Detection of newly antibody-defined epitopes on HLA class I alleles reacting with antibodies induced during pregnancy.

The determination of HLA mismatch acceptability at the epitope level can be best performed with epitopes that have been verified experimentally with informative antibodies. The website-based International Registry of HLA Epitopes (http://www.epregistry.com.br) has a list of 81 antibody-verified HLA-ABC epitopes but more epitopes need to be added. Pregnancy offers an attractive model to study antibody responses to mismatched HLA epitopes which can be readily determined from the HLA types of child and mother. This report describes a HLAMatchmaker-based analysis of 16 postpregnancy sera tested in single HLA-ABC allele binding assays. Most sera reacted with alleles carrying epitopes that have been antibody-verified, and this study focused on the reactivity of additional alleles that share other epitopes corresponding to eplets and other amino acid residue configurations. This analysis led in the identification of 16 newly antibody-defined epitopes, seven are equivalent to eplets and nine correspond to combinations of eplets in combination with other nearby residue configurations. These epitopes will be added to the repertoire of antibody-verified epitopes in the HLA Epitope Registry.

Should HLA mismatch acceptability for sensitized transplant candidates be determined at the high-resolution rather than the antigen level?

Defining HLA mismatch acceptability of organ transplant donors for sensitized recipients has traditionally been based on serologically defined HLA antigens. Now, however, it is well accepted that HLA antibodies specifically recognize a wide range of epitopes present on HLA antigens and that molecularly defined high resolution alleles corresponding to the same low resolution antigen can possess different epitope repertoires. Hence, determination of HLA compatibility at the allele level represents a more accurate approach to identify suitable donors for sensitized patients. This approach would offer opportunities for increased transplant rates and improved long term graft survivals.

Update of the HLA class I eplet database in the website based registry of antibody-defined HLA epitopes.

Eplets are small configurations of polymorphic amino acid residues on human leukocyte antigen (HLA) molecules and are considered as essential components of HLA epitopes recognized by antibodies. This report describes a new design of the eplet repertoire in HLA-ABC alleles used in Luminex kits for antibody testing. There were three steps (1): identify all combinations of polymorphic residues with HLA molecular modeling within a 3-Å radius, (2) determine polymorphic residue compositions of 3 Å patches from amino acid sequences of HLA alleles in Luminex panels and (3) annotate eplets from one or more patches present on one allele or shared by the same group of alleles. There are now 270 HLA-ABC eplets in the Registry, of which 219 are in antibody-accessible positions on the molecular surface and 51 are defined solely by residue polymorphisms located below the molecular surface. Each eplet has a list of Luminex and non-Luminex alleles for which mismatch acceptability can be determined.

First report on the antibody verification of HLA-ABC epitopes recorded in the website-based HLA Epitope Registry.

The International Registry of Antibody-Defined HLA Epitopes ( http://www.epregistry.com.br) has been recently established as a tool to understand humoral responses to human leukocyte antigen (HLA) mismatches. These epitopes are defined structurally by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. So-called eplets represent essential components of HLA epitopes and they are defined by polymorphic residues. A major goal is to identify HLA epitopes that have been verified experimentally with informative antibodies. Our analysis has also included data in many publications. As of 1 November 2013, 95 HLA-ABC antibody-verified epitopes have been recorded, 62 correspond to eplets and 33 are defined by eplets paired with other residue configurations. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

First report on the antibody verification of MICA epitopes recorded in the HLA epitope registry.

The International Registry of HLA Epitopes (http://epregistry.com.br) has been recently established as a tool to understand antibody responses to HLA mismatches. These epitopes are defined structurally by three-dimensional molecular modelling and amino acid sequence differences between HLA antigens. A major goal was to identify HLA epitopes that have been verified experimentally with informative antibodies. This report addresses the identification of MICA epitopes. Our analysis included published information about MICA antibody reactivity in sera from sensitized patients as well as data from our own laboratories. This report describes twenty-one MICA epitopes verified with antibodies which have primarily been tested in Luminex assays with single alleles. The epitopes correspond to distinct eplets that are often defined by single residues. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

16th IHIW: a website for antibody-defined HLA epitope Registry.

The concept that HLA antibodies are specific for epitopes rather than HLA antigens is important not only for the determination of mismatch acceptability for sensitized patients but also for a better understanding of the antibody response to an HLA mismatch. Numerous publications describe epitope-specific antibodies, but there is no standardized information about the repertoire of clinically relevant HLA epitopes. Under auspices of the 16th IHIW, we have developed a website-based registry of antibody-verified HLA epitopes. Epitope notations are based on HLA molecular modelling of amino acid residues in polymorphic sequence positions. Informative epitope-specific antibodies had been induced by a transplant, transfusion or pregnancy and were monoclonal antibodies or eluates of sera absorbed with single HLA alleles. Antibody reactivity was determined in binding assays with single-allele panels. Antibody producer/immunizer HLA types enhanced the characterization of specific epitopes. The Registry also includes epitopes described in original research publications. Based on the extent of antibody reactivity information, we assigned epitope status as confirmed (well documented) or provisional (more data are needed). At present, the Registry has 69 HLA-ABC, 53 DRB1/3/4/5, 17 DQ, 8 DP and 22 MICA antibody-verified epitopes and will be updated on a quarterly basis. Laboratories worldwide continue to submit data about previously unreported antibody-specific epitopes. For each epitope, the website shows its amino acid composition and HLA alleles that share the epitope. Links show antibody reactivity patterns, sensitization information and references. Other links show molecular modelling of corresponding structural epitopes and polymorphic residue information for epitope-carrying alleles. The website will also have a link to epitope frequency information in different populations. Search functions will list mismatched epitopes on mismatched alleles for selected HLA types. The HLA Epitope Registry will become a valuable resource for researchers interested in HLA compatibility at the epitope level and investigating antibody responses to HLA mismatches.

The antibody response to an HLA mismatch: a model for nonself-self discrimination in relation to HLA epitope immunogenicity.

Antibodies to HLA mismatches are specific for epitopes rather than antigens. HLAMatchmaker considers each HLA antigen as a string of eplets that represent key elements of epitopes. Certain antibodies are specific for single eplets, but recent studies have demonstrated that epitopes defined by eplet pairs always involve one nonself-eplet and a self-eplet shared between the immunizing antigen and the antibody producer. This suggests an autoreactive component of the alloantibody response to an HLA mismatch and this report expands this concept. During B-cell development, V(H) and V(L) gene rearrangements produce a diversity of Ig receptors that can recognize epitopes on autologous proteins. It is hypothesized that B cells carry low-affinity receptors for self-HLA antigens. Their interactions with self-HLA proteins will not lead to B-cell activation or antibody production. In contrast, exposure to HLA mismatches induces often strong alloantibody responses. The activation of self-HLA-specific B cell by a nonself-eplet may require that the remainder of the structural epitope of the immunizing antigen has considerable structural similarity with one of the antibody producer's alleles. This hypothesis has been tested in molecular modelling studies with six epitopes defined by human monoclonal antibodies. In each case, one allele of the antibody producer had no or few differences with the immunizing allele in antibody-accessible positions defined by a 15 Ångstrom radius of the mismatched eplet. The other alleles of the antibody producer showed significantly greater numbers of residue differences with the immunizer (5.8 ± 2.0 versus 1.0 ± 0.6, P < 0.0001). These data support the concept that HLA antibodies originate from B cells with self-HLA immunoglobulin receptors that recognize mismatched eplets as nonself entities on immunizing antigens. The nonself-self paradigm provides a new insight of HLA epitope immunogenicity and may explain why sensitized patients have antibodies to a restricted number of mismatched epitopes.

Antibody-reactive epitope determination with HLAMatchmaker and its clinical applications.

Antibodies against allogeneic human leukocyte antigen (HLA) molecules are important impediments to the success of different clinical procedures including transplantation and platelet transfusion. In these settings, characterization of the repertoire of immunogenic epitopes is important for permissible mismatch determination and the identification of acceptable mismatches for sensitized patients. HLAMatchmaker is a computer algorithm that considers small configurations of polymorphic residues referred to as eplets as essential components of HLA epitopes. This review critically elaborates the concepts underlying the HLAMatchmaker and describes the usefulness of HLAMatchmaker in the clinical setting. Recent developments have increased our understanding of structural basis of HLA antigenicity (i.e. reactivity with specific antibody) and immunogenicity (i.e. its ability to induce an antibody response).

Three Ia species with different structures and alloantigenic determinants in an HLA-homozygous cell line.

Three distinct molecular subsets with different structures and alloantigenic determinants were identified in human Ia antigens from cells of an HLA-Dw7 homozygous cell line. The subsets carried DR7 specificity, BR4X7 supertypic specificity and MB2 supertypic specificity, respectively, and were immunospecifically separated by the use of operationally monospecific alloantisera. These specificities showed HLA-linked segregation in families and they were distributed in the population according to different but partially overlapping patterns. On peptide mapping analysis, the three subsets showed marked differences in the beta-chains. The alpha-chains of DR7 and BR4X7 subsets were very similar to each other, whereas the alpha-chains of MB2 subset were distinctive from those of DR7 and BR4X7. These data indicate the presence of a minimum of three HLA-linked loci; DR locus, a locus that encodes BR4X7, and a locus that encodes MB2, and substantiate the three-loci concept for the genetic control of human I antigens.

The complexity of HLA-DS molecules. A homozygous cell line expresses multiple HLA-DS alpha chains.

Ia molecules expressed by an HLA-DRw6 homozygous cell line were immunoprecipitated with anti-Ia allosera and monoclonal antibodies and analyzed by 2-D gel electrophoresis. The DRw6 homozygous cell line was shown to express two DS beta chains; this observation extends our previous finding that a DR5 homozygous cell line expresses two DS beta chains and suggests that the expression of at least two DS beta chains by DR homozygous cell lines is a generalized phenomenon. The data presented here document for the first time that a DR homozygous cell line expresses at least two DS alpha chains. Therefore, this cell line expresses at least two DS molecules with the potential for the expression of four DS molecules. In agreement with previous reports, the cell line was shown to express two DR beta chains and one DR alpha chain that combine to form two DR molecules. The molecular specificities of two MB1 allosera and two MB1 -like monoclonal antibodies were also compared in these studies. Both MB1 allosera isolated a single DS molecule, while the MB1 -like monoclonal antibodies isolated at least two DS molecules. Therefore, these studies document for the first time that anti-Ia reagents which are specific for the MB1 or MB1 -like determinants in population studies do not recognize the same Ia molecules in immunochemical studies. The data presented here for the expression of at least two DS alpha chains and the location of the MB1 allodeterminant on only one of multiple DS molecules are in agreement with recent studies at the gene level.

Demonstration of a third structurally distinct human Ia beta chain by two-dimensional gel electrophoresis.

Previous studies have indicated that HLA-DR homozygous cell lines express two Ia alpha and Ia beta chains that combine to form at least two Ia molecules. This report demonstrates by two-dimensional gel electrophoresis the existence of a third structurally distinct human Ia beta chain on DR2 and DR5 cell lines. This suggests that at least five separate genes control the expression of Ia molecules on HLA-DR homozygous cell lines.

Correlations between Terasaki's HLA class II epitopes and HLAMatchmaker-defined eplets on HLA-DR and -DQ antigens.

Human leukocyte antigen (HLA) class II-specific antibodies increase the risk of transplant failure, and their characterization must consider epitopes rather than antigens. There are two strategies to determine HLA epitope structure. Terasaki's group has analyzed antibody reactivity patterns with single antigen panels with a computer program based on shared amino acid residues of reactive alleles. HLAMatchmaker is a theoretical algorithm that predicts HLA epitopes on the HLA molecular surface from stereochemical modeling of epitope-paratope interfaces of antigen-antibody complexes. Our epitope repertoire is based on so-called 'eplets' representing 3-A patches of at least one polymorphic residue on the molecular surface. This report describes how 49 of 53 Terasaki's HLA-DR epitopes correspond to HLAMatchmaker-defined eplets. Most of them are equivalent to single eplets (n = 33) or two or more possible eplets (n = 10), but six had corresponding eplet pairs. There were 10 cases whereby eplets have permissible residue combinations, and in 5 cases, we found that eplet specificity might be influenced by nearby hidden residues. We could assign corresponding eplets to 17 of 18 Terasaki's HLA-DQ epitopes. This study demonstrates how the HLAMatchmaker interpretation of amino acid residues shared between antibody-reactive antigens can increase our understanding of the structural basis of HLA epitopes.

Correlations between Terasaki's HLA class I epitopes and HLAMatchmaker-defined eplets on HLA-A, -B and -C antigens.

Although the determination of human leukocyte antigen (HLA) antibody specificity has traditionally been directed toward HLA antigens, there is now increasing attention to structurally defined HLA epitopes. An understanding of the HLA epitope repertoire is important to acceptable mismatching for sensitized patients and to a new epitope-based matching algorithm aimed to reduce antibody-mediated rejection. There are two strategies to determine the HLA epitope repertoire. Terasaki's group has used an empirical method to analyze the reactivity of single allele Luminex panels with mouse monoclonal antibodies (mAbs) and absorbed/eluted alloantibodies with a computer program based on shared residues in the amino acid sequences of reactive alleles. HLAMatchmaker is a theoretical algorithm that predicts HLA epitopes on the HLA molecular surface from stereochemical modeling of epitope-paratope interfaces of antigen-antibody complexes. Our epitope repertoire is based on so-called 'eplets' representing 3-A patches of at least one polymorphic residue on the molecular surface. A comparative analysis has shown that 81/103 Terasaki's HLA class I epitopes are equivalent to individual eplets (n = 50) or pairs of eplets (n = 31) separated far enough to serve as potential contact sites for two complementarity-determining regions of antibody. An additional 12 Terasaki's epitopes (TerEps) correspond to eplets with permissible residue combinations that do not seem to affect epitope specificity. We could not identify corresponding eplets for the remaining 10 TerEps, including 8 that might be considered xeno-epitopes defined by mouse mAbs. Conversely, HLAMatchmaker has 38 additional eplets in well-exposed surface positions that do not have equivalent TerEps, and for many of them, we have found specific antibodies. These findings strengthen the concept that eplets are essential basic units of HLA epitopes and that they provide a better understanding of HLA immunogenicity (i.e. ability to induce an antibody response) and antigenicity (i.e. reactivity with specific antibody).

Thymus adenylate cyclase activity during murine leukemogenesis.

Activity of thymus adenylate cyclase was more than three times higher in leukemic AKR mice than in nonleukemic AKR mice and CBA mice. Preleukemic AKR mice that had no evidence of leukemia but were expected to soon develop the disease exhibited similarly elevated activities of thymus adenylate cyclase.

Second update of the International Registry of HLA Epitopes. I. The HLA-ABC Epitope Database.

The International Registry of HLA Epitopes (http://www.epregistry.com.br) is a website-based resource for HLA epitopes important in transplant rejection and platelet transfusion refractoriness. Its primary goal is to document epitopes that are verified experimentally with specific antibodies. Such epitopes can be defined by single eplets and by eplets paired with certain polymorphic residues within a 15-Å radius, the dimension of the corresponding structural epitope. This report is an update of the HLA-ABC repertoire including descriptions of 72 antibody-verifications of epitopes defined by eplets and/or eplet pairs. The newly updated version 2.0 EpRegistry shows also the polymorphic residue compositions of structural epitopes corresponding to eplets shared between groups of alleles. At present, 151 eplets have not been antibody-verified, and we ranked them with a so-called ElliPro score as a potential predictor of immunogenicity. Sixty eplets with low ElliPro scores might be considered non-epitopes incapable of inducing specific antibodies.

Inhibitory effect of growth hormone on Gross passage A virus-induced thymus leukemia in C3Hf mice.

Administration of ovine growth hormone to young C3Hf mice inhibited Gross passage A virus-induced leukemogenesis as manifested by a delayed onset and a lower incidence of thymus leukemia. These results can be interpreted that growth hormone inhibited thymus-dependent leukemogenesis perhaps through thymotrophic influences which prevented or delayed the thymus involution believed to be essential for leukemia change. In female but not in male Gross passage A virus-infected mice, development of a thymus-independent leukemia appeared to be promoted by growth hormone.

Changes in lymphoid cyclic adenosine 3':5'-monophosphate metabolism during murine leukemogenesis.

Cyclic adenosine 3':5'-monophosphate (cyclic AMP) levels were slightly increased in preleukemic AKR mouse thymus cells, compared with nonleukemic thymus cells, but were markedly reduced in leukemic cells. Adenylate cyclase activity rose during the preleukemic and leukemic phases of leukemogenesis. Although the drop of epinephrine-induced stimulation of thymus adenylate cyclase in the preleukemic phase was probably age related, there was an additional decrease of adenylate cyclase activation by epinephrine in leukemic cells. Cyclic AMP phosphodiesterase activity was slightly higher in preleukemic cells and more than fourfold AKR thymus. These observations suggest that cyclic AMP phosphodiesterase is largely responsible for the low levels of cyclic AMP in leukemic cells. Significant changes in cyclic AMP metabolism are already detectable before neoplastic cells may be found in the thymus.