HLA Class I-specific Antibodies Research Articles

HLA-C*04:09N is expressed at the cell surface and triggers peptide-specific T-cell activation.

The null allele HLA-C*04:09N differs from HLA-C*04:01 in a frameshift mutation within its cytoplasmic domain, resulting in translation of 32 additional amino acids that are assumed to prevent cell surface expression. However, we recently identified a multiple myeloma-reactive T-cell receptor (TCR) that appeared to recognize antigen presented on HLA-C*04:09N and encouraged us to ask whether HLA-C*04:09N, albeit not easily detectable at the cell surface, can present antigen sufficient for T-cell activation. We generated two HLA-class I-deficient cell lines, re-expressed HLAC* 04:09N, detected HLA expression by flow cytometry, and tested for T-cell activation using a cytomegalovirus peptide- specific HLA-C*04:01-restricted TCR. In both cell lines, HLA-C*04:09N expression was detectable at the cell surface and could be enhanced by IFN-γ exposure. Recombinant HLA-C*04:09N expression was sufficient for T-cell activation in vitro, which could be blocked by an HLA-class I-specific antibody, suggesting HLA-TCR interaction at the cell surface. Peripheral blood mononuclear cells isolated from an individual who physiologically expressed HLA-C*04:09N triggered peptide-specific T-cell activation, confirming our results with cells with natural HLA expression levels. In conclusion, we present peptide-specific HLA-C*04:09N-restricted T-cell activation and suggest consideration of this allele in the appropriate clinical context, such as allogeneic stem cell transplantation, or in the setting of cellular therapy.

Long-Term Persisting Donor-Derived Human Leukocyte Antigen Antibody as a Possible Passenger Lymphocyte Syndrome Following Lung Transplantation: A Case Report

Herein, we reported the transfer of donor-derived antihuman leukocyte antigen (HLA) antibodies in 2 recipients after lung transplantation. Case 1: A 39-year-old woman with pleuroparenchymal fibroelastosis underwent a single brain-dead donor lung transplantation. Antibody screening 36 days after transplantation demonstrated high levels of de novo nondonor HLA class I-specific antibodies. The antibody screening in the donor serum revealed that the donor demonstrated a largely overlapping antibody profile. Importantly, the donor serum also included high-level HLA-specific antibodies against the recipient HLA-specific antigens, which were not detected in the recipient sera after transplantation. Donor-derived anti-HLA antibodies were still detected in the recipient 39 months after transplantation, without causing any complications such as graft-vs-host disease. Case 2: A 47-year-old woman underwent living-donor lobar lung transplantation for pulmonary complications after bone marrow transplantation with a right lower lobe from her husband and the left lower lobe from her sister. On postoperative day 39, the recipient's anti-HLA-class I antibody profile was found to be similar to that of the highly sensitized left lung donor. These donor-derived anti-HLA antibodies remained to be produced in the recipient 66 months after transplantation, without the development of complications.

Long-Term Persisting Donor-Derived Anti-Human Leukocyte Antigen (HLA) Antibody as a Possible Passenger Lymphocyte Syndrome Following Lung Transplantation

<h3>Introduction</h3> A routine screening for the presence of HLA-specific antibodies after solid organ transplantation, produced by recipient B cells or plasma cells, is important for the management of antibody-mediated rejection. A novel possibility has recently been suggested in a few clinical case reports, where some de novo HLA-specific antibodies could continue to be produced by donor passenger B lymphocytes/plasma cells. We herein report the transfer of donor-derived anti-HLA antibodies in two recipients following lung transplantation. <h3>Case Report</h3> Case 1: A 39‐year‐old woman with pleuroparenchymal fibroelastosis underwent single lung transplantation with a left lung from a brain-dead donor. Antibody screening 36 days after transplant showed high levels of de novo non-donor HLA class I-specific antibodies. Then antibody screening in the donor serum identified that the donor showed a largely overlapping antibody profile to that found in the recipient. Importantly, the donor serum also included high-level HLA-specific antibodies against the recipient HLA-specific antigens, which were not detected in the recipient sera after transplantation. The donor-derived anti-HLA antibodies have still been detected in the recipient at 39 months after transplantation, which have never caused any complications such as graft-versus-host disease (GVHD). Case 2: A 47‐year‐old woman underwent living donor lobar lung transplantation for pulmonary complications after bone marrow transplantation with the right lower lobe from her husband and the left lower lobe from her sister. On postoperative day 39, the recipient anti- HLA-class I antibody profile was found to be similar to that of the high-sensitized left lung donor. These donor-derived anti-HLA antibodies have remained to be produced in the recipient at 66 months after transplantation without development of complications such as GVHD. <h3>Summary</h3> This case report suggests that alloreactive passenger B cells or plasma cells located within the highly sensitized donor lungs could lead to the early production of de novo anti-HLA-specific antibodies in the lung transplant recipients. Although there have been no overt clinical manifestations in either of the patients so far, the clinical significance of transfer of donor HLA-specific allosensitization should be elucidated.

Infusion of Ex Vivo Expanded Allogeneic Cord Blood-Derived Natural Killer Cells in Combination with Autologous Stem Cell Transplantation for Multiple Myeloma: Results of a Phase I Study

Background: Multiple myeloma (MM) is an incurable disease thought to be characterized by immune dysregulation and exhaustion, whereby proliferation of malignant plasma cells is not checked by the native immune system. Long term remissions in some patients after allogeneic stem cell transplant (SCT) suggest a graft versus myeloma effect; however the treatment-related toxicity limits the widespread use of this modality. Allogeneic natural killer (NK) cells are active in various hematologic malignancies and may have a role against MM, without concomitant graft versus host disease (GVHD). Umbilical cord blood is a potential source for allogeneic NK cells and ex vivo expanded umbilical cord blood-derived NK (CB-NK) cells demonstrate activity comparable to that of peripheral blood-derived NK cells. We describe here the results of a phase I, first-in-human study of ex vivo expanded allogeneic CB-NK cells in conjunction with high dose chemotherapy and autologous SCT.Methods: Patients with symptomatic MM who were appropriate candidates for high dose chemotherapy and autologous SCT were eligible. CB units with at least 4/6 match at HLA-A, B and DR were chosen for each patient. When possible, CB units with potential NK alloreactivity were prioritized. On day (-19) CB units were thawed and mononuclear cells (MNCs) were isolated by ficoll density gradient centrifugation. MNCs were cultured in a gas permeable bioreactor with irradiated (100 Gy) K562-based aAPCs expressing membrane bound IL-21 "Clone 9.mbIL21" (2:1 feeder cell:MNC ratio) and IL-2 (100 IU/mL). On day 7, cells were CD3-depleted via immunomagnetic depletion and remaining cells were re-stimulated with aAPC feeder cells and cultured for an additional 7 days. NK cell purity was determined after 14 days of culture. Due to pre-clinical data demonstrating synergy between lenalidomide and NK cells, patients received lenalidomide (10 mg orally daily) from days (-8) to day (-2). Melphalan 200 mg/m2 was given intravenously on day (-7). Freshly expanded CB-NK cells were infused on day (-5). Autologous peripheral blood progenitor cells (PBPC) were infused on day (0).Results: 12 patients have been enrolled thus far with 3 patients each on the following CB-NK cell dose levels: 5 e6 NK cells/kg, 1 e7 NK cells/kg, 5 e7 NK cells/kg and 1 e8 NK cells/kg. 11/12 patients had at least 1 high-risk feature of progressed/relapsed disease (n=7), high-risk cytogenetics (n=2) or International Staging System III disease (n=3). Successful NK expansion to target dose was achieved in all patients with median NK purity (CD56+/CD16+/CD3-) of 98.9% (96.8-99.7). Expanded cells demonstrated cytotoxicity against classic K562 and MM cell line targets. There were no infusional toxicities and no occurrence of GVHD. One patient (1 e8 NK cells/kg) failed to engraft due to a poor PBPC graft quality; this patient was rescued with a back-up autologous PBPC graft. There have been no other significant adverse events and no second primary malignancies. 11/12 patients are evaluable beyond day 100. Best response has been 8/12 nCR or better, 2/12 VGPR and 1/12 PR. 4/12 patients have progressed at a median of 330 days. By DNA microsatellite chimerism analysis, donor CB-NK cells were detected in 2 patients at the 1 e7 NK cells/kg dose and all 3 patients in the 1 e8 NK cells/kg dose, for at least 5 days after infusion. By a more sensitive flow cytometric chimerism assay using HLA class I-specific antibodies for donor or recipient, donor CB-NK cells were detected in 3 evaluable patients at doses of 1 e7 NK cells/kg, 5 e7 NK cells/kg and 1 e8 NK cells/kg for at least 12 days after infusion. Further analysis of these cells indicated persistence of an activated phenotype (NKG2D+/NKp30+) in vivo.Conclusion: CB-NK cells can be activated and expanded to clinical scale. This is the first clinical study of CB-NK cells for MM. When infused in the setting of myeloablative chemotherapy, up to 1 e8 allogeneic CB-NK cells/kg are well tolerated with no infusional toxicities or GVHD. These cells can persist for at least 12 days in vivo and demonstrate an active phenotype. Though clinical data are early, responses are encouraging in this high-risk patient population. Further updated data will be presented at the annual meeting. DisclosuresOff Label Use: Lenalidomide with high dose chemotherapy and autologous stem cell transplantation. Kaur:UT MD Anderson Cancer Center: Employment. Orlowski:Millennium Pharmaceuticals: Consultancy, Research Funding; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Genentech: Consultancy; Onyx Pharmaceuticals: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding; Acetylon: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding; Forma Therapeutics: Consultancy. Cooper:Intrexon: Equity Ownership, Patents & Royalties, Research Funding; ZIOPHARM Oncology: Employment, Equity Ownership, Patents & Royalties, Research Funding. Lee:Cyto-Sen: Equity Ownership; Ziopharm: Equity Ownership; Intrexon: Equity Ownership.

OR31: CASE STUDY: DOA: DONOR ORIGINATING ANTIBODY; A CASE OF PASSIVE ANTIBODY TRANSFER

Aim To investigate the unexplained HLA antibody specificity observed in a post-transplant serum specimen from a lung transplant recipient. Methods Antibody (Ab) evaluation was performed using phenotype and single antigen panels tested on the Luminex ® platform (Lifecodes, One Lambda). Results Patient JD is a 47 year old female with a history of cystic fibrosis. The patient received a bilateral lung transplant on October 3, 2013 from a deceased donor at Johns Hopkins Hospital. A serum specimen drawn immediately prior to transplant was negative for IgG, HLA class I-specific antibody and demonstrated very weak reactivity for IgG, class II-specific antibody. At day T + 4, a post-transplant serum specimen exhibited a substantial antibody increase. Although the timing of antibody appearance indicated an anamnestic response, the specificity was inconsistent with the patient’s historical HLA-specific antibody and appeared to be specific for non-donor antigens. Repeated testing and testing with additional assays confirmed the initial results. Transplanted lungs can be accompanied by substantial donor lymphoid tissue. We therefore tested donor serum for HLA specific antibody. Tests of the deceased donor serum revealed strong HLA-specific antibody nearly identical to the pattern observed with the recipient’s Day T + 4 post-transplant serum. Upon further investigation, we found that, in fact, the donor has been the recipient of a kidney transplant. The deceased donor’s previous transplant information was obtained. The antibody detected in the donor serum was specific for multiple mismatched antigens present in the HLA phenotype of the deceased donor’s kidney transplant. Tests of sera obtained monthly from the lung recipient have shown a steady reduction in antibody strength and breadth. The pattern and the course of the antibody over time suggest that the antibody was of donor origin. Conclusion In this case, having access to the deceased donor’s serum allowed us to investigate the anomalous HLA-specific antibody testing results. Retrieving the deceased donor’s past medical history listed in UNET provided evidence of the transfer of donor antibody-producing cell to the patient JD.

Alloreactivity-Based Medical Conditions

Alloreactivity is a response of the immune system to individual antigenic differences within species. These responses in general occur following exposure to alloantigens as a consequence of medical intervention (such as transfusion or tissue transplantation) or during pregnancy. Responses to alloantigens form a basis of a broad spectrum of medical conditions, such as graft rejection, graft versus host disease, reaction to blood (products), or biopharmaceuticals and fetal and neonatal diseases (thrombocytopenia, hemolytic anemia, hemochromatosis, biliary atresia, and glomerulopathy). On the other hand, normal pregnancies are often accompanied by strongly tolerogenic responses to fetal alloantigens. Diverse immunologic components, including T cells, antibodies (B cells), and NK cells, promote alloreactivity, and this complexity makes the pathogenesis and tolerogenesis of these conditions distinct and unique. This special issue contains seven manuscripts touching on various aspects of alloreactivity in medicine. Blocking CD40-CD40L interaction can induce acceptance of cardiac allografts. However, the blockade is less efficient under inflammatory conditions. In “Glucocorticoid-induced TNFR-related protein reverses cardiac allograft acceptance induced by CD40-CD40L blockade,” Krill et al. demonstrate that stimulation of GITR (a cell surface molecule displayed by effector and regulatory T cells) can initiate cardiac graft rejection irrespective of the presence or absence of CD40-CD40L blockade, defining thus at least one possible molecular mechanism of resistance to CD40-CD40L suppression. The balance between the effector and regulatory T cells is critical for development of graft-specific immune responses in general. Franzese et al. review the usefulness of this balance in predicting or establishing the diagnosis of graft rejection in “Regulatory T cells in the immunodiagnosis and outcome of kidney allograft rejection.” Allogeneic hematopoietic stem cell transplantation is used for treatment of autoimmune diseases, multiple organ transplantation, consequences of supralethal irradiation and advanced malignancies and other serious conditions refractory to conventional methods. In “New allogeneic hematopoietic cell transplantation method: hematopoietic cell transplantation plus thymus transplantation for intractable diseases,” Hosaka demonstrates that allogeneic hematopoietic stem cell transplantation combined with the same donor thymus transplantation has a superior effect producing more efficient T cell function and reduced graft versus host disease. Redzovic et al., in “Mucins help to avoid alloreactivity at the maternal fetal interface,” review roles of mucin-1 and tumor associated glycoprotein-72 in regulating immune responses. Parallels are drawn between the ability of these molecules to promote tumor growth, invasion, and metastasis and to prevent trophoblast invasion. Removing glycoproteins TAG-72 and Muc 1 from the eutopic implantation site likely contributes to better control of trophoblast invasion by T cells and NK cells. This enables tolerance to paternal antigens of the fetus and normal course and outcome of the pregnancy. Three manuscripts explore distinct aspects of alloantibody formation. Preformed donor HLA class I-specific antibodies are a risk factor for rejection of kidney, heart, and lung grafts, but the role of these antibodies is less clear in liver transplantation, where rejection rates are lower. Yoshizawa et al., in “Significance of semiquantitative assessment of preformed donor-specific antibody using luminex single bead assay in living related liver transplantation,” describe a quantitative assay for donor specific antibodies that has a predictive value for living donor liver transplantation. In “Nuclear antigens and auto/alloantibody responses: friend or foe in transplant immunology,” Nakano et al. review current thinking about the role of nuclear antigens and antibodies specific for nuclear antigens in initiation and regulation of immune responses and graft rejection. Finally, transfusion of allogeneic red blood cells may in some recipients induce alloantibodies. Tatari-Calderone et al. in “The association of CD81 polymorphisms with alloimmunization in sickle cell disease” suggest that genetic elements in the gene encoding B-cell molecule CD81 may be predictive markers of alloimmunization. The huge breadth of the field of alloreactivity-based medical conditions allows the special issue only to scratch under the surface of any specific topic. Our hope, however, is that this issue will help raise awareness of the immunological basis of these conditions and will foster further efforts to better understand their pathogenesis. Ultimately, this should help us design better treatments or prevent the development of these medical conditions altogether.

The kinetics of donor HLA class I-specific antibody absorption following a combined split liver and kidney transplant

Hyperacute rejection of a transplanted liver is rare even when the recipient has circulating donor-specific alloantibodies (DSA). There is also evidence that a transplanted liver may provide immunological protection for other organs transplanted from the same donor. We monitored the kinetics of circulating DSA in a highly sensitized recipient of a combined split liver and kidney transplant and demonstrated a reduction in antibody titres immediately after liver perfusion. The absorption of DSA was not compromised by the smaller liver mass transplanted. DSA titres remained low at 3 months post-transplant, and the recipient did not experience antibody-mediated rejection.

Functional assessment of immune markers of graft rejection: a comprehensive study in live‐related donor renal transplantation

A better understanding of the immunobiological processes and predictors of graft rejection holds promise for the development of potential therapeutic strategies and also individualization of immunosuppression. The objective of this study is to analyze the clinical relevance of immune parameters such as antidonor antihuman leukocyte antigen (anti-HLA) antibodies, monitoring of cytokines and their receptors on the graft outcome following live-related donor renal transplantation. Flow cytometry-based methods were used to detect antidonor antibodies (flow cytometry crossmatch, FCXM) and intracellular cytokines. Enzyme-linked immunosorbent assay (ELISA) methods were employed to detect anti-HLA class I and class II antibodies and quantitative serum-soluble interleukin-2 receptor (sIL-2R) levels. The data revealed that patients with HLA class I-specific IgG antibody experienced higher acute rejection (AR) episodes at 1 yr in comparison to the antibody negative group (82% vs. 56%, p = 0.01). On the contrary, donor-specific class II antibodies (B+) did not have any influence on the graft survival. However, 15 recipients having both T- and B-cell antidonor antibodies (T+B+) had significantly poor graft survival (60%) as compared to the antibody-negative group (T-B-, 82%, p = 0.05). Additionally, patients having non-donor but HLA-specific antibodies (FCXM-/ELISA+) had poor graft survival as compared to the antibody-negative group (64% vs. 88%, p < 0.05). Further, patients undergoing AR episodes had significantly higher expression of IFN-gamma-producing T cells (19.16 +/- 7.4% median 17.50) as compared to their pre-transplant levels (5.68 +/- 1.63%, Median 5.20) and the non-rejecter group (5.97 +/- 4.39%, median 4.3, p = 0.0004). Similarly sIL-2 was significantly increased in AR episodes during the first month of transplantation (292 +/- 131.5 pmol/L) as compared to those with well-functioning grafts (p = 0.01) and healthy controls (p = 0.001). Evaluation of antidonor antibodies by flow cytometry is found to be relatively more sensitive and a better predictor of graft outcome. Further monitoring of cytokine expression profile of primed peripheral T-helper cells and quantitative analysis of sIL-2R offer additional valuable diagnostic and prognostic tools for follow-up of transplant subjects and a better alternative for functional assessment of immunosuppression.

Extensive chimerism in liver transplants: Vascular endothelium, bile duct epithelium, and hepatocytes

The transplanted liver has been shown to be particularly capable of inducing tolerance. An explanation may be the presence of chimerism. Cells of donor origin have been found in recipient tissues after transplantation of any solid organ. Evidence for the presence of cells of recipient origin within the transplanted liver is very limited. We investigated whether nonlymphoid cells of recipient origin can be found within human liver allografts. Five male patients who received a liver transplant from a female donor and 11 patients who received an HLA-I mismatched liver transplant were studied. We confirmed our observations with two different techniques in combination with double-staining techniques. To identify male cells in female liver transplants, we used in situ hybridization for sex chromosomes. To identify specific HLA class I antigens of recipient origin, we used immunohistochemistry with HLA class I-specific antibodies. Double staining was performed to discriminate different cell lineages and inflammatory cells. Endothelial cells of recipient origin were found in 14 of 16 donor livers. Bile duct epithelial cells of recipient origin were found in 5 of 16 cases. Hepatocytes of recipient origin were seen in only 1 of the 5 studied sex-mismatched donor livers. Our study provides evidence that cells of recipient origin can replace biliary epithelial cells, endothelial cells, and hepatocytes within the human liver allograft. This is consistent with the concept that circulating pluripotent progenitor cells exist, capable of differentiating into endothelial cells, epithelial cells, and hepatocytes. (Liver Transpl 2003;9:552-556.)

Is there MHC Class II restriction of the response to MHC Class I in transplant patients?

In this study, we evaluated distinct HLA-DRB1 alleles to determine class II restriction of the production of HLA-A2-specific antibodies in renal transplant patients. Data from 217 renal transplant patients who received an HLA-A2-mismatched renal graft were analyzed with regard to HLA-A2 humoral responsiveness. High-resolution DNA typing of class II HLA-DR alleles was performed by polymerase chain reaction-sequence-specific primer. Patients who had one of the following eight HLA-DRB1 alleles were included in the study: -*0101, -*0301, -*0401, -*0701, -*1101, -*1301, -*1401, and -*1501. Serum samples were screened posttransplantation with the standard complement-dependent cytotoxicity procedure. In addition, recombinant HLA-A2 monomers (the "MonoLISA" assay) were used as a target for the detection of HLA-A2 group-specific antibodies. The following HLA-A2 amino acid positions (termed "epitopes") that are responsible for the induction of an antibody response were defined: 74H, 65-66GK, 62G, 114H, 142-145TTKH, and 107W-127K. The definition of the "HLA-DR permittors" of anti-HLA-A2 response was based on a "class II restriction table" designed for this purpose. Prediction of immunogenic and/or nonimmunogenic HLA-A2 peptides was based on an MHC database. The HLA-DRB1-*0101 and -*1401 alleles had a trend toward a positive correlation with the production of HLA class I-specific antibodies against the HLA-A2 shared (public) epitopes 65-66GK and -62G, respectively. Only the DRB1-*1501 allele had higher trend toward a positive correlation with the production of antibodies against the HLA-A2 private (74H) epitope. In 42 patients with the HLA-DRB1-*1501 allele, 11 (26%) patients produced HLA-specific antibodies against the HLA-A2 group of epitope(s). Moreover, in these patients, spreading of the alloreactivity against "other" HLA antigens was detected. Many of these other HLA antigens did not belong to HLA-A2 group but had newly defined shared epitopes with this group. Furthermore, the epitope prediction, based on an MHC database, revealed differences in the ligation strength (score) to the HLA allele (class I and II) for a specific HLA-A2 peptide in the 42 patients (responders and nonresponders). The data presented in this paper suggest that the HLA class II allele and the type of the bound allopeptide may influence the humoral and cellular response. The immunogenicity of these allopeptides could be predicted with an MHC database (high-scored peptide=activating peptide and low-scored peptide=suppressor peptide). In the future, production of synthetic peptide analogues, on the basis of these predictions, could be used for induction of T-cell anergy and/or tolerance. In the short term, algorithms, on the basis of our approach, could be tested for influence on graft survival and allosensitization in current high-quality data sets.

Epitope analysis of HLA class I donor specific antibodies in sensitized renal transplant recipients.

The goal of this study was to evaluate the epitope specificity of donor-specific HLA class I antibodies detected in the serum of alloimmunized from a previous renal graft patients. A total of 410 serum samples from 87 patients who had lost a previous graft, were collected every 4 months during a 2-year follow-up period. All recipients and donors were typed for class I HLA-antigens by a standard lymphocytotoxicity technique. To define the specificities of the HLA class I antibodies, two techniques were used in parallel: the antihuman globulin augmented CDC (AHG-CDC) technique and an ELISA technique. The mismatched HLA-antigens and the detected HLA class I antibodies were categorized as intra-cross-reactive group mismatches (intra-CREGs-MMs) and other-CREG-MMs. For each sensitized patient actual and at risk epitope specificities were defined. Thirty-eight patients (43.7%) had developed IgG HLA class I-specific antibodies with stable specificities against mismatched alloantigens from the previous graft. A total of 60 antibody reactivity patterns and 82 specificities against private and public epitope were recognized. Patients with only intra-CREGs-MMs produced HLA class I-specific antibodies less frequently than patients with only other-CREG-MMs, although the difference was nearly statistically significant (P=0.053). All HLA class I donor-specific antibodies were considered to have specificities against the private epitopes of the mismatched graft HLA-antigens. In the cases where HLA class I alloreactivity was spreading to more than one donor antigens, we considered that the detected antibodies had specificities against the private and the shared between the alloantigens epitope(s). No epitope-specific antibodies were detected against shared epitopes between the mismatched alloantigens and the HLA-antigens of the patients. In 11/38 cases (28.9%) HLA class I alloreactivity spreading to non-graft antigens was detected. These antibodies were directed against HLA-antigens that share epitope(s) and have strong serological reactivity with the immunogenic alloantigens. Our data show that a small number of private and public alloepitopes seem to be responsible for antibody production in patients sensitized from a previous graft. A detailed description of these HLA-epitopes, in the context of clinical graft complications, may lead to an improved organ allocation strategy.

Detection of HLA antibodies by using flow cytometry and latex beads coated with HLA antigens.

Detection of HLA class I antibodies in sera is needed in various clinical situations. The standard method for detecting HLA class I antibodies is the complement-dependent lymphocytotoxicity (CDC) assay, but solid-phase assays are now available. This study assessed the ability of a flow cytometric assay using latex beads coated with HLA class I antigens to detect HLA class I-specific antibodies. The CDC assay was compared with the pooled-bead assay for the detection of HLA class I antibodies. Thirty-one randomly selected serum samples previously tested by CDC assay were tested with pooled beads and analyzed by flow cytometry. Twenty-seven additional serum samples, chosen by clinical criteria and CDC assay results, were tested against the pooled beads. Next, samples from six patients from whom three or more serum samples were drawn on consecutive days were tested with both methods. Finally, serum samples that were proved positive by both methods were tested with selected beads coated with antigens from a single person. Among the randomly selected serum samples, there was 90-percent agreement between the two assays. There was 96-percent agreement between the two assays of the 27 samples that were selected by clinical criteria and CDC assay results. Testing the sera with individual beads suggested that the HLA class I antibodies react with beads expressing the corresponding HLA antigen and beads expressing antigens in the same cross-reactive group. The pooled-bead assay can be used as an alternative method for detecting HLA class I antibodies. However, if the specificity of the HLA class I antibody is required, another assay must be used.

Bypassing hybridoma technology: HLA-C reactive human single-chain antibody fragments (scFv) derived from a synthetic phage display library (HuCAL) and their potential to discriminate HLA class I specificities.

The generation of discriminative, monospecific anti-HLA antibodies used to be a difficult endeavor. Phage display technology, using single-chain antibody fragments (scFv) offers a powerful alternative obtaining target-specific, genetically stable reagents. Most of scFv obtained to date have been enriched by panning phage libraries to solid-phase coupled antigens. In the present study, HLA-C-specific scFv were isolated using a synthetic phage library in combination with a Cw*0602 overexpressing cell line. ScFv from this procedure precipitated HLA-Cw*0602 heavy chains from whole cell lysates. Flow cytometry analysis revealed that scFv stained HLA-Cw*0602-positive cells, but not cells expressing HLA alleles Cw*0302, Cw*0802, A*0201, B*2705, or Gm1*01011, indicating the specificity of scFv. Similarly they showed an ability to discriminate Cw*0602-positive from Cw*0602-negative peripheral blood lymphocytes (PBL). The results of our study demonstrate the feasibility to genetically engineer single-chain HLA-class I-specific antibodies, by phage display technology. This approach might be a valuable tool to develop a broad range of novel monospecific antibodies against HLA-class I specificities.

A limited number of HLA epitopes are recognized from HLA class I-specific antibodies detected in the serum of sensitized patients

The goal of this study was to evaluate the epitope specificity of HLA class I-specific antibodies detected in the serum of sensitized patients awaiting retransplantation. The study group consisted of 22 sensitized from previous graft patients, who produced stable IgG HLA class I-specific antibodies. A total of 60 serum samples were screened and analyzed by two techniques in parallel: the antihuman globulin augmented CDC (AHG-CDC) technique and an ELISAtechnique. All recipients and donors were typed for class I HLA antigens by a standard lymphocytotoxicity technique. The epitope identification was based on class I HLA antigens sequencing, where the multiple immunogenic epitopes are differentially shared among various HLA antigens. The unique epitope configuration on one HLA antigen represents the private epitope of the specific HLA antigen while epitopes shared by more than one HLA antigen represent public determinants. In some HLA antigens (HLA-A1), more than one private epitope has been defined, while in others (HLA-B35, -B51), the private epitopes are not yet known. In a total of 36 antibody reactivity patterns, the majority of the definable IgG HLA class I-specific antibodies corresponded to the A-locus (75%), and only 25% had specificities against the B-locus antigens, although the number of incompatibilities concerning both loci were almost identical (29 for the HLA antigens of the A-locus and 26 for those of B-locus). All patients produced HLA class I-specific antibodies with specificities against the private epitopes of the immunogenic mismatched HLA antigen(s). In 6/21 cases (28.6%), HLA class I alloreactivity spreading to nongraft HLA antigens was detected and 9 public (shared) immunogenic alloepitopes were recognized. In conclusion, appling the epitope analysis of HLA class I-specific antibodies produced by sensitized from previous graft patients, we were able to define the immunogenic alloepitopes. We consider that the immunogenic alloepitopes, during transplantation course, are mainly private epitopes of mismatched HLA antigens and, in certain cases, shared epitopes between the donor alloantigens and other HLA antigens. This knowledge may offer the potential of transplanting sensitized patients through improved donor selection.

A novel strategy for the detection and definition of HLA-specific antibodies in patients awaiting renal transplantation.

Conventional testing for HLA-specific antibodies employs complement-dependent cytotoxicity (CDC) which is labour intensive and dependent on a supply of viable lymphocytes. Our strategy to minimise CDC screening is initially to screen sera by ELISA (Quikscreen) to detect HLA Class I-specific antibodies. Negative sera are then screened by flow cytometry (FCS) using lymphoblastoid cell line pools to detect HLA Class II-specific antibodies. Only Quikscreen- or FCS-positive sera are then tested by CDC and, when indicated, with an ELISA kit (PRA-STAT) for specificity definition. Of 3680 sera, 886 (24.1%) were Quikscreen positive. Of the 2794 Quikscreen-negative sera, 374 (13.4%) were FCS positive. Therefore, only 1265 of the 3680 (34.3%) sera contained HLA-specific antibodies requiring specificity definition. This novel screening strategy has significantly reduced the CDC workload of the laboratory whilst enabling the detection of additional HLA-specific antibodies.

HLA-specific antibodies in highly sensitized patients can cause a positive crossmatch against pig lymphocytes.

The presence of IgG HLA-specific antibodies in the serum of patients awaiting transplantation indicates T- and B-cell priming and would result in acute rejection of a poorly matched human allograft. Recent advances in xenotransplantation, with the amelioration of hyperacute rejection using transgenic pig kidneys, may benefit such patients. However, accelerated cellular rejection might result from the primed T-cell recognition of antigenic epitopes shared between pig and human MHC molecules. We have compared the reactivity of IgG antibodies from 8 nonsensitized (NS) and 13 highly sensitized (HS) patients with human and pig lymphocytes by flow cytometry. Xenoreactive natural antibodies (XNA) were absorbed with pig red blood cells, and HLA class I-specific antibodies were further absorbed with pooled human platelets. Before XNA absorption, 20 of the 21 patients had a positive IgG crossmatch with pig lymphocytes, and there was no difference between NS and HS patients. In contrast, after XNA absorption, none of the 8 NS patients were positive, compared with 9 of the 13 HS patients (mean of the median channel fluorescence values of 7.7 and 86.5, respectively; P=<0.001). For XNA-absorbed HS patient sera, 20 of 30 (67%) pig lymphocyte crossmatch combinations were positive, with a mean median channel fluorescence value of 125 (range 31 to 294) compared with 9.5 (range 7 to 13) for the 10 crossmatch-negative combinations. Platelet absorption resulted in a concomitant reduction in antibody binding to pig lymphocytes in three of six HS patient sera, indicating that HLA class I-specific antibodies are responsible, at least in part, for the positive crossmatch. These results suggest that some IgG HLA-specific antibodies can bind to pig lymphocytes, analogous to a positive crossmatch with allogeneic donors.

Detection of HLA-specific antibodies by PRA-STAT and their association with transplant outcome.

The aim was to investigate the correlation between renal transplant outcome and the presence of HLA-specific antibodies detected using the ELISA kit PRA-STAT as compared with complement-dependent cytotoxicity (CDC). 295 sera from 95 renal transplant recipients (99 transplants) were investigated for the presence of HLA-specific antibodies using both PRA-STAT and CDC. The patients were divided into group I (49 transplants failed within 1 month) and group II (50 successful transplants). The concordance between PRA-STAT and CDC for the detection of HLA class I-specific antibodies was 87.8% (259 of 295). For 19 sera, antibodies were detected only by PRA-STAT; for 17 sera, antibodies were detected only by CDC. No donor-specific antibodies were detected by either technique for patients in group II. For four group I patients (six sera), donor-specific IgG antibodies were detected only by PRA-STAT (one before, three after transplant) and all four transplants failed. For five other group I patients (six sera), donor HLA-specific antibodies were detected only by CDC (one before, four after transplant) and all five transplants failed. The antibodies detected before transplant by CDC were shown to be IgM alloantibodies. This study showed that PRA-STAT could detect HLA-specific IgG antibodies relevant to transplant outcome that were not detected by CDC. However, it could not detect IgM alloantibodies that were also shown to be important. PRA-STAT is therefore a useful addition to a histocompatibility laboratory's screening repertoire only when used in conjunction with other techniques.

The HLA-E gene encodes two differentially regulated transcripts and a cell surface protein.

Abstract An HLA-E-specific oligonucleotide probe was used to study the expression of HLA-E. This probe detects two HLA-E transcripts, 1.8 and 2.7 kb in size, which are present in varying ratios in all tissues and cell lines investigated. We demonstrate that alternative poly(A) site usage accounts for the differential regulation of the two HLA-E mRNA species. Sequence analysis of three cDNA clones, representing the two transcripts of HLA-E, and of an HLA-E gene encoded by cosmid cd3.14, revealed identity of gene and cDNA in the 3' untranslated region. S1 nuclease protection assays confirmed that the two HLA-E transcripts are not alternative splicing products. Introduction of cd3.14, together with human beta 2 m into the murine myeloma cell line P3X63-Ag8.653, resulted in a cell surface expression of an HLA-class I heavy chain detectable by indirect immunofluorescence whereas transfection into the human beta 2m expressing mouse L cell line, J27 was negative with regard to cell surface expression. Cell surface labeling of transfectants and immunoprecipitation with a monomorphic HLA class I-specific antibody or an antibody against human beta 2m confirmed the presence of an HLA-E H chain on the cell surface. These results indicate that the HLA-E gene codes for a class I H chain that can be expressed on the cell surface.

Antigenic determinants shared between HLA-A, ?B, ?C antigens and H-2 class I molecules modified by bovine beta-2 microglobulin

The specificity of the mouse class I-specific antibody COB6-3 was examined in detail. It was found to react with the mouse class I molecules H-2Db, Kd, and Qa-2, and with human HLA-A, -B, -C antigens. The specificity pattern of COB6-3, despite its different origin, was similar to that of the monomorphic HLA class I-specific antibody W6/32. Cross-inhibition studies show that on human cells the antigenic determinants recognized by the two antibodies are situated close together and may be identical. On mouse cells, reactivity of both antibodies was generated upon replacement of mouse beta-2 microglobulin (B2m) with its bovine counterpart, but differences in specificity were observed using human B2m.

Anti-HLA class I antibodies inhibit the T cell-independent proliferation of human B lymphocytes.

The role of major histocompatibility complex-encoded class I molecules in the proliferation of human B lymphocytes is presently unclear. This question was addressed by investigating the effect of three individually derived anti-HLA class I monoclonal antibodies (mAb) on purified human B cells (less than 1.5% T cells) stimulated by either the T-independent mitogen Staphylococcus aureus or the phorbol ester, phorbol-12-myristate-13-acetate. The three anti-HLA class I antibodies, whether specific for gene products of the HLA-A locus (mAb 131), HLA-B locus (mAb 4E), or HLA-A, -B, and -C locus (mAb W6/32), inhibited S. aureus-induced proliferation by 70 to 90%. This inhibition was significant over a 5-day culture period, was not altered by the addition of exogenous interleukin 2 or B cell growth factor, and was not due to nonspecific cytotoxicity. In addition, the inhibition of proliferation was unchanged when the mAb were added 12 hr after the initiation of culture. The proliferative response was not affected by either of the control antibodies OKB7 and R3-367. In contrast with S. aureus-stimulated B cells, phorbol-12-myristate-13-acetate-induced proliferation was resistant to the inhibitory activity of HLA class I-specific antibodies. These results suggest that HLA class I molecules are involved in human B lymphocyte proliferation and may regulate a critical event preceding the upregulation of protein kinase C activity.