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.     

Analysis of the CD1 cluster in sheep.

The anti-CD1 monoclonal antibodies submitted to the 1st International Workshop on Leucocyte Differentiation Antigens of Cattle, Sheep and Goats were tested for their reactivity on sheep skin, thymus and lymph node and for their reactivity with sheep efferent and afferent lymph and peripheral blood. With the exception of 20-27 they all stained that same cell populations. The antibodies precipitated molecules with a heavy chain of 46,000 apparent molecular weight and a light chain of 14,000 apparent molecular weight. VPM5 and CC14 antigens were purified by affinity chromatography. All the antibodies cross-reacted with these molecules. The results show that 20-27 recognises the same molecules as the other antibodies and suggest that 20-27 is a pan CD1 monoclonal antibody and the other monoclonal antibodies are homologues of the human CD1b molecules.     

Distribution of MHC-II and CD1 molecules in the skin of lambs and changes during experimentally-induced contact hypersensitivity

The presentation of antigen to specific T-cell populations is a crucial event during the elicitation phase of contact hypersensitivity (CHS). Significant changes in CD4(+) T-cell and gammadelta T-cell populations occur in the skin of sheep 48h after re-exposure to dinitrochlorobenzene but the expression of antigen presentation molecules such as MHC-II and CD1 at this stage of the hypersensitivity response has not been investigated. In the present study, a panel of monoclonal antibodies recognising CD1 and MHC-II subtypes was used in combination with computer assisted morphometric analysis to estimate the distribution of antigen presentation molecules in the superficial and deep dermis of the ears of lambs during the elicitation phase of CHS. The MHC-II molecules showed predominantly a perivascular and peri-appendageal distribution in the dermis and there were scattered MHC-II(+) cells in the basal and suprabasal layers of the epidermis. The CD1w2(+) (CD1b-like) molecules were present on distinct cells that were scattered evenly through the dermis, whereas CD1w3(+) (CD1c-like) molecules were almost exclusively detected on or in close association with the vascular endothelium. There was a significant increase in the presence of MHC-DQ(+) cells in the superficial dermis of dinitrochlorobenzene-treated animals compared with both an untreated control group and a vehicle-treated control group. However, MHC-DQ/DR(+) and CD1w3(+) cells only showed a significant increase compared with the vehicle-treated control group. The present study shows that the distribution of molecules involved in antigen presentation to CD4(+) T-cells and gammadelta T-cells changes during the elicitation phase of CHS in sheep, and suggests a role for MHC-DQ molecules on antigen presenting cells. However, the changes in distribution and expression of MHC-II and CD1 subtypes argue against a prominent role for a CD1-dependent pathway for T-cell recognition in the clinical cutaneous hypersensitivity response in sheep. Based on the expression of MHC-II molecules and CD1c molecules, we also suggest a potential role for endothelial cells in antigen presentation during the clinical dermatitis reaction.     

Production and characterization of monoclonal antibodies specific for sheep IgG subclasses IgG1 or IgG2

Monoclonal antibodies specific for sheep IgG subclasses IgG1 or IgG2 were produced using conventional cell fusion techniques. Monoclonal antibodies detected by preliminary screening assays were further characterized and their specificity verified by titration of ascites in radioimmunoassay or passive haemagglutination using pure sheep IgG1 or IgG2. Further evidence for the subclass specificity of the antibodies was obtained from immunoelectrophoretograms of sheep serum or purified proteins developed with monoclonal antibodies. Reaction of monoclonal antibodies with various IgG fragments showed that the determinants recognised were located on the pFc' portion of the heavy chain.     

Lymphocyte antigens of sheep: identification and characterization using a panel of monoclonal antibodies

A panel of monoclonal antibodies has been developed and used to identify and characterize the lymphocyte antigens of sheep. These studies have shown that sheep lymphocyte antigens display similar, if not identical, tissue distributions to their analogues in other species. Some of the major sheep antigens, including CD5, CD4, CD8, SBU-T19, Pgp-1, LCA and the MHC antigens, are described in detail.     

Transfection of genes encoding lymphocyte differentiation antigens: applications in veterinary immunology

The work conducted so far in this laboratory has demonstrated the application of the use of genes encoding lymphocyte differentiation molecules, in the isolation of homologous genes from other mammalian species, by the technique of cross-species DNA hybridization. The studies have also highlighted the use of transfection as a means of obtaining expression of genes, either from total genomic DNA or cloned in plasmids, which encode lymphocyte antigens. Preliminary work presented in this paper demonstrates the application of these technologies in the isolation and expression of genes for lymphocyte antigens from species in which the gene products have not been fully defined. We favour this approach because it may allow isolation and definition of important immunological molecules independently of the existence of specific antibodies. It therefore seems the most direct way to avoid the frustrating randomness in production of anti lymphocyte subset-specific monoclonal antibodies, and to shorten the time and effort needed to define the specificities of such reagents. Furthermore, the cDNA clones isolated from alternate species (in this case the bovine) have a use in classical immunological studies apart from the application of antibodies made to their products in veterinary immunology. That is, comparisons of the DNA sequences of lymphocyte differentiation antigens from different species provide much important information about structural or functional elements of evolutionarily conserved proteins involved in generation of immune responses.     

Analysis of serum and lymphocyte surface IgM of healthy and immunodeficient horses with monoclonal antibodies

Nine monoclonal antibodies which reacted with equine immunoglobulin (Ig)M and not other equine Ig and serum proteins were prepared. Cells producing antibodies (C 1.9) which precipitated with IgM and bound to staphylococcal protein A were triple-cloned (C 1.9/3.2) and the antibodies further characterized. Monoclonal antibody C 1.9/3.2 reacted with an IgM determinant present on serum IgM from horses of several breeds. Studies with 125I-labeled IgM revealed the presence of this determinant on all IgM molecules. The monoclonal antibody enabled quantitation of IgM in presuckling foal and adult horse sera, using rocket electrophoresis. This procedure was used because presumably it gives a positive precipitation reaction over a wide range of antigen-antibody ratios. The C 1.9/3.2 monoclonal antibody recognized an exposed mu-chain determinant on live B lymphocytes, as determined by immunofluorescence. Also, IgM-containing cells could be identified in acetone-fixed frozen sections of lymphoid tissue. Sera from several other species carry the determinant identified by C 1.9/3.2, suggesting that the reagent may be useful for IgM studies in other species.     

Porcine CD5 gene and gene product identified on the basis of inter-species conserved cytoplasmic domain sequences

Analysis of published CD5 amino acid sequences identified conserved sequences with potentially immunogenic epitopes. To obtain anti-porcine CD5, synthetic peptides representing conserved sequences identified in mouse, human, cattle and sheep CD5 cytoplasmic tail domains were linked to KLH and used to immunize rabbits. Anti-synthetic peptide serum reacted with an antigen extracted from porcine lymphocyte membrane which was consistent in size (67 kDa) with CD5. Murine monoclonal anti-porcine wCD5 (b53b7) and the anti-CD5 synthetic peptide serum react with the same ligand confirming that porcine wCD5 has conserved amino acid sequences similar to those of CD5 of several species. Analysis of porcine genome for CD5 gene sequences by PCR was conducted to verify the presence of CD5-like genes. Oligomeric primers were designed to identify CD5-like sequences by polymerase chain reaction in pigs and other species. Amplified DNA similar in size to that predicted for CD5 elements were amplified from a variety of animal genomes including that of pig. The porcine-derived fragment was cloned and shown to be 96% similar to mouse CD5. The use of published CD sequences for prediction of immunogenic peptides has provided a complimentary alternative to the more traditional approaches to production of CD-specific antibodies.     

Kidney-derived proteins in urine as biomarkers of induced acute kidney injury in sheep

Acute kidney injury (AKI) is a life-threatening condition for which an early diagnosis is problematic. The aim of the present study was to identify kidney-derived urinary proteins specific to AKI in sheep. AKI was induced in six sheep by an overdose of ketoprofen. Six untreated sheep served as controls. Urine samples were collected for up to 24 h after drug administration and pooled according to time and treatment. Tissue samples from kidney were taken immediately after euthanasia. Urinary proteins were separated by two-dimensional gel electrophoresis (2DE) and the proteins of interest were identified by mass spectrometry. Calbindin-D28k, retinol-binding protein 4 and CD1d were identified in ketoprofen-treated sheep, but not in controls. In addition, calbindin-D28k and CD1d were localized in kidney tissues by immunohistochemical staining. These preliminary results suggest that urinary calbindin-D28k and CD1d represent potential useful biomarkers of AKI, at least in sheep.     

Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspecies comparative immune system analyses. This study describes cross-reactive mAbs that recognize leukocyte differentiation molecules (LDMs) and major histocompatibility complex antigens on DS and BHS leukocytes. Characterization of multichannel eosinophil autofluorescence in this study permitted cell-type specific gating of granulocytes for evaluating LDMs, specifically on neutrophils, by single-label FC. Evaluation of relative abundances of LDMs by flow cytometry revealed greater CD11a, CD11b, CD18 (β₂ integrins) and CD 172a (SIRPα) on DS neutrophils and greater CD14 (lipopolysaccharide receptor) on BHS neutrophils. Greater CD25 (IL-2) was identified on BHS lymphocytes following Concavalin A stimulation. While DS and BHS have similar total peripheral blood leukocyte counts, BHS have proportionately more neutrophils.     

Cloning and comparison of bighorn sheep CD18 with that of domestic sheep, goats, cattle, humans and mice

Previously, we have shown that CD18, the beta-subunit of beta(2)-integrins, serves as a receptor for leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica on bovine leukocytes. Anti-CD18 monoclonal antibodies (mAbs) inhibit Lkt-induced cytolysis of bighorn sheep (Ovis canadensis) leukocytes suggesting that CD18 may serve as a receptor for Lkt on the leukocytes of this species as well. Confirmation of bighorn sheep CD18 as a receptor for Lkt, and elucidation of the enhanced Lkt-susceptibility of bighorn sheep polymorphonuclear leukocytes (PMNs), necessitates the cloning and sequencing of cDNA encoding bighorn sheep CD18. Hence, in this study we cloned and sequenced the cDNA encoding CD18 of bighorn sheep, and compared with that of other animal species. The cDNA of bighorn sheep CD18 has an open reading frame (ORF) of 2310bp. CD18 sequences obtained individually from peripheral blood mononuclear cells (PBMCs) and PMNs were identical to each other. Comparison of the deduced 770-amino acid sequence of CD18 of bighorn sheep with that of domestic sheep, goats, cattle, humans and mice revealed 99, 98, 95, 82 and 80% identity, respectively. Availability of cloned bighorn sheep CD18 cDNA should allow the molecular characterization of M. haemolytica Lkt-receptor interactions in bighorn sheep and other ruminants that are susceptible to this disease.     

Sheep leukocyte molecules: a review of their distribution, structure and possible function

Sheep leukocyte molecules and the subsets of cells they mark have only recently been identified. In this review, the leukocyte molecules which have been characterized in sheep are described in light of recent data derived from studies in man, mouse, rat and cattle. In addition, some applications of monoclonal antibodies to studies on the immune system of sheep are discussed, with particular emphasis on the thymus and lymphocyte recirculation.     

ras p21 expression in ovine pulmonary carcinoma

We have adapted an enzyme-linked immunoblot assay (ELIBA) for the detection of a c-ras proto-oncogene and oncogene protein products in human cell lines and tumors of 21,000 daltons molecular weight (p21ras) to studies of tissues derived from sheep. In the ELIBA, a double antibody system is used in which p21ras proteins are initially immunoprecipitated from protein extracts with monoclonal antibodies, and subsequently identified using additional anti-ras antibodies. Binding is identified with a non-radioactive enzyme-linked colorimetric detection system. In the present study, the ELIBA system was used to study twenty-seven ovine lung specimens, representing normal lung, inflammatory, and neoplastic lesions. We detected p21ras protein expression in every tissue examined, but the nature and amount of the protein product varied significantly among the tissues examined. Some tissues expressed multiple ras species. Broncho-alveolar carcinoma specimens were most likely to express c-Ki-ras proteins. Mutant proteins of c-N-ras and c-Ki-ras were detected in several bronchoalveolar carcinoma specimens, based on migrational differences between mutant and normal proteins in 15% polyacrylamide gels. The results of this study demonstrate the utility of the ELIBA system for detection of c-ras expression in ovine lung tissues, and demonstrate the ability of the system to discriminate specific ras protein species. The prognostic significance of ras expression in sheep pulmonary carcinoma has yet to be determined.     

鸡败血支原体黏附素GapA单克隆抗体的研制及免疫特性鉴定

本研究通过对鸡败血支原体（MG）强毒株黏附素GapA的N端（98aa-322aa）高变区作为免疫原免疫小鼠．利用Bac-to-bac系统构建GapAN重组杆状病毒，感染Sf9昆虫细胞，以作为检测抗原，筛选获得5株抗GapAN端的单克隆抗体：3D4、5810、185、1D7和4B3。经部分免疫学特性鉴定，5株单抗具备良好的免疫反应性，并且在免疫荧光试验及Western-blot检测过程中发现这些单抗针对的抗原表位存在明显差异，这些单抗的成功制备为下一步深入研究MG的黏附特性奠定了基础。     

Sequence analyses of feline B7 costimulatory molecules

Using RT-PCR amplifications with mRNA from mitogen-stimulated feline peripheral blood mononuclear cells, cDNA of feline B7-1 (CD80) and B7-2 (CD86) were cloned. The cDNA were sequenced and putative translated protein sequences compared with known counterpart sequences. Hydrophilicity patterns of the feline CD80 and CD86 which were only 26.8% identical at the amino acid sequence were very distinct from each other, but similar to the putative human CD80 and CD86 proteins, respectively. The feline CD80 gene encoded a protein of 292 amino acids and the CD86 gene encoded a protein of 329 amino acids. Amino-terminal signal sequences, extracellular Ig V- and Ig C-like domains, transmembrane domains, and carboxyl cytoplasmic domains were identified in both molecules. Although the most conserved domain among the CD80 sequences was the Ig C-like domain, the most conserved domain among the CD86 sequences was the Ig V-like domain. Among the known sequences, the bovine CD80 and the porcine CD86 sequences available for comparisons were identified as most closely related to the feline CD80 (63.3%) and CD86 (67.5%), respectively. The mouse molecules were the least identical (43.6 and 43.6%, respectively) with the feline CD80 and CD86 proteins. The human CD80 and CD86 molecules were 56.3 and 57.0% identical with the feline molecules.     

Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink.