurred in 24/73 (33%) and class II HLA antibodies occurred in 3 (4%). Among patients with RBC antibodies, 16/30 (53%) had HLA antibodies, while 9/43 (21%) patients without RBC antibodies had HLA antibodies (OR 4.32 [1.6-12.1]). In a multivariate analysis, antibodies to RBC antigens were an independent predictor of HLA alloimmunization (P = 0.041). The association of RBC and HLA immunization was strongest among patients with no history of chronic transfusion therapy. CONCLUSIONS: This analysis is the first description of HLA alloimmunization in pediatric SCD patients who receive primarily leukoreduced RBC transfusions and demonstrates that HLA alloimmunization tendency is associated with antibodies to RBC antigens.

Donor-reactive HLA antibodies in renal allograft recipients: considerations, complications, and conundrums.

Whether sensitized patients wait for a compatible crossmatch with a deceased donor, enter a paired exchange program with the hope of finding a compatible living donor, or go through a desensitization protocol depends on a number of factors, not the least of which is the overall philosophy of the transplant center. Centers such as ours take the position that donor-directed antibodies detected by solid phase assays (even those that are "weak") present an unacceptable risk factor to the patient. This philosophy is predicated on the biologic role of the immune system, specifically that antibodies were generated in response to a non-self (allo) antigen and that a successful immune response eliminates that which caused its stimulation. Although obviously an oversimplification, this philosophy mandates a comprehensive evaluation of HLA antibodies in sensitized recipients. This article addresses the challenges and conundrums associated with human leukocyte antigen antibody identification.

Monitoring after renal transplantation: recommendations and caveats.

This commentary discusses the strengths and weaknesses of a recent study reported by Kobayashi and colleagues, in which paired serum samples from 297 renal transplant recipients who underwent transplantation between December 1972 and September 2004 were tested for the presence of human leukocyte antigen (HLA) antibody at two discrete time points following surgery (2004 and 2006). Urine samples collected on the same day as the serum samples, were also measured for urinary protein levels. The results of the study suggest that patients with a positive change in HLA antibody status in the post-transplantation period (i.e. from negative to positive) plus a positive urine protein test were at increased risk of developing graft dysfunction (i.e. increased serum creatinine level). Solely on the basis of these data, the authors advocate that patients should be monitored annually for de novo production of HLA antibodies and for an increased urinary protein level by use of simple and cost-effective approaches to assess the risk of graft dysfunction or failure. While the proposal is an intriguing one, this commentary identifies several issues and limitations regarding post-transplantation monitoring that should be considered.

Adult reserve stem cells and their potential for tissue engineering.

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher orders. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50-70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

Regulated expression of human histocompatibility leukocyte antigen (HLA)-DO during antigen-dependent and antigen-independent phases of B cell development.

Human histocompatibility leukocyte antigen (HLA)-DO, a lysosomal resident major histocompatibility complex class II molecule expressed in B cells, has previously been shown to be a negative regulator of HLA-DM peptide loading function. We analyze the expression of DO in human peripheral blood, lymph node, tonsil, and bone marrow to determine if DO expression is modulated in the physiological setting. B cells, but not monocytes or monocyte-derived dendritic cells, are observed to express this protein. Preclearing experiments demonstrate that approximately 50% of HLA-DM is bound to DO in peripheral blood B cells. HLA-DM and HLA-DR expression is demonstrated early in B cell development, beginning at the pro-B stage in adult human bone marrow. In contrast, DO expression is initiated only after B cell development is complete. In all situations, there is a striking correlation between intracellular DO expression and cell surface class II-associated invariant chain peptide expression, which suggests that DO substantially inhibits DM function in primary human B cells. We report that the expression of DO is markedly downmodulated in human germinal center B cells. Modulation of DO expression may provide a mechanism to regulate peptide loading activity and antigen presentation by B cells during the development of humoral immune responses.