Monoclonal antibodies to sheep MHC class I and class II molecules: biochemical characterization of three class I gene products and four distinct subpopulations of class II molecules

The availability of a panel of monoclonal antibodies to sheep MHC class I and class II gene products has allowed for the first time an assessment of the relative complexity of the sheep MHC. By using four monoclonal antibodies to MHC class I, and seven monoclonal antibodies to MHC class II molecules together with one-dimensional SDS-PAGE, sequential immunoprecipitation and 2-dimensional gel analysis, three class I gene products and four distinct subsets of class II molecules have been identified. Sheep class I molecules showed heterogeneity on 2-dimensional gels and as in mouse and man, represented the products of at least three different non-allelic class I genes. Interestingly, the sheep beta 2 microglobulin molecule also displayed heterogeneity, consistent with either two primary gene products or allelic variation. Four sheep class II monoclonal antibodies identified distinct, non-overlapping subsets of sheep class II molecules of Mr 32-36 K (alpha chain) and 25-28 K (beta chain). These class II molecules were co-expressed on sheep B lymphocytes and represented the primary products of different sheep MHC class II genes. The class II molecules within three of these subsets displayed allelic polymorphism essentially restricted to their beta polypeptides, while the fourth subset of class II molecules showed allelic variation in both their alpha and beta polypeptides. The results of this study represent the first evidence for gene duplication and heterogeneity within the sheep MHC. The identification of three primary class I gene products and four distinct subsets of class II molecules suggests three class I loci and up to four distinct class II subregions within the sheep MHC. Potentially large numbers of allelic variants of these different gene products may be expressed in normal sheep.     

Molecular cloning, expression pattern, and 3D structural analysis of the MHC class IIB gene in the Chinese longsnout catfish (Leiocassis longirostris)

Major histocompatibility complex (MHC) class I and class II molecules encode glycoproteins which mediate the specificity of the vertebrate adaptive immune response. In this study, MHC class IIB gene from the Chinese longsnout catfish (Leiocassis longirostris) was cloned and sequenced, which encoded a predicted protein of 248 amino acids (28.06 kDa) containing a signal peptide, a beta 1 domain, a beta 2 domain, a connecting peptide, a transmembrane region, and a cytoplasmic tail. Using PCR with primers designed from known fish MHC class IIB sequences followed by elongation of the 5' and 3' ends using rapid amplification of cDNA ends (RACE), the full-length cDNA of longsnout catfish MHC class IIB was identified to be 1293 bp, consisting of a 26 bp 5'-terminal untranslated region (UTR), a 520 bp 3'-UTR, and a 747 bp open reading frame (ORF) bearing characteristics of the immunoglobulin C-type 1 (IGc1) family. The deduced amino acid sequences of the Chinese longsnout catfish MHC class IIB gene had 58-75% identity with those of other fishes. Six class IIB alleles were identified from five individuals. At most two different alleles observed in each individual may infer the existence of a single locus of class IIB gene in the Chinese longsnout catfish genome. An extensive study of polymorphism was examined in 60 individuals. A total of 11 haplotypes of exon 2 were detected in the sampled Chinese longsnout catfish. The rates of nonsynonymous substitutions (d(N)) occurred at a higher frequency than that of synonymous substitutions (d(S)), suggesting the polymorphism of exon 2 seemed to be maintained by the balancing selection. By using long PCR technique, the genomic sequence was further identified to be 2345 bp in length, which contained six exons and five introns. Interestingly, a 98 bp intron 5 cut the 3'-UTR into two parts. Real-time quantitative RT-PCR demonstrated high expression of MHC IIB in gills, spleen, head kidney, and intestine, moderate expression in liver and stomach, and low or negligible expression in heart. Homology modelling has been applied to create a 3D model of longsnout catfish MHC class IIB, with features evaluated and discussed to investigate its interaction with CD4 participating in antigen recognition. The present findings will provide valuable information for understanding structure, function, expression, and molecular polymorphism of MHC class IIB in adaptive immunity of the Chinese longsnout catfish and teleost.     

Bovine T lymphocyte response to bovine herpesvirus-1: cell phenotypes, viral recognition and acid-labile interferon production

The phenotype of bovine cells that proliferate to bovine herpesvirus (BHV-1) were identified by peanut agglutinin (PNA) and monoclonal antibodies with specificity for Pan T cells (B29), a T cell subset (B24), and an MHC class II (Ia-like) antigen (R-1). PNA+ cells but not PNA- cells separated by fluorescent activated cell sorting responded to BHV-1. Virally-activated T cells expressed MHC class II antigens, and possibly antigen specific receptors for virus. Binding of radiolabeled virus increased six-fold beyond the expected value for activated cells suggesting specificity of the lymphocytes for the virus. Finally, cells that responded to BHV-1 produced high levels of acid sensitive interferon. Taken together these results characterize the phenotypes of bovine lymphocytes that interact with BHV-1.     

Down-regulation of MHC class II receptors of DH82 cells, following infection with Ehrlichia canis

In order to evaluate whether infection with E. canis alters the expression of major histocompatibility complex (MHC) class I and/or MHC class II receptors, and by doing so alters the immune response to the organism, flow cytometry was performed on DH82 cells infected with Ehrlichia canis (90% infection) and on uninfected DH82 cells of the same passage, using anti-canine MHC class I and II antibodies. MHC class II expression was evident in 47.6 and 46.2% (mean 46.9%) of uninfected DH82 cells using two different anti-MHC class II antibodies, while no MHC class II expression was evident in DH82 cells infected with E. canis. The present results indicate that infection of DH82 macrophages with E. canis down-regulates their MHC class II receptors. These results suggest a possible mechanism by which E. canis evades the immune system.     

Identification of two independent MHC class II antigens in a bovine lymphoblastoid cell line

Bovine major histocompatibility complex (MHC) class II antigens were investigated using monoclonal antibodies (MoAbs) with known MHC class II specificities in other species. Thirty-four MoAbs were tested for reactivity with bovine peripheral blood mononuclear (PBM) cells and the bovine lymphoblastoid cell line, BL3, by flow cytometry. Twenty-seven of 31 MoAbs tested, reacted with BL3 cells, and 22 of 25 MoAbs tested with PBM cells were reactive. MoAbs that reacted with BL3 cells were used to immunoprecipitate class II molecules from BL3 lysate labeled with [35S]methionine. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, many MoAbs were found to immunoprecipitate a single band of approximately 31,000 relative mass (Mr). MoAbs yielding successful immunoprecipitations and with known antigen specificity in other species were then used in sequential immunoprecipitations and two dimensional (2-D) non-equilibrium pH gradient electrophoresis (NEPHGE). The HLA-DR specific MoAb H4 and the predominantly HLA-DQ specific MoAb CC11.23 were used to identify the presence of two independent antigens in BL3 cell lysate. These class II molecules consist of alpha and beta chains.     

Class II major histocompatibility complex expression and cell size independently predict survival in canine B-cell lymphoma

Background: Class II major histocompatibility complex (MHC) is an independent predictor of outcome in human B‐cell lymphoma. We assessed class II expression together with other markers for their impact on prognosis in canine B‐cell lymphoma. Hypothesis: Low class II MHC expression, large cell size, and expression of CD34 will predict a poorer outcome in canine B‐cell lymphoma. Expression of CD5 and CD21 on tumor cells also may be associated with outcome. Animals: One hundred and sixty dogs with cytologically confirmed lymphoma. Methods: Patient signalment, treatment type, and flow cytometry characteristics were analyzed for their influence on outcome. A multivariable predictive model of survival was generated using 2/3 of the patients and validated on the remaining 1/3 of the dataset. Results: Class II MHC expression had a negative association with mortality and relapse. Treatment type also influenced relapse and mortality, whereas cell size and patient age was only associated with mortality. CD34, CD21, and CD5 expression was not associated with disease outcome. The constructed model performed variably in predicting the validation group's outcome at the 6‐month time point. Conclusions and Clinical Importance: Low levels of class II MHC expression on B‐cell lymphoma predict a poor outcome, as in human B‐cell lymphoma. This finding has implications for the use of dogs to model human lymphomas. Class II expression, cell size, treatment, and age can be combined to predict mortality with a high level of specificity.     

Characterization of bovine haemopoietic progenitor cells using monoclonal antibodies and fluorocytometry

Monoclonal antibodies against bovine leucocyte cell surface differentiation antigens were used in combination with a fluorescence activated cell sorter to enrich bovine haemopoietic progenitor cells present in bone marrow cell populations prior to in vitro culture. After two sequential centrifugations of the bone marrow cell suspension through Ficoll-Paque, the interface fraction was stained with a cocktail of monoclonal antibodies directed against mature monocytes/macrophages, granulocytes and lymphocytes. Using appropriate electronic window settings on a FACStar Plus, cells with a high 90 degrees light scattering property (granular cells), a low forward light scattering property (erythrocytes and reticulocytes) and cells positive for monoclonal antibodies specific for lineage-restricted leucocyte markers were removed and the negative cell fraction collected. These negatively-selected cells were stained with monoclonal antibodies specific for a pan-leucocyte or a MHC class II marker and the positive cell population was collected in a second sort and subsequently submitted to culture. All erythroid and granulocyte/macrophage colony forming cells expressed MHC class II antigens, as well as the pan-leucocyte antigen. These same progenitors did not bind any of a variety of monoclonal antibodies directed against lineage-specific antigens on lymphocytes, granulocytes or monocytes/macrophages, although they did bind monoclonal antibodies recognizing MHC class I antigens. Between 85% and 91% of the isolated cells seeded were capable of forming erythroid or granulocyte/macrophage colonies within 5 to 10 days, thus increasing the plating efficiency of these cell types in bone marrow populations by at least 60 fold.     

MHC class II expression by follicular keratinocytes in canine demodicosis--an immunohistochemical study

MHC class II proteins present fragments of extra cellular antigen to stimulate CD4(+) T lymphocytes. Aim of this study was the detection of MHC class II antigens on different cutaneous cells in canine demodicosis. Histopathological and immunohistochemical examination of skin biopsies from 44 dogs with demodicosis is reported. The control group consisted of skin biopsies taken from 10 necropsied dogs without obvious skin lesions. The immunohistological assessment of the MHC class II expression revealed MHC class II proteins on different cell types of infiltrating inflammatory cells, i.e. APCs (antigen-presenting cells), macrophages, T lymphocytes and B lymphocytes. The plasma cells, however, only showed expression in 32 (73%) of 44 cases. Generally it was noticeable that most plasma cells but never all of them expressed MHC class II. Neutrophils, mast cells and eosinophils were MHC class II negative. Furthermore, in 39 biopsies (89%) from dogs with demodicosis MHC class II positive follicular keratinocytes were found. The control group did not show MHC class II expression on epithelial cells. Concerning the endothelial cells, a total of 25 biopsies (57%) showed MHC class II expression in which different vascular plexuses were affected by staining. This examination shows that MHC class II expression in the skin of dogs suffering form demodicosis is elevated. Especially the MHC class II expression by follicular keratinocytes seems to be conspicuous. We hypothesize that this is in association with the development and the maintenance of follicular inflammation.     

Detection of protein binding to a glucocorticoid response element-like sequence in a chicken major histocompatibility complex class II promoter

The glucocorticoid response element in gene promoters mediates regulation of gene expression by glucocorticoids. The major histocompatibility (MHC) class II genes, crucial for immunoresponsiveness, are among those modulated by glucocorticoids. A GRE-like sequence has been located in the promoter of a chicken MHC class II promoter. DNase footprinting revealed protein binding by the GRE-like sequence when nuclear extract from chicken T or B cell lines were used. Gel shift assays detected multiple binding activities in the lymphocyte cell lines, but little binding in the macrophage cell line. Relative band intensity differed among the lymphocyte cell lines. By using a mutant GRE oligonucleotide, most of the binding activities were demonstrated to be specific to the GRE. This study suggests a role of the GRE-like sequence in regulating chicken MHC class II genes and provides further evidence for the previously reported influence of glucocorticoids on chicken MHC class II expression which may be the molecular basis of glucocorticoid immunomodulation.     

A single amino acid deletion in the antigen binding site of BoLA-DRB3 is predicted to affect peptide binding

Two bovine MHC class II alleles, BoLA-DRB3*0201 and BoLA-DRB3*3301, contain a three base pair deletion which results in the deletion of a lysine (K beta 65) in the antigen recognition site (ARS). Modelling of BoLA-DRB3*0201 with the conserved lysine K beta 65 and BoLA-DRB3*0201 without K beta 65 indicated that this deletion altered the peptide specificity of the ARS, and may impact on the immune response. To test this hypothesis, the presence of K beta 65 was analysed in a sample of cattle vaccinated with the commercial cattle tick vaccine (TickGARD). Homozygous deletion of K beta 65 was significantly associated with high response to TickGARD (P<0.05). Screening of the TickGARD antigen identified a potential T cell epitope that is recognised better by animals that are homozygous for the K beta 65 deletion. This study provides evidence that changes in the ARS of MHC class II molecules may be associated with the well recognised animal to animal variation in magnitude of vaccine response.     

Influence of age and parity on the distribution of cells expressing major histocompatibility complex class II, CD4, or CD8 molecules in the endometrium of mares during estrus

OBJECTIVE: To evaluate effect of age and parity on distribution and number of cells expressing major histocompatibility complex (MHC) class II, CD4, or CD8 molecules in the endometrium of mares during estrus. ANIMALS: 32 gynecologically healthy mares, categorized as young (3 to 8 years; n = 17) or old (9 to 16 years; 15) and nulliparous (n = 6), nulliparous embryo donors (16), or parous (10). PROCEDURES: Endometrial specimens collected from the uterine body and horns during estrus were stained by use of the avidin-biotin-peroxidase method, using monoclonal antibodies against equine MHC class II, CD4, and CD8 molecules. Labeled cells in the stratum compactum within 5 randomly selected fields at 400x magnification (total area = 0.31 mm2) were counted, and numbers were compared among groups and between locations. RESULTS: Age did not affect cell numbers within the 3 cell subsets examined. Numbers in each subset were higher in the uterine body than in the horns, although the difference was not significant for cells expressing MHC class II. Significantly more cells expressing MHC class II molecules were detected in the uterine body of nulliparous and parous mares than in embryo donors, whereas in the horns, these cells were significantly higher in number only in parous mares. Parity did not affect number of CD4+ or CD8+ cells. CONCLUSIONS AND CLINICAL RELEVANCE: The increased likelihood for endometritis to develop in mares as they age cannot be explained by a decrease in number of cells expressing MHC class II, CD4, or CD8 molecules within the endometrium. However, greater number of cells within these 3 subsets detected in the uterine body, compared with the horns, during estrus suggests a local readiness to act against microorganisms or semen introduced during mating or insemination.     

Requirement for MHC class II positive accessory cells in an antigen specific bovine T cell response

Purified populations of bovine antigen presenting cells (APCs) and T cells have been isolated from peripheral blood and characterised using various monoclonal antibodies (mAbs) for cell surface markers. Bovine APCs were found in an adherent cell fraction and were non-specific esterase positive, phagocytic and expressed bovine major histocompatibility complex (MHC) class II determinants, all of which are typical macrophage characteristics. T cells were rigorously depleted of accessory cell function before being used in an antigen presenting cell assay. The generation of T helper cells in response to the soluble antigen, ovalbumin, was entirely dependent upon a critical number of APCs. Further the proliferative response was inhibited by several mAbs to bovine MHC class II molecules. Thus the interaction between bovine APCs and helper/inducer T lymphocytes (TH/I) appears to be similar to that in other species.     

Upregulation of major histocompatibility complex class II antigens in hepatocytes in Doberman hepatitis

Major histocompatibility complex (MHC) class II antigen expression in hepatocytes and its correlation with mononuclear cell infiltration into the liver were studied using immunohistochemical techniques in 38 Dobermans with Doberman hepatitis (DH). Liver biopsy samples were obtained from 18 dogs at the subclinical stage. Autopsy samples were taken from 6 DH dogs euthanized for a reason other than DH, from 14 dogs euthanized because of advanced liver failure and from 6 control Dobermans. Upon examination of the control liver samples, no expression of MHC class II antigens was detected in hepatocytes. By contrast, in 15 of the 18 DH biopsies (83%) and in all 20 DH autopsy liver samples, hepatocytes expressed MHC class II molecules. MHC class II expression was either cytoplasmic or membranous and occurred in conjunction with lymphocyte infiltration. A correlation between the inflammatory reaction and the expression of MHC class II in hepatocytes suggests that the aberrant expression of MHC class II in hepatocytes is induced by cytokines. Hepatocytes presenting a putative MHC class II molecule-associated autoantigen could thus become the target of an immune attack mediated by CD4+ T cells. In addition, corticosteroid treatment was observed to significantly decrease MHC class II expression in DH hepatocytes. Inappropriate MHC class II expression in hepatocytes and mononuclear cell infiltration are suggesting an autoimmune nature for chronic hepatitis in Dobermans.     

Immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors

Immunohistochemical and histochemical stains are useful adjunct techniques in the diagnosis of canine cutaneous round cell tumors, which can appear histologically similar. We applied a panel of monoclonal antibodies (recognizing tryptase, chymase, serotonin for mast cells; CD1a, CD18, MHC class II for histiocytes; CD3 for T lymphocytes; CD79a for B lymphocytes and plasma cells) and one histochemical stain (naphthol AS-D chloroacetate for chymase activity) to formalin-fixed, paraffin-embedded sections of canine cutaneous mast cell tumors, histiocytomas, lymphosarcomas, plasmacytomas, and unidentified round cell tumors. Of 21 tumors with a histologic diagnosis of mast cell tumor, 7/7 (100%) grade I, 6/7 (85.7%) grade II, and 3/7 (42.9%) grade III tumors were diagnosed as mast cell tumors based on positive staining for tryptase antigen and chymase activity. Mast cells were positive for both tryptase antigen and chymase activity, indicating equal efficacy of tryptase immunohistochemistry and chymase histochemistry. Chymase was detected immunohistochemically in both tumor and nontumor cells, while serotonin was not detected in most mast cell tumors, and thus, neither was useful in the diagnosis of mast cell tumors. Immunohistochemistry to detect CD18 and MHC class II was equally effective in staining histiocytomas, although lymphosarcoma must be ruled out through the use of CD3 and CD79a immunohistochemistry. Immunohistochemistry using three different monoclonal antibodies to human CD1a showed no cross-reactivity in canine histiocytomas and was not useful. A final diagnosis was obtained for 4/5 (80%) of the unidentified tumors, indicating the usefulness of multiple stains in poorly differentiated round cell tumors.     

Immunophenotype of pulmonary cellular infiltrates in sheep with visceral caseous lymphadenitis

Pulmonary lesions associated with Corynebacterium ovis were analyzed with an indirect immunoperoxidase staining technique using monoclonal antibodies. The predominant cells in abscess walls and surrounding lung parenchyma were large macrophages which expressed major histocompatibility complex (MHC) class II molecules on their surfaces. T lymphocytes were prominent in the same sites in the naturally occurring lesions, and SBU-T4-positive ("helper/inducer") cells were the major subset of lymphocytes (mean T4/T8 ratio = 3.5). B lymphocytes and granulocytes comprised minor populations of infiltrating cells. These results implicate activated macrophages and MHC class II-restricted T lymphocytes in the pathogenesis of established C. ovis infections in sheep.     

Use of Monoclonal Antibodies for Immunohistochemical Study of Bovine Spleen on Frozen Sections

Many monoclonal antibodies reactive with bovine leukocyte differentiation antigens are now available. Immunohistochemical staining on frozen sections using these monoclonal antibodies permits study of the functional morphology of bovine spleen. This study confirms accepted notions (B and T dependent-zones) and supplies complementary data about the repartition of CD4 and CD8 cells. γδ cells. MHC (Major Histocompatibility Complex) II expression, and macrophages.     

Major histocompatibility class II expression in the normal canine cornea and in canine chronic superficial keratitis

OBJECTIVE: To compare the expression of major histocompatibility complex (MHC) class II antigen in the corneas of normal dogs and dogs affected with chronic superficial keratitis (CSK). METHODS: MHC class II expression was determined in frozen sections of normal canine cornea and cornea from lesions of CSK by immunohistochemistry using a monoclonal antibody directed against the canine MHC class II molecule. Langerhans cell phenotype was determined morphologically and by histochemical determination of ATPase activity. To determine the influence of gamma interferon on expression of MHC class II molecules by corneal cells, corneal explants were cultured with the cytokine and MHC class II expression determined as above. RESULTS: Numerous MHC class II-expressing cells were demonstrated within the stroma and epithelium of the normal corneal limbus and conjunctival epithelium while very little MHC class II expression was detected in the central region of normal canine cornea. In limbal and conjunctival epithelium, cells expressing MHC class II antigen showed ATPase activity, suggesting that they were Langerhans cells. Corneas from dogs with CSK showed MHC class II expression associated with stromal cells, some of which exhibited a dendritic morphology while most were lymphocytic. Corneal epithelial cells within the lesion also aberrantly expressed MHC class II. Corneal explants expressed MHC class II to varying degrees after differing periods of incubation with the cytokine gamma interferon. CONCLUSIONS: While the normal central cornea has little MHC class II expression, aberrant expression occurs in CSK, associated with secretion of gamma interferon by infiltrating CD4-expressing lymphocytes. Although this change is likely to be a secondary feature of the CSK lesion, increased MHC class II expression may play a part in perpetuating the corneal inflammation seen in the disease.     

Immunohistochemical evaluation of mononuclear infiltrates in canine lupoid onychodystrophy

Claw biopsy samples of 11 dogs with lupoid onychodystrophy were evaluated. They were stained with hematoxylin and eosin and with antibodies against CD 3 as a T-cell marker, BLA 36 and HM 57 (CD 79alpha) as B-cell markers, and lysozyme, Mac 387, and major histocompatibility complex (MHC) class II as a marker for histiocytes using an immunoperoxidase and avidin-biotin technique. Inflammatory cells were counted in five high-power fields. The inflammatory infiltrate comprised predominantly B cells and T cells. Macrophages were typically only present in small numbers. CD 3, BLA 36, lysozyme, and MHC class II preserved significant antigenicity during formalin fixation and short decalcification for 24-48 hours, whereas CD 79alpha and particularly Mac 387 seemed to be more susceptible to denaturation by the decalcification process.     

Characterisation of ovine alveolar macrophages: regulation of surface antigen expression and cytokine production.

Regulation of ovine alveolar macrophage function by recombinant interferon gamma (rIFN gamma) and lipopolysaccharide (LPS) was investigated. Ten units per millilitre of rIFN gamma increased surface expression of MHC class I and class II (DR alpha, DP alpha, and DQ alpha) molecules but not other surface antigens examined. The upregulation of MHC class II expression was specifically blocked by rIFN gamma specific monoclonal antibodies and determination of a dose/response curve established that the minimum concentration of rIFN gamma required for increased class II expression was 0.1 U ml-1 and for increased class I expression, 1 U ml-1. Northern blot analysis indicated that rIFN gamma mediated increases in surface MHC class I and class II expression were due to increased levels of specific mRNA. Using Northern blot analysis and homologous human cDNA probes we failed to detect mRNA encoding the cytokines IL-1 alpha, IL-1 beta, and TNF alpha in RNA extracted from freshly isolated macrophages or macrophages cultured in medium alone. Exposure of macrophages to LPS increased production of all three cytokines although kinetics of upregulation varied. TNF alpha mRNA was induced to maximal levels within 1 h, declining thereafter. IL-1 alpha mRNA was detected at 1 h post stimulation with a maximal level at 5 h, but none at 24 h. In contrast, IL-1 beta mRNA was not detected until 5 h after stimulation with a low level remaining at 24 h. Dose response analysis indicated that LPS concentrations of 100 pg ml-1 induced detectable levels of TNF alpha mRNA while levels as low as 10 pg ml-1 induced secretion of bioactive IL-1. Analysis of the kinetics of secretion of bioactive IL-1 from LPS stimulated macrophages indicated that levels peaked at 24 h post stimulation.