Definition of supertypes for HLA molecules using clustering of specificity matrices

Major histocompatibility complex (MHC) proteins are encoded by extremely polymorphic genes and play a crucial role in immunity. However, not all genetically different MHC molecules are functionally different. Sette and Sidney (1999) have defined nine HLA class I supertypes and showed that with only nine main functional binding specificities it is possible to cover the binding properties of almost all known HLA class I molecules. Here we present a comprehensive study of the functional relationship between all HLA molecules with known specificities in a uniform and automated way. We have developed a novel method for clustering sequence motifs. We construct hidden Markov models for HLA class I molecules using a Gibbs sampling procedure and use the similarities among these to define clusters of specificities. These clusters are extensions of the previously suggested ones. We suggest splitting some of the alleles in the A1 supertype into a new A26 supertype, and some of the alleles in the B27 supertype into a new B39 supertype. Furthermore the B8 alleles may define their own supertype. We also use the published specificities for a number of HLA-DR types to define clusters with similar specificities. We report that the previously observed specificities of these class II molecules can be clustered into nine classes, which only partly correspond to the serological classification. We show that classification of HLA molecules may be done in a uniform and automated way. The definition of clusters allows for selection of representative HLA molecules that can cover the HLA specificity space better. This makes it possible to target most of the known HLA alleles with known specificities using only a few peptides, and may be used in construction of vaccines. Supplementary material is available at .     

Acetylcholine receptor peptide recognition in HLA DR3-transgenic mice: in vivo responses correlate with MHC-peptide binding.

HLA DR3 is an MHC molecule that reportedly predisposes humans to myasthenia gravis (MG). Though MG is an Ab-mediated autoimmune disease, CD4+ T cells are essential for the generation of high-affinity Abs; hence the specificities of autoreactive CD4+ T cells are important. In this study we report the HLA DR3-restricted T cell determinants on the extracellular region sequence of human acetylcholine receptor alpha subunit. We find two promiscuous determinants on this region 141-160 and 171-190 as defined by their immunogenicity in HLA DR3-, HLA DQ8-, and HLA DQ6-transgenic mice in the absence of endogenous mouse class II molecules. We also studied the minimal determinants of these two regions by truncation analysis, and the MHC binding affinity of a set of overlapping peptides spanning the complete sequence region of human acetylcholine receptor alpha subunit. One of the peptide sequences strongly immunogenic in HLA DR3-transgenic mice also had the highest binding affinity to HLA DR3. Identification of T cell determinants restricted to an MHC molecule known to predispose to MG may be an important step toward the development of peptide-based immunomodulation strategies for this autoimmune disease.     

HLA serum screening based on an heuristic solution of the set cover problem.

A computer program initially written by the Milwaukee Blood Bank has been modified to use a new algorithm for the assignment of HLA specificities to antisera. The assignment is based on the reactions of cells with known specificities. Specificities which are present only on cells which do not react are first ruled out. This step is followed by one or more steps in which the 'least reactive' specificities are ruled out. The rationale for the algorithm is discussed and an example is presented.     

HLA-DR polymorphism in a Senegalese Mandenka population: DNA oligotyping and population genetics of DRB1 specificities.

HLA class II loci are useful markers in human population genetics, because they are extremely variable and because new molecular techniques allow large-scale analysis of DNA allele frequencies. Direct DNA typing by hybridization with sequence-specific oligonucleotide probes (HLA oligotyping) after enzymatic in vitro PCR amplification detects HLA allelic polymorphisms for all class II loci. A detailed HLA-DR oligotyping analysis of 191 individuals from a geographically, culturally, and genetically well-defined western African population, the Mandenkalu, reveals a high degree of polymorphism, with at least 24 alleles and a heterozygosity level of .884 for the DRB1 locus. The allele DRB1*1304, defined by DNA sequencing of the DRB1 first-domain exon, is the most frequent allele (27.1%). It accounts for an unusually high DR13 frequency, which is nevertheless within the neutral frequency range. The next most frequent specificities are DR11, DR3, and DR8. Among DRB3-encoded alleles, DR52b (DRB3*02) represents as much as 80.7% of all DR52 haplotypes. A survey of HLA-DR specificities in populations from different continents shows a significant positive correlation between genetic and geographic differentiation patterns. A homozygosity test for selective neutrality of DR specificities is not significant for the Mandenka population but is rejected for 20 of 24 populations. Observed high heterozygosity levels in tested populations are compatible with an overdominant model with a small selective advantage for heterozygotes.     

HLA-typing analysis following allogeneic bone grafting for sinus lifting

According to the Brazilian Association of Organ Transplants, in 2015, 19,408 bone transplants were performed in Brazil, over 90% by Dental Surgeons. The surgical technique itself has a respectable number of reports regarding its clinical efficacy, as measured by long-term survival of dental implants in grafted areas. Uncertainty remains, however, as to whether fresh frozen grafts from human bone donors remain immunologically innocuous in the body of the host. Six male with no previous medical history of note, including systemic diseases, surgery or blood transfusion were selected. These patients underwent reconstructive procedures (sinus lifting) using fresh frozen human bone from a tissue bank. All patients had venous blood samples collected prior to surgery and 6 months after the procedure. Anti-HLA analysis for the detection of HLA (human leukocyte antigen) antibodies was performed using methods such as the LABScreen PRA Class I and Class II, LABScreen Single Antigen Class I and Class II, Luminex Platform. Reactive individuals to the screening tests (LABScreen PRA) were further investigated to determine the specificity of the antibodies detected (LABScreen Single Antigen) with a cutoff value of median fluorescence intensity ≥500. As a result, it was observed that two patients (33%) were positive in screening tests, one presenting with anti-HLA Class I and II sensitization and the other with anti-HLA class II. The specificity analysis showed that the patients sensitized to HLA class II presented 4 specificities, 3 of which immunologically relevant. In the second individual, 23 specificities were identified, 6 of which immunologically important for HLA class I and 4 specificities for HLA class II, 3 of these were immunologically important. All specificities detected had average fluorescence. These findings are suggestive that sinus-lifting procedures with allogeneic bone can induce immunological sensitization.     

Global dynamics of a reaction and diffusion model for Lyme disease

This paper is devoted to the mathematical analysis of a reaction and diffusion model for Lyme disease. In the case of a bounded spatial habitat, we obtain the global stability of either disease-free or endemic steady state in terms of the basic reproduction number R?. In the case of an unbounded spatial habitat, we establish the existence of the spreading speed of the disease and its coincidence with the minimal wave speed for traveling fronts. Our analytic results show that R? is a threshold value for the global dynamics and that the spreading speed is linearly determinate.     

MHC-peptide specificity and T-cell epitope mapping: where immunotherapy starts

The evaluation and characterization of epitope-specific human leukocyte antigen (HLA)-restricted memory T-cell reactivity is an important step for the development of preventive vaccines and peptide-based immunotherapies for viral and tumor diseases. The past decade has witnessed the use of HLA-restricted peptides as tools to activate strong immune responses of na?ve or memory T cells specifically. This has fuelled an active search for methodological approaches focusing on HLA and peptide associations. Here, we outline new perspective on the emerging opportunity of evaluating HLA and peptide restriction by using novel approaches, such as quantitative real-time PCR, that can identify epitope specificities that are potentially useful in clinical settings.     

Serologic dissection of HLA-D specificities by the use of monoclonal antibodies

To study the gene products of the HLA complex, we produced two monoclonal antibodies, termed HU-18 and HU-23. They were active in complement-dependent cytotoxicity and detected B-cell alloantigens encoded by a locus (or loci) linked to HLA. When three types of HLA-DR4 homozygous B-cell lines with different HLA-D specificities were tested for reactivity with HU-18 and HU-23, they displayed distinct reaction patterns depending on the HLA-D specificities they possessed: EBV-Wa (HLA-DYT homozygous), negative for both HU-18 and HU-23; KT2 and KOB (HLA-DKT2 homozygous), positive only for HU-18; and ER (HLA-Dw4 homozygous), positive for both. These differential reaction patterns were further confirmed by testing against a panel of 17 HLA-DR4-positive peripheral blood lymphocytes with known HLA-D specificities. Thus, these monoclonal antibodies allow us to identify HLA-DYT, HLA-DKT2, and HLA-Dw4 solely by serologic methods. This is the first clearcut serologic identification of these three HLA-DR4-associated HLA-D specificities, which have been indistinguishable by conventional serology and identified only by cellular techniques. It is hoped that immunochemical investigations using HU-18 and HU-23 will advance our understanding of the HLA-D region on a molecular level.     

The peptide-binding specificity of HLA-A*3001 demonstrates membership of the HLA-A3 supertype

Human leukocyte antigen class I (HLA-I) molecules are highly polymorphic peptide receptors, which select and present endogenously derived peptide epitopes to CD8+ cytotoxic T cells (CTL). The specificity of the HLA-I system is an important component of the overall specificity of the CTL immune system. Unfortunately, the large and rapidly increasing number of known HLA-I molecules seriously complicates a comprehensive analysis of the specificities of the entire HLA-I system (as of June 2008, the international HLA registry holds >1,650 unique HLA-I protein entries). In an attempt to reduce this complexity, it has been suggested to cluster the different HLA-I molecules into “supertypes” of largely overlapping peptide-binding specificities. Obviously, the HLA supertype concept is only valuable if membership can be assigned with reasonable accuracy. The supertype assignment of HLA-A*3001, a common HLA haplotype in populations of African descent, has variously been assigned to the A1, A3, or A24 supertypes. Using a biochemical HLA-A*3001 binding assay, and a large panel of nonamer peptides and peptide libraries, we here demonstrate that the specificity of HLA-A*3001 most closely resembles that of the HLA-A3 supertype. We discuss approaches to supertype assignment and underscore the importance of experimental verification.     

Chemical, physical-chemical, and immunological properties of papain-solubilized human transplatation antigens.

Papain-solubilized HLA-A, -B, and -C antigens have been isolated from cadaveric spleens. The isolated material was homogeneous and comprised subunits with the apparent molecular weights 33,000 and 12,000. Amino acid analyses of a mixture of HLA antigen heavy chains obtained from a great number of spleens with different HLA antigen phenotypes revealed a composition that is very similar to that of individual HLA-A and -B antigens. Likewise, the NH2-terminal 30 residues of the HLA-antigen heavy chain mixture were virtually identical with that recorded for individual specificities. The circular dichroism spectra for the isolated HLA antigens and for free beta2-microglobulin revealed similarities with spectra recorded for immunoglobulin chains and domains. The HLA-antigen heavy chain may contain an appreciable amount of beta structure. Antibodies raised against free beta2-microglobulin react better with beta2-microglobulin in free form than when bound to the HLA-A, -B, and -C antigen heavy chains. This is due to the fact that free beta2-microglobulin can bind a maximum of four Fab fragments simultaneously, whereas the HLA-antigen-associated beta2-microglobulin can bind only two Fab fragments without dissociating from the heavy HLA-antigen subunit.     

Identification of nonlinear models of biological membranes by the method of fixed voltage

A mathematical substantiation of the method suggested by Hodkin and Huxley in 1952 for the identification of nonlinear systems is presented. A procedure for the application of this method was developed, which involves creating the structure of a mathematical model, carrying out a series of tests with specially chosen signals, and finding the unknown parameters. The basic requirements to admissible sets of entrance and target signals and the operator of the system were determined. It was shown that it should be quite continuous, the minimal number of unknown parameters and the minimal complexity of structure of the operator should provide the required quality of approximation. The merits and demerits of the mathematical models of Hodkin-Huxley and Noble, and the procedures used for their creation are discussed. The structure of the operator for the identification of mathematical models of excitable membranes when a large number of membrane currents is considered is offered. It was found that nonlinear electric properties of biological membranes can be identified using tests with other kinds of "fixed" parameters, for example, the method of "fixed" current, the fixed linearly increasing voltage, and others.     

HLA class II restriction of T helper cell response to cytomegalovirus (CMV). I. Immunogenetic control of restriction

All three HLA class II families (DR, DQ, and DP) are involved in restriction of helper T cell (Th) recognition of nominal antigens including CMV. Only limited studies have been described previously to determine whether restricting determinants of DR and especially DQ are subtypic to the serologically defined DR and DQ specificities, and to what extent restricting determinants are associated with Dw specificities defined in alloresponses. In the present report, we describe a large number of CMV-specific Th clones derived from two different individuals who are seropositive for CMV. Clones were classified as being DR-, DQ-, or DP-reactive based on blocking with monoclonal antibodies. DR- and DQ-restricted clones were then examined in panel studies using antigen-presenting cells (APC) expressing the Dw subtype of the restricting DR-DQ haplotype, as well as APC expressing different Dw subtypes associated with the serologically defined specificity. Unrelated specificities were also included. Our findings show that not only for DR but for DQ as well, the primary restricting determinants appear to be subtypic to the serologically defined antigen; furthermore, subtype restriction for both DR and DQ is very closely associated with single Dw specificities. In several cases in which cross-reactivity among restricting Dw specificities was observed in association with a given DR or DQ haplotype, a molecular basis could be suggested to explain the cross-reacting determinants. A small minority of the clones appeared to be CMV specific, but was restricted by a determinant(s) that is either monomorphic or minimally polymorphic.     

Hereditary C2 deficiency associated with immune complex disease.

A patient presenting with a syndrome probably due to immune complex deposition was investigated and found to possess an inherited C2 complement deficiency. Family studies indicated that the deficiency was transmitted as an autosomal recessive trait. HLA typing for the HLA-A and HLA-B specificities and HLA-D specificities indicated a close linkage between the HLA and C2 genes, as has been described elsewhere. The HLA-A and B locus specificities HLA-AW25 and HLA-B18 were coded for by each of the two chromosomes carrying the C2(0) gene. However, the two chromosomes differed at the HLA-D locus, as one coded for HLA-DW2 whilst the other did not. This case, therefore, provides a unique haplotype and may be of importance in mapping the C2(0) locus, as it suggests that the gene order on chromosome 6 is HLA-D, C2(0), HLA-B, HLA-A. Extensive complement component assays indicated that utilization of complement in the patient was occurring via the alternate complement pathway. It is suggested that, as a result of the C2 deficiency, infections with viruses and other agents could lead to an immune complex disease due to an impaired capacity to effectively eliminate circulating complexes.     

Serological typing of HLA-D: Predictive value in mixed lymphocyte cultures (MLC)

Locally produced antisera and antisera received through the Seventh International Histocompatibility Workshop exchange were investigated for specific B-cell cytotoxic activity in a panel of 95 unrelated HLA-D-typed donors. A number of sera formed clusters defining eight B-cell specificities which were strongly associated (p<0.001) to the HLA-D determinants Dw1–8. In panel investigations, only four triplets occurred. In five HLA recombinant families, these B-cell specificities followed the HLA-B-D chromosomal region, and in one —B/D recombination, DRw1 traveled with —Dw1. In MLCs between panel donors sharing zero, one, or two HLA-D-related B-cell specificities, significantly weaker MLC stimulation was observed with increasing compatibility, the median relative responses being 100, 52, and 17 percent, respectively. It is concluded that B cell-specificities HLA-DRw1–7 and WIA8 are probably coded for by HLA-D; they are excellent markers for the HLA-D determinants, which can thus be typed for by serological means; and serological typing for HLA-D has great value in predicting the outcome of MLCs.     

The polymorphisms of HTC-defined HLA specificities in the Shanghai Chinese population

With reference sera and homozygous typing cells (HTCs) of 3rd Asia-Oceania Histocompatibility Workshop Conference, 56 healthy unrelated subjects in Shanghai were typed for HLA-A, B, C, DR, DQ, and Dw. This paper presents the results of HLA-Dw typing, its relationship to serological class II antigens, and the distribution of Dw in the population. The polymorphism patterns of Chinese Dw specificities were quite different from those in Caucasoids and Japanese. The predominant Dw phenotypes detected in Shanghai Chinese were Dw 2, Dw 3, DKT 2, Dw 7 c, (Dw7 + Dw 17) and Dw 23 (DB 5). And significant correlations were observed between Dw 1 and DR 1, Dw 2 and DR 2, Dw 3 and DR 3, Dw 7 c and DR 7, DB 7 and DRw 8, as well as Dw 23 and DR 9. SMY 129, a novel Dw specificity defined by local HTCs and co-studied by the laboratories joined for Dw typing in 3rd AOHWC showed its correlation with DR 5. Nevertheless, more than fifty percent of Dw specificities could not be assigned in the four correspondent designated serological antigens, DR 2, DR 5, DRw 8 and DR 9, respectively, which, together with other blank Dw specificities, gave a total blank Dw gene frequency as high as 43.2% in the population. It was suggested by further analysis that novel Dw specificities might be identified more effectively if efforts would be concentrated on DR 5 and DR 9, two antigen families which, in some way, might represent the characteristics of HLA system in Chinese. Besides, certain HTC-defined antigens, e.g. Dw 3 and the DR 4-related Dw specificities, have been revealed to be in linkage disequilibrium with other DR antigens in addition with the correspondent designated ones, resulting in some unique haplotype combinations in Shanghai Chinese. It seems to us that the particular patterns of polymorphisms of serum- and cell-defined HLA class II antigens would be helpful to elucidate the mechanisms by which certain diseases are in association with HLA in Chinese in a different manner as compared with that in Caucasoids.     

A Common Minimal Motif for the Ligands of HLA-B*27 Class I Molecules

CD8⁺ T cells identify and kill infected cells through the specific recognition of short viral antigens bound to human major histocompatibility complex (HLA) class I molecules. The colossal number of polymorphisms in HLA molecules makes it essential to characterize the antigen-presenting properties common to large HLA families or supertypes. In this context, the HLA-B*27 family comprising at least 100 different alleles, some of them widely distributed in the human population, is involved in the cellular immune response against pathogens and also associated to autoimmune spondyloarthritis being thus a relevant target of study. To this end, HLA binding assays performed using nine HLA-B*2705-restricted ligands endogenously processed and presented in virus-infected cells revealed a common minimal peptide motif for efficient binding to the HLA-B*27 family. The motif was independently confirmed using four unrelated peptides. This experimental approach, which could be easily transferred to other HLA class I families and supertypes, has implications for the validation of new bioinformatics tools in the functional clustering of HLA molecules, for the identification of antiviral cytotoxic T lymphocyte responses, and for future vaccine development.     

Identifying HLA supertypes by learning distance functions

MOTIVATION: The development of epitope-based vaccines crucially relies on the ability to classify Human Leukocyte Antigen (HLA) molecules into sets that have similar peptide binding specificities, termed supertypes. In their seminal work, Sette and Sidney defined nine HLA class I supertypes and claimed that these provide an almost perfect coverage of the entire repertoire of HLA class I molecules. HLA alleles are highly polymorphic and polygenic and therefore experimentally classifying each of these molecules to supertypes is at present an impossible task. Recently, a number of computational methods have been proposed for this task. These methods are based on defining protein similarity measures, derived from analysis of binding peptides or from analysis of the proteins themselves. RESULTS: In this paper we define both peptide derived and protein derived similarity measures, which are based on learning distance functions. The peptide derived measure is defined using a peptide-peptide distance function, which is learned using information about known binding and non-binding peptides. The protein derived similarity measure is defined using a protein-protein distance function, which is learned using information about alleles previously classified to supertypes by Sette and Sidney (1999). We compare the classification obtained by these two complimentary methods to previously suggested classification methods. In general, our results are in excellent agreement with the classifications suggested by Sette and Sidney (1999) and with those reported by Buus et al. (2004). The main important advantage of our proposed distance-based approach is that it makes use of two different and important immunological sources of information-HLA alleles and peptides that are known to bind or not bind to these alleles. Since each of our distance measures is trained using a different source of information, their combination can provide a more confident classification of alleles to supertypes.     

Systems-level modeling of neuronal circuits for leech swimming

This paper describes a mathematical model of the neuronal central pattern generator (CPG) that controls the rhythmic body motion of the swimming leech. The systems approach is employed to capture the neuronal dynamics essential for generating coordinated oscillations of cell membrane potentials by a simple CPG architecture with a minimal number of parameters. Based on input/output data from physiological experiments, dynamical components (neurons and synaptic interactions) are first modeled individually and then integrated into a chain of nonlinear oscillators to form a CPG. We show through numerical simulations that the values of a few parameters can be estimated within physiologically reasonable ranges to achieve good fit of the data with respect to the phase, amplitude, and period. This parameter estimation leads to predictions regarding the synaptic coupling strength and intrinsic period gradient along the nerve cord, the latter of which agrees qualitatively with experimental observations.     

Monoclonal rat anti-MHC alloantibodies detectHLA-linked polymorphisms in humans

Two monoclonal rat anti-MHC alloantibodies detect a polymorphic determinant expressed on the peripheral lymphocytes of normal human donors. The pattern of cytotoxicity observed with these antibodies correlated with theHLA type of the individual; no HLA-A-locus specificities showed significant associations, and all of the HLA-B-locus specificities showing significant association were members of the Bw6 supertype. Family studies established that the determinant detected by the monoclonal antibodies is linked toHLA. These studies therefore provide an alternative basis for the production of monoclonal antibodies to polymorphic HLA determinants based on the conservation of polymorphic MHC determinants between man and rats.