Dissection of the D-region of the human major histocompatibility complex by means of induced mutations in a lymphoblastoid cell line

This paper describes part of a mutagenic dissection of the human D-region. Twenty-six human lymphoblastoid cell mutants that had lost expressions of HLA-DR were created with a two-step procedure: (i) A mutant from which one entire haplotype had been physically deleted by gamma-rays was isolated by means of immunoselection against cells expressing a specific HLA-B antigen. (ii) This heterozygous deletion mutant was irradiated with gamma-rays or treated with ICR 191, a frameshift mutagen, and mutants that no longer expressed the remaining DR1 antigen were selected with a monoclonal antibody directed against a monomorphic DR determinant. Monoclonal antibody GENOX 3.53 was used to show that four of the gamma-ray induced DR-null mutants did not express the cis-linked MB1/MT1 locus. Since MB1/MT1 was still expressed in the other 16 gamm-ray induced and 6 ICR 191-induced DR-null mutants, the separate loss of expression of MB1/MT1 and DR1 is strong evidence that the DR1 and MB1/MT1 alloantigens are under separate genetic control in the cells we used. Since DR-null mutants bound SB2-specific monoclonal antibody ILR1, whether or not they expressed MB1/MT1, the results mean that gamma-rays resolved the genetic determinants for DR1, MB1/MT1, and SB2. Additional complexity of determinants encoded by D-region genes is indicated by the following results. The amount of MB1/MT1 antigen that was detected with ELISA tests for binding of GENOX 3.53 antibody to cells varied inversely with the number of expressed copies of DR or of a locus near DR. This could result from an increased amount of MB1/MT1 antigen or from increased binding accessibility of GENOZ 3.53-reactive antigen in DR-null mutants. Monoclonal antibodies CC 11.23 and CC 6.4 displayed patterns of binding to parental and diverse mutant cells that differed from that of GENOX 3.53, suggesting the existence of at least one additional D-region antigen that is neither SB, DR, nor MB/MT.     

Genetics and mode of presentation of antigenic determinants: influence on immunologic responsiveness in transplantation

The role of specialized antigen-presenting cells in the generation of alloreactions is discussed. Methods to bypass the stimulation of effector cells reactive to alloantigens and which might be used to activate suppressor T cells responsive to class I and II molecules are described. These nations are considered in the context of recent observations in cellular immunology.     

Localization of the recombination points in a family with two DR/DP recombinations

In a family with a maternal DR/GLO recombination, cellular DP typing showed it to be located between DR and DP. RFLP studies done during the 9th international histocompatibility workshop gave anomalous segregation patterns of DPA and DPB bands that could be interpreted as being due to a second, paternal DR/DP recombination. This assumption was confirmed later by PCR-SSO typing. A more precise mapping has been done by new markers showing the maternal recombination to be within the TAP2 locus and the paternal recombination to be between DQB1 and DQB3. This supports earlier suggestions of a hot spot of recombination in the TAP region. The recombinations involve parental haplotypes that presently show DR/DP linkage disequilibrium in the French population and it is proposed that DR/DP recombinations occur randomly while B/DR recombinations preferentially occur on haplotypes without strong linkage disequilibrium. Existing DR/DP linkage disequilibria in a given population will thus be broken down with time. The mixed lymphocyte culture response towards an isolated DP difference was tested in this and another DR/DP recombinant family. It showed that an alloresponse towards DP may be highly variable and this suggests that it might be important to define the rules for the strength of this reaction and the possible implications for allotransplantation.     

Molecular genetic studies on DNA polymorphism of the HLA class II genes associated with human longevity

Okinawan Japanese are well known for their longevity; the population rate of centenarians in Okinawa is about 3.8 times higher than that of the whole Japan, where the average life expectancies both among men and among women are the highest in the world. In this study, we analyzed HLA class II alleles of Okinawan centenarians by the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method for the purpose of clarifying the presence of primary genetic factors in the major histocompatibility complex (MHC) region associated with human longevity. DRB1*1401, DQB1*0503, DQA1*0101=0104 and DQA1*05 were significantly increased in the centenarians. The significant increase of HLA-DQB 1*0503 and/or DQA 1*0101=0104 in the centenarians can be explained by a linkage disequilibrium with DRB 1*1401, or vice versa. Further, the tendency was observed toward increase with respect to DRB 1*0101 and DRB1*1201. These data suggest that several alleles of the HLA-DRB1 and/or HLA-DQ genes are involved in human longevity.     

On a key postulate of T-cell receptor restrictive function: the V-gene loci act as a single pool encoding recognition of the polymorphic alleles of the species major histocompatibility complex

The proposition that single Valpha or Vbeta gene segments specify the recognition of the allele-specific determinants expressed on the major histocompatibility complex-encoded restricting elements of the species has as its consequence a totally different picture of the functioning of the T-cell receptor. This commentary justifies this assumption and outlines some of its most important consequences.     

DM influences the abundance of major histocompatibility complex class II alleles with low affinity for class II-associated invariant chain peptides via multiple mechanisms

DM catalyses class II-associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation-sensitive anti-class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles. Evidence from analysis of cellular class II levels after cycloheximide treatment and from pulse-chase experiments indicates that DM increases the half-life of affected alleles. Unexpectedly, the pulse-chase experiments also revealed an early effect of DM on assembly of these alleles. The allelically variant feature that correlates with susceptibility to these DM effects is low affinity for CLIP; DM-dependent changes in abundance are reduced by invariant chain (CLIP) mutants that enhance CLIP binding to class II. We found evidence that DM mediates rescue of peptide-receptive DR0404 molecules from inactive forms in vitro and evidence suggesting that a similar process occurs in cells. Thus, multiple mechanisms, operating along the biosynthetic pathway of class II molecules, contribute to DM-mediated increases in the abundance of low-CLIP-affinity alleles.     

An HLA-DR11/DQ3 haplotype with a DRB1*0301 sequence motif in the third hypervariable region of the HLA-DR beta-1 chain: molecular and serological analysis of its generation in a European Caucasian family

The formation of a new human leukocyte antigen (HLA)-DRB1 allele (DRB1*0340) has been detected during the routine testing of a European Caucasian blood and potential stem cell donor and his family. HLA typing of the donor with two polymerase chain reaction - sequence specific oligonucleotides (PCR-SSO) systems yielded inconclusive results. HLA typing of the family members including sequence-based typing of DRB1 in both directions after haplotype-specific amplification showed that the allele had most likely formed by a double crossover event in exon 2 of the DRB1 gene. The HLA haplotype containing the new allele was most probably derived from the father, who was typed as HLA-DRB1*0301,*1101 and DRB3*0101,*0202. The comparison of the sequences of the paternal DRB1 and DRB3 alleles with the exon 2 sequence of the DRB1*0340 showed that it had most likely formed through an uptake of at least the sequence part codons 58–77 of DRB1*0301 (donor) by DRB1*1101 (acceptor). We suppose that the recombination sites are located in the sequences from codons 38–57 and codons 78–88. At the protein level, more than 50% of the alpha-helical structure of the DRB1*1101 chain is replaced by a DRB1*0301-derived sequence with the exchange of several amino acids. Serological typing of the allele showed HLA-DR3. However, one monoclonal anti-DR11 of five DR11-reactive antibodies reacted positive, which might indicate residual immunogenic epitopes of DRB1*1101. HLA alleles that are most similar to HLA-DRB1*0340 are DRB1*030501, *0317, *0329 and *1107 with at least four amino acid differences in exon 2. In conclusion, HLA-DRB1*0340 is a new allele with unique properties compared with other known HLA-DRB alleles with regard to antigenicity, T-cell receptor-binding and peptide-binding possibilities.     

Enhancement of thyroid allograft survival following organ culture: II. Induction of Recipient Peripheral Tolerance

Hyperbaric oxygen culture (HOC) prolongs endocrine graft survival and decreases major histocompatibility complex (MHC) class I surface expression. If graft prolongation were the result of passenger cell inactivation and decreased class I expression, then simultaneous transplantation of both a nontreated and a HOC-treated graft should result in rejection of the nontreated graft and acceptance of the HOC-treated graft. Simultaneous transplant of a nontreated and a HOC-treated thyroid allograft beneath the kidney capsule of recipient mice resulted in prolonged survival of both grafts in three strain combinations differing at class I (K^K , D^d, D^b). In vitro analysis of the recipient splenic population revealed the presence of primed donor-specific cytotoxic T cells. These results suggest that recipient tolerance was not because of anergy or clonal deletion. Splenectomy at the time of transplant, revealed that both graft prolongation and the induction of recipient tolerance were dependent on the spleen. Finally, analysis of graft infiltrating cells reveals the presence of CD8⁺ cells but no CD4⁺ cells in tolerant recipients, whereas graft infiltrating cells from rejecting recipients contained both populations. The results suggest that active peripheral tolerance can be generated following transplantation of a HOC-treated allograft and that tolerance results from redirection of the recipients immune response.     

Characterization of a monoclonal anti-Bw4 antibody (Tü109): Evidence for similar epitopes on the Bw4 and Bw6 antigens

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tü109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tü109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tü109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tü109 ascites fluid, however, extra-reactivity on all HLA-Bw6⁺ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6⁺ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tü109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.     

Integration of microsatellite characteristics in the MHC region: a literature and sequence based analysis

Reviews of microsatellite markers in the human leukocyte antigen region have been very useful in addressing the needs of the immunogenetics community. Nevertheless, characterization of the same microsatellite loci in different laboratories can lead to seemingly contradictory results, particularly in terms of nomenclature. Here we provide an update of previous reports, as well as a standardized characterization of primers for microsatellites located within the major histocompatibility complex (MHC). A uniform and extended inventory of 378 primer pairs from published reports was performed as well as a standardized characterization of the corresponding microsatellite loci according to the extended full-length consensus sequence of MHC region. The literature-based approach was complemented by a sequence-based analysis of each reported microsatellite locus. Iterative electronic polymerase chain reaction runs and an original algorithm that characterizes patterns of repeats within sequence were used. The sequence of primers was corrected according to the consensus sequence. Table of synonymous names for individual microsatellite loci is provided.     

Cell surface glycoproteins of rabbit lymphocytes: characterization with monoclonal antibodies

The structural characteristics of antigens recognized by a panel of monoclonal antibodies prepared against a rabbit T-lymphocyte cell line have been investigated. Those antigens which could be isolated using immunoadsorbents prepared from the monoclonal antibodies had mol. wts of 42,000, 90,000 and 120,000. The 42,000 mol. wt molecule is similar or identical to a rabbit class I major histocompatibility complex antigen and its characterization has been reported elsewhere. Three different 90,000 mol. wt proteins can be distinguished by their reactivity with lectins and by sequential immunoprecipitation. The 120,000 mol. wt protein is a very abundant surface glycoprotein that appears to be a specific marker for T-cells in the rabbit. It is the immunodominant antigen in a lentil lectin bound glycoprotein pool. Over half of the antibodies were directed against this antigen. All antigens detected by the panel of monoclonal antibodies have been detected on normal lymphoid cells.     

Absence of the OKT4 epitope on blood T cells and thymus cells in a patient with thymoma, hypogammaglobulinemia, and red blood cell aplasia

Human helper/inducer T-lymphocytes that express the T4 antigen are important in the regulation of B and T cell functions. Several epitopes of the T4 molecule have now been recognized; however, the precise role of these molecules in the function of helper/inducer T cells is unclear. We studied a patient with thymoma, hypogammaglobulinemia, and red blood cell aplasia whose blood lymphocytes and thymus cells did not express the epitope recognized by OKT4 monoclonal antibody but did display the T4 epitopes recognized by OKT4A and Leu3A monoclonal antibodies. The absence of the OKT4 epitope on the patient's thymus cells suggested that the abnormality occurred during early T cell differentiation. The patient had intact delayed hypersensitivity to 4/4 antigens, and his blood lymphocytes proliferated normally to phytohemagglutinin, concanavalin A, pokeweed mitogen, tetanus toxoid, and allogeneic cells. The patient's T cells demonstrated augmented suppressor activity that was localized to the OKT8+ population rather than to the unusual T4 subset. Irradiation abrogated suppressor activity and rendered his T cells capable of providing help for polyclonal B cell differentiation. The data emphasize the limitations of OKT4 as the sole reagent for characterizing the subset of human helper/inducer cells and demonstrate that the expression of the T4 epitope recognized by OKT4 monoclonal antibody is not required for certain helper/inducer T cell functions in vitro and in vivo.     

Biochemical characterization of a p43,12 complex: comparison with human and murine class I molecules

A monoclonal antibody designated as C21 reacting with a p43,12 complex was developed against human thymocytes. It stained predominantly the early hematopoietic cells of the lymphoid lineage and also thymocytes, peripheral B-cells and activated T- and B-cells similarly to OKT10. The heavy chain of this antigen was a glycoprotein of Mr 43,000 (p43). Sequential immunoprecipitation with C21 and OKT10 antibodies indicated that they both reacted with an identical heavy-chain molecule. This observation was further documented by two-dimensional analysis. Monoclonal antibody C21 was used to probe a p43,12 complex further. Structural polymorphism of the p43 heavy chain isolated from T- and B-cells of different individuals was not detected by chymotryptic peptide mapping, although molecules from these cell types possessed a different charge on two-dimensional gels. An unusual observation was made regarding this complex on MOLT4 cells. The light chain co-precipitated from these cells was 12,000 daltons and had a pI distinct from that of beta 2-microglobulin but similar to the pI of the beta t molecule. Comparison between chymotryptic peptide maps of the p43 heavy chain and those of the human and murine class I molecules such as HLA, T6, H-2K, Qa-2 and TL revealed no apparent homology. We have shown, however, that the peptide backbone of p43, as studied by both tunicamycin treatment of cells and endoglycosidase F digestion of immunoprecipitates, was identical in size to that of murine Qa-1. These results suggest that the p43 antigen may be homologous to murine Qa-1 or another class I antigen encoded in the murine TL:Qa region.     

Gluten-sensitive enteropathy coincides with decreased capability of intestinal T cells to secrete IL-17 and IL-22 in a macaque model for celiac disease

Celiac disease (CD) is an autoimmune disorder caused by intolerance to dietary gluten. The interleukin (IL)-17 and IL-22 function as innate regulators of mucosal integrity. Impaired but not well-understood kinetics of the IL-17/22 secretion was described in celiac patients. Here, the IL-17 and IL-22-producing intestinal cells were studied upon their in vitro stimulation with mitogens in class II major histocompatibility complex-defined, gluten-sensitive rhesus macaques. Pediatric biopsies were collected from distal duodenum during the stages of disease remission and relapse. Regardless of dietary gluten content, IL-17 and IL-22-producing cells consisted of CD4 + and CD8 + T lymphocytes as well as of lineage-negative (Lin −) cells. Upon introduction of dietary gluten, capability of intestinal T cells to secrete IL-17/22 started to decline (p < 0.05), which was paralleled with gradual disruption of epithelial integrity. These data indicate that IL-17/22-producing cells play an important role in maintenance of intestinal mucosa in gluten-sensitive primates.     

HLA-inferred extended haplotype disparity level is more relevant than the level of HLA mismatch alone for the patients survival and GvHD in T cell-replate hematopoietic stem cell transplantation from unrelated donor

Serious risks in unrelated hematopoietic stem cell transplantation (HSCT) including graft versus host disease (GvHD) and mortality are associated with HLA disparity between donor and recipient. The increased risks might be dependent on disparity in not-routinely-tested multiple polymorphisms in genetically dense MHC region, being organized in combinations of two extended MHC haplotypes (Ehp). We assessed the clinical role of donor-recipient Ehp disparity levels in N?=?889 patients by the population-based detection of HLA allele phase mismatch. We found increased GvHD incidences and mortality rates with increasing Ehp mismatch level even with the same HLA mismatch level. In multivariate analysis HLA mismatch levels were excluded from models and Ehp disparity level remained independent prognostic factor for high grade acute GvHD (p?=?0.000037, HR?=?10.68, 95%CI 5.50–32.5) and extended chronic GvHD (p?<?0.000001, HR?=?15.51, CI95% 5.36–44.8). In group with single HLA mismatch, patients with double Ehp disparity had worse 5-year overall survival (45% vs. 56%, p?=?0.00065, HR?=?4.05, CI95% 1.69–9.71) and non-relapse mortality (40% vs. 31%, p?=?0.00037, HR?=?5.63, CI95% 2.04–15.5) than patients with single Ehp disparity. We conclude that Ehp-linked factors contribute to the high morbidity and mortality in recipients given HLA-mismatched unrelated transplant and Ehp matching should be considered in clinical HSCT.