Overview of the Second International Workshop to define swine cluster of differentiation (CD) antigens

The aim of the Second International Swine Cluster of Differentiation (CD) Workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. At the summary meeting of the workshop in July, 1995, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human and ruminant CD. Swine CD numbers will now be given to clusters of mAb to swine orthologues of human CD molecules when homology is proven by (1) suitable tissue distribution and lymphoid cell subset expression, (2) appropriate molecular mass of the antigen recognized by the mAbs, and (3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on the human gene products. In some cases, this reactivity would not be fully proven, mainly due to the lack of cloned gene products; for these CD antigens, the respective clusters will be assigned by the prefix ‘w' which will lead to ‘wCD' antigens. As a result of the Second International Swine CD Workshop the assignment of 16 mAb to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, CD18a, wCD21, wCD25) was confirmed, and 2 mAb to existing swine workshop clusters (SWC). More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb and 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). Finally, the demarcation of two new SWC molecules in swine, SWC8 (2 mAb) and SWC9 (2 mAb) was confirmed.     

Overview of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The aim of the Third International Workshop on Swine Leukocyte Differentiation Antigens (CD workshop), supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters, using nomenclature in accordance with human and ruminant CD nomenclature, as agreed at the summary meeting of the Second International Swine CD Workshop in Davis, 1995: only mAb with proven reactivity for the orthologous porcine gene product or cross-reactivity for the human gene products, were given the full CD nomenclature, all other allocations were prefixed with "w". As in previous workshops, the overall organization was entrusted to the chair and first author, with support by the chair of the previous workshop and second author. In addition to the existing 26 pig leukocyte CD/SWC determinants established in previous workshops, this workshop established/confirmed another 11 CDs for pig leukocytes, identified by a total of 21 mAb: CD11R1 (2 mAb), CD11R2 (1 mAb), CD11R3 (4 mAb), wCD40 (1 mAb), wCD46 (4 mAb), wCD47 (3 mAb), wCD49d (1 mAb), CD61 (1 mAb), wCD92 (1 mAb), wCD93 (1 mAb) and CD163 (2 mAb).     

Summary of workshop findings for porcine myelomonocytic markers.

About 65 monoclonal antibodies (mAb) including 17 internal controls were analyzed for their ability to recognize and bind to various cells of the myelomonocytic lineage. Flow cytometry (FCM) utilizing both single and double staining, and immunoprecipitation (IP) assays were used in the analysis. About 38 of the mAb were reactive with myelomonocytic cells, resulting in nine clusters of interest. Although the exact identity of many of the molecules on the cells bound by the mAb remains undetermined, information obtained about the mAb analyzed in this workshop should be helpful in further identifying various populations of myelomonocytic cells and their stages of differentiation. Out of 12 mAbs with potential CD11 specificity, seven were assigned to three different swine specific alpha chains of the CD11/CD18 integrin heterodimer, the assignment of the remaining four was tentative. One antibody had a binding specificity consistent with SWC3 and one with SWC8. CD14 expression on pig cells was characterized with a panel of CD14-positive antibodies, two of these antibodies were assigned to swine CD14. Two antibodies were assigned to CD163. Further work is required to determine the antigens recognized by many of the other mAb.     

Summary of workshop findings for porcine T-lymphocyte-specific monoclonal antibodies.

Fifty-seven monoclonal antibodies (mAb) selected after the first round analyses in the Third International Swine CD workshop for their possible reactivity with T-lymphocyte specific antigens were further analysed in a second round. As target cells for flow cytometric analyses served peripheral blood mononuclear cells, nylon-wool enriched T-lymphocytes, thymocytes, splenocytes, and lymphocytes derived from Peyer's patches. These second round analyses revealed 15 different data sets. Together with 22 pre-selected data sets from the first round analyses with the whole panel of monoclonal antibodies, 37 data sets were used for the clustering of the respective mAb. Using the LTDB4 program, 19 preliminary clusters could be defined. Two clusters (C3 and C7) with 4 mAb showed no labelling of resting T-lymphocytes. Seven clusters (C1, C2, C4, C5, C6, C11, and C12) contain mAb (in total: 16 mAb) directed against subsets of CD4(-)CD8(-) T-lymphocytes. These mAb seem to recognise antigens on porcine T-lymphocytes with T-cell receptor (TcR) gamma/delta chains. Three clusters (C8, C9, C10, C13) seem to be artificial. They contain either mAb staining CD4(-)CD8(-) T-lymphocytes and low CD8+ cells (C8, C9), mAb with various reactivity (C10) and mAb with known differences in their reactivity (C13). Cluster C14 contains 3 mAb against the CD4a-epitope, C15 describes mAb directed against porcine CD8c-epitope whereas mAb against CD8a and CD8b-epitopes grouped in C19. The mAb found in C16 seem to recognise CD45R. Cluster C17 is composed of different standards directed against CD2, CD3, CD5 and wCD6. Two additional mAb recognising the CD2a-epitope could be enclosed. C18 contains two mAb directed against SWC2.     

Monoclonal antibodies putatively recognising activation and differentiation antigens.

In the activation/maturation section, 46 monoclonal antibodies (mAbs) were analysed using freshly isolated as well as mitogen activated and recall antigen re-stimulated cells. A total of 10 internal standards as well as 6 antibodies with established reactivity for human cells, reported to cross-react with porcine leukocytes, were included in the panel. The standard antibodies were anti-CD25, CD44, CD45, SLA II, SWC1, SWC2, SWC7 and SWC8 reagents. The test panel contained antibodies with putative reactivity to CD25, SLA II and other mAbs directed against ill-defined targets. Single and double colour surface staining was performed in the attempt to group the mAbs tested into clusters of differentiation. Five new anti-class II reagents, two directed to SLA-DQ and three to SLA-DR, could be added to the previously established ones. One new anti-CD25 as well as two new antibodies with SWC7 and SWC8 specificities, respectively, could also be added to the previously established ones. The identity of the two latter antibodies was also confirmed in other sections of this workshop (B-cell section for SWC7 antibodies and myeloid section for the SWC8 antibodies). The antibody JM2F12, in our hands, has shown strong similarities to the cross-reactive anti human-CD49f reagent. No other clusters were identified, as all remaining antibodies behaved in a different way on different target leukocyte populations. The second purpose of the section was fulfilled: interesting staining profiles of several antibodies on differentiating lymphocytes were recorded and are discussed here.     

Analysis of monoclonal antibodies that recognize γδ T/null cells

Thirty two monoclonal antibodies (mAbs) from the first round of analysis in the Second International Swine CD Workshop were placed together with additional mAb derived from the first workshop in the null cell panel for further evaluation. Preparations of peripheral blood leukocytes, concanavalin A stimulated peripheral blood mononuclear cells, and spleen cells were used in flow cytometric analyses. Nineteen mAbs identified molecules that were not expressed on null cells, not lineage specific, or recognized activation molecules. Sixteen mAbs including control mAbs were identified that were specific for null cells. One of the latter mAbs, 041 (PGBL22A), that recognizes a determinant on a constant region of porcine γδ TcR established the majority of null cells are γδ T cells. Use of this mAb in further comparisons demonstrated the γδ T cell population is comprised of two major subpopulations, one negative and one positive for CD2. Two color analyses demonstrated that 11 of the mAbs formed a broad cluster that included control mAbs 188 (MAC320) that defined the CD2 negative SWC6 cluster in the first workshop and mAb 122 (CC101) that might recognize an orthologue of bovine WC1 and nine mAbs that recognize determinants on one or more molecules with overlapping patterns of expression on subsets of CD2⁻ γδ T cells. Two groups of mAbs formed the previously identified subset clusters SWC4 and SWC5. Two new mAbs formed a third subcluster. Three mAbs did not form clusters. Three mAbs predicted to recognize TcR in the first workshop (020 [PT14A], 021 [PT79A], and 022 [MUC127A]) and mAb PGBL22A were shown to immunoprecipitate a 37, 40 kDa heterodimer.     

Summary of the animal homologue section of HLDA8

The development of reagents against leukocyte differentiation antigens in veterinary species is delayed compared to mouse and men and therefore also the number of existing reagents for the characterisation of leukocytes derived from species with importance in veterinary medicine is restricted. Cross-reactive studies with existing well defined monoclonal antibodies directed against leukocyte differentiation antigens derived from other species are an alternative approach to enhance the panel of reagents in veterinary immunology. This study describes the activities of the animal homologue section in frame of human leukocyte differentiation antigen 8-workshop (HLDA8) were 376 monoclonal antibodies, mainly directed against human leukocytes had been tested for their reactivity with 17 different animal species including non-human primates, ruminants, swine, horse, carnivores, rabbit, guinea pig, chicken and fish. In a first round 182 mAb were selected based on there reactivity in FCM analyses with at least one species for further studies, including multi-colour FCM, and molecular analyses of the antigens. Interesting was the species-overlapping reactivity of mAb directed against distinct clusters: 11 out of 17 species reacted with CD9, 11 of 17 with CD11a, CD14 (11/17), CD18 (13/17), CD21 (7/17), CD29 (10/17), CD44 (13/17), CD45 (9/17), CD47 (10/17), CD49d (13/17), CD61 (6/17), CD86 (7/17), CD91 (5/17), and CD172a (10/17), indicating evolutionary highly conserved epitopes on these surface molecules. Our results suggest the suitability of cross-reactive mAb for the animal model studies. Moreover, these findings contribute to our understanding of the evolution of the immune system.     

Membrane markers of the immune cells in swine: an update

Besides their breeding value, swine are increasingly used as biomedical models. As reported in three international swine clusters of differentiation (CD) workshops and in the animal homologue section of the last workshop for the determination of human leukocyte differentiation antigens (HLDA 8), characterisation of leukocyte surface antigens by monoclonal antibodies and other molecular studies have determined the cell lineages and blood leukocyte subsets implicated in the immune response, including cell adhesion molecules involved in cell trafficking. This review focusses on the current state of knowledge of porcine leukocyte differentiation and major histocompatibility complex (SLA) molecules. Examples of porcine particularities such as the double-positive T lymphocytes with the phenotype CD(4+)CD8(low) and CD(4-)CD8(low) alphabeta T cell subsets and the persistence of SLA class II after T-lymphocyte activation are illustrated, as well as the shared characteristics of the Artiodactyla group, such as the high proportion of gammadelta TcR (T cell receptor) T cells in blood and other lymphoid tissues. Furthermore, discrepancies between swine and humans, such as CD16 expression on dendritic cells and CD11b (wCD11R1) tissue distribution are outlined. The rapidly growing information should facilitate manipulation of the swine immune system towards improving disease control, and open new avenues for biomedical research using the pig as a model.     

Analysis of monoclonal antibodies reacting with molecules expressed on gammadelta T-cells.

Twenty-six monoclonal antibodies (mAbs) selected after the first round of analysis in the Third International Swine Workshop were grouped with additional mAbs from the first and second workshops and mAbs under study for further evaluation. Preparations of peripheral blood leukocytes were used in single and multicolor flow cytometric (FC) analyses. Six mAbs did not react with gammadelta T-cells. Two were negative for all tested specificities. Seven mAbs recognized molecules expressed on gammadelta T-cells that were not lineage restricted. One of these from the first workshop (2B11) yielded a pattern of labeling identical to a mAb under study (PGB73A). Ten mAbs were characterized in previous workshops and known to react with the gammadelta TCR or molecules expressed on subsets of gammadelta T-cells. One belonged to SWC4, two to SWC5, and one to SWC6. Two mAbs from the second workshop recognized a molecule or molecules expressed on subsets of gammadelta T-cells. A new mAb (PPT16) added late to the workshop following a request by the workshop chairs appeared to recognize a determinant expressed on the gammadelta TCR/CD3 molecular complex.     

Summary of workshop findings for antibodies reacting with porcine T-cells and activation antigens: results from the Second International Swine CD Workshop

After initial evaluation of the 176 new and 19 control monoclonal antibodies (mAb) submitted to the Second International Swine CD Workshop, 57 were assigned to the T-cell/activation marker subgroup. These 57 mAb were further analyzed using flow cytometry on whole blood lymphocytes, splenocytes, Peyer's patch lymphocytes, in vitro cell lines, broncho-alveolar lavage cells, Con A and PHA blasts, fetal cell populations, and by 2-color flow cytometry against mAb to porcine CD2, CD4, and CD8. Finally, the molecular weights of the target antigens were characterized when possible. As a result of these analyses, 23 mAb were distributed into 7 CD clusters. Newly confirmed mAb assignments included: two CD2; one CD4; two CD5; one wCD6; and one wCD25. Three new mAb were found that reacted with wCD8, one of which defined a new epitope, wCD8c. For the first time, mAb against porcine CD3 were identified, including 6 mAb that reacted with three different epitopes. Several new mAb reacted with antigens whose expression varied depending on the activation state of the test cell. These will require further characterization in order to assign a CD number.     

猪CD58分子基因克隆、表达及其结构功能预测

CD58在机体免疫系统中具有重要作用,本研究通过对人、绵羊CD58 mRNA序列比对,设计兼并引物,应用反转录PCR技术克隆猪CD58基因,并在大肠杆菌中进行原核表达,同时运用生物信息学方法对其核苷酸序列、编码的氨基酸序列以及蛋白结构进行预测。结果表明:克隆的猪CD58 cDNA全长800 bp,ORF为735 bp;将其在大肠杆菌中进行表达,产物可被CD58抗血清识别;序列比对结果显示猪、羊和人的CD58核苷酸序列及氨基酸序列同源性并不高,但蛋白结构预测表明三者蛋白结构非常相似,尤其是V区三维结构,这是异种动物淋巴细胞和红细胞发生黏附的分子基础。该研究为CD58作为疫苗佐剂或免疫调节药物在临床中的应用、进一步研究CD58分子结构及CD2-CD58复合物激活免疫系统的机理奠定了基础。     

Monoclonal antibody specific for bovine CD 5 antigen which enhances mitogen-induced blastogenesis and IL-2 production.

This study reports on the functional characteristics of a bovine T-cell differentiation antigen recognized by the monoclonal antibody (mAb) 8C11. This mAb has previously been found to react with a 67-kD molecule shared by thymocytes and peripheral blood T cells and to be undetectable on the B cells of healthy animals. This antigen is also largely expressed on the B cells from bovine leukemia virus-infected animals. Molecules with a similar cell distribution have been described in other species (mouse, human, rat and sheep), and were termed CD5 molecules. In order to confirm the CD5-like nature of the target molecule recognized by 8C11, functional T-cell assays were carried out. We report here that this mAb, like its human and murine homologues, enhances the proliferative responses of T cells to mitogens or alloantigens but does not directly stimulate T-cell division. Moreover, we have shown an enhancing effect of this 8C11 mAb on bovine interleukin-2 production by concanavalin A-stimulated bovine peripheral blood mononuclear cells.     

Differentiation of porcine myeloid bone marrow haematopoietic cell populations.

The myeloid panel of monoclonal antibodies (mAbs) submitted to the Third Swine CD Workshop were analysed for reactivity with bone marrow haematopoietic cells (BMHC). Using single and triple immunofluorescence labelling by flow cytometry (FCM), the mAbs were grouped according to their capacity to recognise myeloid cell populations and/or maturation stages. Group 1 consisted of mAbs labelling the majority of myeloid BMHC, including neutrophilic, eosinophilic and monocytic cells. The ligands for SWC3 and CD11b-like mAbs of group 1 showed a maturation-dependent intensity of expression. The other antibodies of group 1 reacted with BMHC to give a sharp, single peak. Group 2 mAbs reacted only with monocytic cells. The anti-human CD49e mAb Sam-1 was the only mAb detecting the majority of monocytic cells, but not other BMHC. The mAbs in group 3 recognised antigens expressed on granulocytes, but not monocytes. The previously identified SWC8 in this group proved to be useful in differentiating major population of BMHC when cells were double labelled with the pan-myeloid SWC3. Other mAbs within group 3, such as MIL4 and TMG6-5 (an anti-human CD11b), only recognised subsets of neutrophils and eosinophils. Group 4 mAbs reacted with the more mature subpopulations of neutrophils and monocytes. Some of these antibodies might prove useful for assessment of cell maturity, such as anti-CD14 and the anti-human CD50 mAb HP2/19.     

Isolation and characterization of immortalized porcine aortic endothelial cell lines

Primary porcine endothelial cells have a limited life span in culture. After four to five passages, they tend to de-differentiate and eventually reach senescence. The aim of this work was to establish immortalized porcine aortic endothelial cell lines (AOCs) to facilitate in vitro studies of different pathological process involving the endothelium. Primary porcine aortic endothelial cells (PAECs) were transfected with a plasmid containing the SV40 genome and selected on the basis of morphological and phenotypical features. Flow cytometry analysis demonstrated uptake of acetylated low density lipoproteins (Ac-LDL) and constitutive expression of SLA class I, CD29, CD31, CD41/61, CD80/86, CD46, SWC3, and LAMP-1 antigens by all analyzed lines and showed little differences to primary cells. The functional similarity between primary and immortalized endothelial cells was demonstrated in a cytotoxicity assay using a human natural killer cell line (NKL) as effector. The AOCs cell lines should be valuable tools for in vitro study of the human immune response against pig endothelial cells. In addition, they would be very useful to gain insight in the pathogenesis of some viral haemorrhagic diseases of pig such as African swine fever (ASF) or classical swine fever (CSF).     

Immunoprecipitation of equine CD molecules using anti-human MABs previously analyzed by flow cytometry and immunohistochemistry

Earlier studies investigating the cross-reactivity of antibodies submitted to the HLDA8 had used flow cytometry as a method of choice to screen mAbs for reactivity with equine leukocytes, including two-color flow-cytometry to characterize the lymphocyte population they detect. In addition, immuno-histochemistry (IHC) was used to detect distribution of positive cells in lymphoid tissue sections. In this study we performed immunoprecipitation (IP) to complement the previous results and add valuable information regarding the molecules detected by the cross-reacting antibodies. Surface molecules from primary equine PBMC or the equine cell line T8888 were biotinylated prior to precipitation to determine the molecular weight of the corresponding molecules in a western blot using streptavidin-AP. 21 out of 24 mAbs precipitated the molecules with a MW corresponding to its human orthologue. Positive mAbs were directed against CD2, CD5, CD11a, CD11b, CD14, CD18, CD21, CD44, CD83, CD91, CD172a, MHCI and MHCII. Three mAbs directed against CD49d, CD163, and CD206 which were unambiguously identified earlier by flow cytometry failed to immunoprecipitate the corresponding CD molecule. MAbs detecting CD molecules which are expressed internally like CD68 and mAbs of IgM class could not be included into this approach.     

Porcine myelomonocytic markers: summary of the second international swine CD workshop

Forty five mAbs submitted to the Second International Swine CD workshop were analyzed by six different laboratories for their possible reactivity with porcine myelomonocytic cells using flow cytometry and immunohistochemistry. As a result of these analyses, a new swine workshop cluster, SWC9, composed of two mAbs that recognize an antigen selectively expressed on mature macrophages, was defined. In addition, several mAbs were identified, allowing the differentiation of granulocytes from monocytes/macrophages, or monocytes from macrophages. Further work is required to identify the antigen recognized by these mAbs. Nevertheless, they should already prove useful for the identification of different stages in the macrophage maturation/differentiation, and will certainly aid analyses on the complexity of the mononuclear phagocyte system in the pig. Finally, the cross-reactivity of three anti-human CD14 mAbs with porcine myelomonocytic cells was established in this workshop.     

Generation of anti‐porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis

T cell‐mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti‐porcine CD69 mAb‐producing mouse hybridomas, 01‐14‐22‐51 (IgG2b–κ) and 01‐22‐44‐102 (IgG2a–κ), both showing fine reactivity with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin‐stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti‐interferon‐γ mAb and anti‐tumor necrosis factor‐α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs.     

Characterization and functional analysis of skin-derived dendritic cells from swine without a requirement for in vitro propagation

A low-density cell population was isolated from skin explants of pigs and characterized as a highly enriched dendritic cell (DC) population based on phenotypical and functional properties. The skin-derived DCs were identified by their characteristic ultrastructural properties as well as by consistent co-expression of the CD1 and SWC3a antigens that clearly differentiate them from other porcine leukocytes. These cells exhibit higher expression of porcine MHC class II (SLAII) and CD80/86 antigens as compared to macrophage/monocyte cells. They consistently expressed the S100 beta antigen at high levels and did not express the lymphoid markers CD3, CD4 or CD8. Within this population of skin-derived DCs there was variable expression of CD11c, CD14 and CD16. Functional characterization of this DC population revealed that they are efficient in uptake and processing of soluble protein antigens and in endocytosis of small (0.02 microm) but not large (2 microm) polystyrene beads. Further, these cells were efficient inducers of primary allogeneic responses and in stimulating antigen-specific and mitogen-induced proliferation and IFN gamma responses in autologous lymphocytes. This study provides important information to further characterize the cutaneous DCs and develop models to analyze the role of these cells in immune responses in vivo.     

Characterization of platelet and soluble-porcine P-selectin (CD62P)

P-selectin (CD62P), an adhesion molecule expressed on activated endothelial cells and platelets, mediates the initial attachment of leukocytes to the stimulated endothelium upon inflammation and the interaction between leukocytes and platelets. A soluble form of P-selectin is present in the serum of healthy individuals as a circulating protein and high levels have been described in various pathological situations. The aim of this study was to characterize P-selectin on porcine platelets and investigate the soluble form of this protein, which are uncharacterized in several animal species including pigs. A new monoclonal antibody (mAb) (SwPsel.1.9) against porcine P-selectin was produced using a mouse cell line transfected with pig P-selectin cDNA. This mAb together with a previously described mAb (P-sel.KO.2.5), produced in our laboratory, was used to develop an ELISA to quantify porcine P-selectin. No significant levels of soluble-porcine P-selectin were observed in healthy animals. However, the total amount of P-selectin measured in porcine platelets was similar to that found in humans. Increased levels of this circulating protein were detected in the plasma from pigs after allograft implantation. In vitro, P-selectin expression on platelet membrane was rapidly induced by PMA and thrombin, as assessed by flow cytometry. However, these activators did not stimulate the release of soluble P-selectin. Analysis of the proteolytic cleavage of this protein from COS-transfected cells revealed that PMA treatment failed to cause the shedding of membrane-bound P-selectin. These data suggest that porcine P-selectin is a suitable marker for inflammation and that the mechanism involved in the generation of circulating P-selectin is not proteolytic release.