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.     

Naturally-occurring anti-thymocyte autoantibody which identifies a restricted CD4+CD8+CD3-/lo/int thymocyte subpopulation exhibits extensive polyspecificity

In this study, naturally-occurring, monoclonal IgM kappa anti-thymocyte autoantibodies from the neonatal inbred Balb/c mouse-derived hybridoma NMT-1 (NMT-1 mAb), previously reported to identify a restricted CD4+CD8+CD3/lo/int thymocyte subpopulation, have been shown to exhibit extensive polyspecificity. Using immunofluorescence, immunoblotting and antibody titration and competition ELISAs, NMT-1 mAbs exhibited polyspecific binding to 12 apparently structurally unrelated self and non-self antigens. The autoreactive component of the polyspecificity profile of NMT-1 mAbs encompassed reactivity to developmentally-related 14.5 and 18.3 kDa Thy-1 glycoforms expressed on a CD4+CD8+CD3-/lo/int thymocyte subpopulation. The autoreactivity profile of NMT-1 mAbs also included recognition of the heavy and light chains of mouse IgG1 and mouse cytokeratins within thymic medullary epithelium and basal epithelial cells of stratified squamous epithelium of mouse tongue, oesophagus, stomach, skin and vagina. Examination of the polyspecificity profile of NMT-1 mAbs was also undertaken using a panel of 23 antigens including heterologous proteins, phospholipids, haptens and bacterial antigens by antibody titration and competition ELISAs. Antibody titration ELISAs demonstrated that NMT-1 mAbs bound nine antigens including bovine carbonic anhydrase, ovalbumin, cardiolipin, phosphatidylserine, the haptens, DNP and FITC and the bacterial antigens including Escherichia coli beta-galactosidase and the toxoids from Corynebacterium tetani and Clostridium diphtheria. Competition ELISAs, based on the inhibition of NMT-1 mAb binding to antigens adsorbed to ELISA plate surfaces by inhibitor antigens in solution, demonstrated that NMT-1 mAb interactions were not dependent on multivalent binding. In these assays, NMT-1 mAbs recognized unmodified (native) epitopes on the solution phase forms of the protein antigens, including E. coli beta-galactosidase and toxoids from Corynebacterium tetani and Clostridium diphtheria, providing further evidence for the hypothesis that the binding of multiple, apparently unrelated, antigens by NMT-1 mAbs occurs via unique polyspecific antigen combining sites.     

Comparison of workshop CD45R monoclonal antibodies with OvCD45R monoclonal antibodies in sheep

The reactivity of the antibovine monoclonal antibodies (mAbs) comprising temporary cluster TC1 was compared with that of two OvCD45R mAbs on sheep cells. Three of the mAbs--CC31, CC99 and CC103--did not cross-react with sheep cells. All the workshop mAbs precipitated two molecules of apparent molecular weight (MW) 200 kDa and 220 kDa while the antisheep CD45R mAb 20-96 precipitated a single band of 220 kDa. Cell surface expression was examined by single colour FACS (fluorescence activated cell sorting) analysis of efferent and afferent lymph cells and peripheral blood lymphocytes and the distribution of the antigens on CD4+, CD8+ and T19+ (WC1) and B cells was determined by two colour fluorescence staining. By cellular distribution and immunohistology the TC1 mAbs could be divided into four distinct groups which differed from a fifth group comprising the two OvCD45R antibodies.     

Generation of monoclonal antibodies to the rabbit interleukin-2 receptor alpha chain (CD25) and its distribution in HTLV-1-transformed rabbit T cells

Rabbits can be infected with human retroviruses such as human T-cell leukemia virus-1 (HTLV-1) and human immunodeficiency virus (HIV), and provide useful animal models to study retroviral diseases such as adult T-cell leukemia and HIV. Previously we have succeeded in generating monoclonal antibodies (mAbs) against rabbit CD4, CD5 and CD11a antigens. To make this animal species more amenable to cellular and molecular studies, we have attempted to extend the panel of mAbs against rabbit CD antigens. Here we report on the generation of three neutralizing mAbs against interleukin-2 receptor alpha chain (IL-2R alpha) (CD25), Kei-alpha 1 (IgG2b), Kei-alpha 2 (IgG2a) and Kei-alpha 3 (IgG1). They specifically recognize the rabbit Mr 55,000 IL-2 binding protein, IL-2R alpha, and completely inhibit both high- and low-affinity IL-2 binding to F648b cells that express IL-2R alpha as well as IL-2R beta. The use of mAb Kei-alpha 1 confirmed that the rabbit IL-2R alpha is not only a low-affinity IL-2R on its own but also an essential component of high-affinity IL-2R as found in other animal species, and that rabbit activated T cells including HTLV-1-transformed cell lines express high levels of the IL-2R alpha. Together with mAbs against various rabbit CD antigens that we reported previously, these neutralizing mAbs to IL-2R alpha will be valuable for studies of human retrovirus infections, such as those induced by HTLV-1 or HIV, in rabbits.     

Effect of permeabilization on peripheral blood lymphocytes of bovine leukemia virus (BLV)-infected cattle

Cell surface proteins serve as markers for immunophenotypic characterization of lymphocyte subsets by appropriate monoclonal antibodies and fluorescence-activated cell sorter (FACS) analysis. By the same method, internal antigens or those that are only partially expressed on the cell surface can be determined after permeabilization of the cells. Peripheral blood lymphocytes obtained from bovine leukemia virus (BLV)-infected cattle and from BLV-free cattle were permeabilized and several lymphocyte populations were examined. BoCD4, BoCD8 and three CD4 CD8-T-cell subsets retained their original frequencies after permeabilization in both groups of animals. The recognition of the B-B2 lymphocyte molecule was only partially expressed on the cell surface of intact lymphocytes and was further revealed on permeabilization. The frequency of permeabilized, but not intact, cells stained with this mAb was significantly higher for BLV-infected cattle than for BLV-free animals (P = 0.006). Reactivities of an anti-heat shock protein (Hsp) 70 were measured before and after permeabilization of PBLs. Similar increased cell frequencies were obtained for both groups of bovines. These data indicate that flow cytometry studies should be conducted on both permeabilized and intact cells for a better assessment of protein expression on the cell surface, as well as in the cytoplasm.     

Identification of two distinct populations of dendritic cells in afferent lymph that vary in their ability to stimulate T cells

Immunofluorescent staining and flow cytometric analysis of dendritic cells from cattle afferent lymph has established that within the afferent lymph veiled cells (ALVC) there are two phenotypically distinct, major populations. One is CD11a+, CD5+, CD21- and expresses the bovine WC10 (workshop cluster 10) molecule and the Ag recognized by mAb CC81 but is not recognized by mAbs CC149 and IL-A24. The second ALVC subpopulation is CD11a-, CD5-, CD21+/-, workshop cluster 10- and is not recognized by mAb CC81 but is recognized by mAb CC149. Thus, the two populations, which can be identified by staining for CD11a, are defined by the differential expression of a number of Ag. The ALVC populations had differing capacities to stimulate T cells. CD11a- ALVC were more effective at stimulating proliferative responses in allogeneic CD4+ T cells and CD8+ T cells. This was not related to binding of CTLA4Ig or CD40L fusion proteins, implying similar levels of expression of their ligands, CD80 and CD86 or CD40. Both subsets were able to present OVA to resting memory CD4+ T cells, indicating that both were able to take up and process soluble native protein. In contrast, the CD11a- ALVC were more effective in presenting respiratory syncytial virus Ag to resting CD4+ T cells. Considering the central role of dendritic cells in the initiation of immune responses in naive animals, the two cell types may have different roles in the induction of primary responses induced following infection or immunization.     

Cross-reactivity of workshop antibodies with cells from domestic and wild ruminants

Reactivities of the monoclonal antibodies (mAbs) from the workshop panel with cells from cattle, sheep, goats, Cape buffalo (Syncerus caffer) and waterbuck (Kobus defassa) were tested. One hundred and sixty-nine mAbs reacted with bovine cells and 111 with sheep cells; 86 were shown to react with goat cells, 71 with buffalo cells and 70 with waterbuck cells. Some mAbs cross-reacted with all five ruminants tested, and are likely to react with epitopes that are conserved in other ruminant species. Such mAbs will obviate the need to produce mAbs panels to leukocyte antigens of other ruminants.     

Identification of the sheep homologue of the monocyte cell surface molecule--CD14

An ovine monocyte/macrophage cell surface antigen was recognized by three mouse monoclonal antibodies (mAbs) VPM65, VPM66 and VPM67. These mAbs also reacted with bovine cells. The antibodies immunoprecipitated a single, glycosyl-phosphatidylinositol-linked polypeptide of M(r) 55,000 which, when deglycosylated, was reduced to M(r) 53,000. They reacted strongly with peripheral blood monocytes, alveolar macrophages and peripheral blood granulocytes, and weakly with afferent lymph dendritic cells. They also reacted with macrophages in many different tissues but were non-reactive with lymphocytes. Competitive flow cytometry shows that these three mAbs recognize the same or a closely related epitope of a single antigen. An antigen-specific capture ELISA using the anti-human CD14 mAb (TUK4) revealed that all four mAbs associate with the same antigen. These data demonstrate that the mAbs react with the ovine homologue of the lipopolysaccharide (LPS)-LPS binding protein receptor, CD14.     

Dual effects of LPS antibodies on cellular uptake of LPS and LPS-induced proinflammatory functions

Human phagocytes recognize bacterial LPS (endotoxin) through membrane CD14 (mCD14), a proinflammatory LPS receptor. This study tested the hypothesis that anti-LPS Abs neutralize endotoxin by blocking cellular uptake through mCD14. Ab-associated changes in the uptake and cellular distribution of FITC-LPS were assessed by flow cytometry and laser scanning confocal microscopy in human CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and human peripheral blood monocytes. LPS core- and O-side chain-specific mAbs inhibited mCD14-mediated LPS uptake by both cell types in the presence of serum. O-side chain-specific mAb concurrently enhanced complement-dependent LPS uptake by monocytes through complement receptor-1 (CR1) and uptake by CHO-CD14 cells involving another heat-labile serum factor(s) and cell-associated recognition molecule(s). Core-specific mAb inhibited mCD14-mediated uptake of homologous and heterologous LPS, while producing less concurrent enhancement of non-mCD14-mediated LPS uptake. The modulation by anti-LPS mAbs of mCD14-mediated LPS uptake was associated with inhibition of LPS-induced nuclear factor-kappaB (NF-kappaB) translocation and TNF-alpha secretion in CHO-CD14 cells and monocytes, respectively, while mAb enhancement of non-mCD14-mediated LPS uptake stimulated these activities. LPS-specific Abs thus mediate anti-inflammatory and proinflammatory functions, respectively, by preventing target cell uptake of LPS through mCD14 and augmenting uptake through CR1 or other cell receptors.     

Specific regulation of VLA-4 and alpha 4 beta 7 integrin expression on human activated T lymphocytes

Modulation of VLA integrins was studied in several human T cell clones upon specific and nonspecific cellular activation. Human activated T lymphocytes down-regulated both alpha 4 beta 1 and alpha 4 beta 7 integrins upon specific recognition of alloantigens (cytotoxic T cells) or in the presence of Staphylococcus enterotoxin B (superantigen recognizing noncytotoxic T cells). In contrast, the expression of other membrane integrins, such as VLA-1 and VLA-5 integrins, was not modified. Down-regulation of alpha 4 beta 1 and alpha 4 beta 7 integrins was observed as early as 3 h after stimulation, lasted later than 72 h and was partially inhibited by cytochalasin D. Interestingly, neither target cells nor NK cells modulated CD49d expression after interaction with T cells of K562, respectively, suggesting that CD49d expression was linked to specific T cell activation. The down-regulation of the CD49d chain in T cell clones stimulated with immobilized anti-CD3 mAbs confirmed the role of TCR-mediated activation in CD49d regulation. However, the CD3-independent cellular aggregation induced by soluble anti-CD43 mAb was also able to strongly down-regulate alpha 4 beta 1 and alpha 4 beta 7. The present work shows the first evidence that CD49d subunit-bearing integrin expression is distinctly regulated from other integrins after Ag or superantigen recognition by human activated T cells. CD49d modulation may be relevant for the traffic and tissue localization of locally activated T cells during immune responses.     

Anti-bull sperm monoclonal antibodies: I. Identification of major antigenic domains of bull sperm and manifestation of interspecies cross-reactivity

The overall objective of this series of experiments is to generate immunological markers that may elucidate bull sperm surface changes in vitro. Here we report the initial experiments of the study, involving the production and characterization of monoclonal antibodies (mAbs) again bull sperm. BALB/c mice were immunized with phosphate-buffered saline (PBS)-washed whole bull sperm, and their spleen cells were fused with NS-1 myeloma cells in two separate cell fusion experiments, resulting in the generation of 15 mAbs. The mAbs were specific to antigens of either the posterior tail or the head regions of bull sperm and detected five major domains of antigen localization in the bull sperm (apical crescent, equatorial band, principal acrosomal, whole head, and posterior tail). Eleven of the 13 head-specific mAbs recognized intra-acrosomal antigens, whereas 2 mAbs recognized antigens that were localized in the plasma membrane. One mAb specific to the tail region was of the IgM class; the remaining 14 mAbs were of the IgG class. They were all sperm specific, with no cross-reactivity to bovine oocytes or to any of the 12 bovine somatic tissues tested. The mAbs were not species specific, however, because 11, 10, 2, and 1 of the 15 mAbs cross-reacted with sheep, pig, mouse, and human sperm, respectively. None of the mAbs cross-reacted with rooster sperm. The cognate antigens of the 11 tested mAbs were of testicular origin, but several of them showed enhanced binding to epididymal sperm. In western blot analysis, 3 of the 13 mAbs tested identified more than one protein band (40-200 kDa). Seven others recognized proteins of > or = 200 kDa, whereas three mAbs recognized no proteins.     

B-90063, a novel endothelin converting enzyme inhibitor isolated from a new marine bacterium, Blastobacter sp. SANK 71894

We raised mAbs to whole L5178Y leukemia/lymphoma (LL) cells to identify adhesion proteins involved in adherence between LL cells and marrow stromal cells. One mAb, 4C, and its subclones 4C.1 and 4C.2 inhibited adherence of L5178Y LL cells to MLT. a nontransformed murine marrow stromal cell line. These MoAbs are directed against CD45RA. Control anti-CD45 mAbs and isotype mAbs were non-inhibitory. Other anti-CD45 mAbs, M1/9.3, RA3-3A1/6.1 and RA3-2C2/1 do not compete with mAb 4C.1 for binding to the L5178Y cell surface, but mAb 4C.1 competes for binding of mAb RA3-2C2/1. Effects of mAb 4C on tyrosine-phosphatase activity of CD45 in L5178Y cells are minimal, suggesting direct involvement of CD45 as an adhesion protein.     

Spiritual recovery movements and contemporary medical care

At the Eleventh International HLA Histocompatibility Workshop, numerous anti-HLA class II monoclonal antibodies (mAb) were tested. For several of the polymorphic mAb, one epitope for binding has been mapped within the antigen-binding site of the class II molecules. Screening of the available bovine DRB3 and DQB exon 2 sequences revealed that some of the key amino acid (AA) motifs of these epitopes were present in cattle as well, and the question was raised whether this sharing of key AA motifs might cause interspecies cross-reactivity. Eight polymorphic anti-HLA class II mAb (seven anti-HLA DRB1 and one anti-HLA DQB) were selected for analysis of their reactivity towards bovine lymphocytes. In addition, the monomorphic anti-HLA class II mAb, 7.5.10.1, was selected for analysis, as this mAb was described to detect class II polymorphism in cattle. Flow cytometry and lymphocyte microcytotoxicity testing revealed that five of the polymorphic anti-HLA mAb were reactive with bovine lymphocytes. Furthermore, the anti-bovine reactivity of 7.5.10.1 was confirmed. These findings were supported by biochemical analysis. The anti-bovine reaction of the anti-HLA mAb did not correspond with the expected reaction, which was based on the presence of the AA, postulated to be responsible for recognition. Therefore, we suggest that the patterns of reactivity of the anti-HLA mAb are not always determined by one epitope.     

Nitric oxide synthesis is depressed in Bos indicus cattle infected with Trypanosoma congolense and Trypanosoma vivax and does not mediate T-cell suppression

Infection with African trypanosomes causes the diseases sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. Suppression of cellular immune responses is a feature of trypanosomiasis in bovine, human, and murine hosts. Some aspects of immunosuppression in the murine model are mediated by nitric oxide (NO) produced by gamma interferon (IFN-gamma)-activated macrophages. We have investigated whether a similar mechanism is responsible for T-cell unresponsiveness in bovine trypanosomiasis. Bovine monocytes and macrophages from uninfected cattle and activated in vitro with IFN-gamma produced NO; however, this response was down-regulated in infected cattle. Similarly, the expression of inducible NO synthase messenger RNA was depressed in macrophages of infected cattle. Proliferation of mononuclear cells of trypanosome-infected cattle cultured with mitogen or trypanosome antigens was unchanged by the addition of an NO synthase inhibitor. Lymphocytes of infected cattle secreted interleukins with T-cell growth factor activity after in vitro activation with mitogens but not after activation with trypanosome antigens. Although lymph node cells secreted IFN-gamma after in vitro activation, ex vivo expression of mRNA was depressed. In contrast, the level of expression of interleukin 10 mRNA was higher during infection. We conclude that NO is not involved in the loss of T-cell proliferative function associated with trypanosomiasis in cattle and that, in contrast to the mouse model, the capacity of monocytes and macrophages to produce NO is actually down-regulated in infected cattle.     

Lake Vera revisited: parity and survival rates of Anopheles punctipennis at the site of a malaria outbreak in the Sierra Nevada foothills of California

FTY720, a novel immunosuppressant, sequesters circulating mature lymphocytes, especially T cells, within lymph nodes and Peyer's patches by accelerating lymphocyte homing, and thereby causes lymphocyte depletion in the blood. The FTY720-induced acceleration of lymphocyte homing appears to be mediated by lymphocyte homing receptors including CD62L, CD49d/beta7, and CD11a/CD18. In this study, expressions of CD62L, CD49d and CD11a on T cells in the peripheral blood, lymph nodes and Peyer's patches were analysed by flow cytometry in rats given FTY720 (1 mg/kg) orally. FTY720 markedly decreased the number of peripheral blood T cells, while not affecting CD62L, CD49d and CD11a expressions at 1-3 hr after administration. In contrast, both the frequency of CD62L-positive T cells and intensity of CD62L expression on T cells were increased in Peyer's patches but not lymph nodes at 3 hr after administration of FTY720. CD49d and CD11a expressions on T cells were unaffected by FTY720 in both Peyer's patches and lymph nodes at the same point in time. On the other hand, analysis of lymphocyte homing with calcein-labelled lymphocytes and anti-CD62L monoclonal antibody (mAb) confirmed that FTY720 predominantly increased CD62L-dependent lymphocyte homing to Peyer's patches. These findings indicate that FTY720 increases the frequency of CD62L-positive T cells by accelerating CD62L-predominant homing in Peyer's patches.     

Bovine cells infected in vivo with Theileria annulata express CD11b, the C3bi complement receptor

Bovine cells from cattle infected with Theileria annulata were phenotyped with monoclonal antibodies recognizing bovine leukocyte antigens. Macroschizont-infected, transformed cell lines prepared from peripheral blood mononuclear cells of cattle, infected with sporozoites, were assessed by flow cytometry; parasitized cells in tissues from infected cattle were examined by immunocytochemical techniques. Co-expression of markers for different cell lineages by the cell lines precluded a definite conclusion as to their phenotypic origins. For, while the pattern of leukocyte antigens expressed by these in vivo-derived schizont-infected cells, which included CD11b, was indicative of a myeloid origin, the possibility that they were NK cells could not be excluded. The monoclonal antibody (MAb) IL-A15, which recognizes CD11b, reacted with a high proportion of parasitized cells in sections of tissues from infected cattle at all stages of acute disease. Mononuclear cells infected with parasites at all stages of differentiation, from macroschizont to microschizont, expressed CD11b. Such parasitized cells occurred throughout the lymphoid tissues, being found in the thymus, spleen and lymph nodes, particularly the prescapular node draining the site of infection, the hepatic, mesenteric and precrural nodes, as well as in the reticulo-endothelial tissue of the liver, kidney, lung, abomasum, adrenal and pituitary glands. These observations provided the first evidence for a myeloid origin for the parasitized T. annulata cells found in infected bovine tissues and blood and suggested a mechanism whereby schizonts could transfer from cell to cell during mechanical infection with schizont-infected cells.     

Cross-reactivity of human leucocyte differentiation antigen monoclonal antibodies on porcine cells

Thirty-six subpanels of monoclonal antibodies (mAbs) supplied to the Fifth International Workshop on Human Leucocyte Differentiation Antigens were assayed on porcine peripheral blood leucocytes for cross-reactivity. Sixty-two of the 752 mAbs-stained porcine cells. These mAbs identified 30 different CD groups and will be valuable reagents in the field of porcine immunology.     

Monoclonal antibodies recognising differentiation antigens on porcine B cells

Nineteen monoclonal antibodies (mAbs) submitted to the porcine CD Workshop were analysed on porcine B cells from different lymphoid tissues by flow cytometry and immunohistochemistry. Six mAbs only bound to pig B cells and three of these were assigned cluster numbers, 76-7-4 (No. 001):CD1, K231-3B2 (No. 027):CD25 and CC51 (No. 100):CD21. Of the three remaining B cell positive mAbs, one was assigned to a swine cluster, CC55 (No. 101):SWC7, a second, LIG4 (No. 123), recognised cell surface IgM and the third, MUC106A (No. 072), remained unassigned.     

A novel LFA-1 activation epitope maps to the I domain

A panel of 21 alpha-subunit (CD11a) and 10 beta-subunit (CD18) anti-LFA-1 mAbs was screened for ability to activate LFA-1. A single anti-CD11a mAb, MEM-83, was identified which was able to directly induce the binding of T cells to purified ICAM-1 immobilized on plastic. This ICAM-1 binding could be achieved by monovalent Fab fragments of mAb MEM-83 at concentrations equivalent to whole antibody, was associated with appearance of the "activation reporter" epitope detected by mAb 24, and was completely inhibited by anti-ICAM-1 and LFA-1 blocking mAbs. The epitope recognized by mAb MEM-83 was distinct from that recognized by mAb NKI-L16, an anti-CD11a mAb previously reported to induce LFA-1 activation, in that it was constitutively present on freshly isolated peripheral blood mononuclear cells and was not divalent cation dependent for expression. The ICAM-1 binding activity induced by mAb MEM-83 was, however, dependent on the presence of Mg2+ divalent cations. Using an in vitro-translated CD11a cDNA deletion series, we have mapped the MEM-83 activation epitope to the "I" domain of the LFA-1 alpha subunit. These studies have therefore identified a novel LFA-1 activation epitope mapping to the I domain of LFA-1, thereby implicating this domain in the regulation of LFA-1 binding to ICAM-1.