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.     

Response of bovine gammadelta T cells to activation through CD3

Since the T cell receptor of γδ T cells is associated with CD3 molecules, it is a reasonable postulate that signal transduction through CD3 would occur in γδ T cells as it does in β T cells. However, while a small percentage of bovine γδ T cells divided in cultures of peripheral blood mononuclear cells (PBMCs) in response to stimulation by anti-CD3 monoclonal antibody (mAb) the majority of viable γδ T cells at the end of the culture period had not. This was assessed by carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis here and previously [Res. Vet. Sci. 69 (2000) 275]. When intracytoplasmic staining for interferon-γ (IFN-γ) was also used here to assess activation through CD3, a small proportion of γδ T cells (approximately 14%) produced IFN-γ during the first 4 h of culture and by 72 h of culture that number had doubled. By comparison, a much larger proportion of CD4 and CD8 T cells stimulated with anti-CD3 mAb divided and although the percentage of CD4 and CD8 T cells that produced IFN-γ at 4 h was similar to that of γδ T cells, by 72 h the majority of CD4 and CD8 T cells were IFN-γ⁺. Addition of IL-2 did not increase the proportion of γδ T cells that responded to anti-CD3 stimulation by cell division. To test the hypothesis that γδ T cells were inhibited from responding by other mononuclear cell populations within PBMC, monocytes were removed from the PBMC or γδ T cells were purified by magnetic-bead sorting. Only a small distinct population of the sorted cells underwent multiple cell divisions in response to anti-CD3 mAb and removal of monocytes resulted in only a moderate increase in γδ T cell replication. The anti-CD3 mAb stimulation system may provide a useful system to evaluate the difference in the requirements for activation and clonal expansion for γδ T cells versus β T cells.     

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.     

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.     

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.     

Report on the analyses of mAb reactive with porcine CD8 for the second international swine CD workshop

Based on an analysis of their reactivity with porcine peripheral blood lymphocytes (PBL), only three of the 57 mAbs assigned to the T cell/activation marker group were grouped into cluster T9 along with the two wCD8 workshop standard mAbs 76-2-11 (CD8a) and 11/295/33 (CD8b). Their placement was verified through the use of two-color cytofluorometry which established that all three mAbs (STH101, #090; UCP1H12-2, #139; and PG164A, #051) bind exclusively to CD8⁺ cells. Moreover, like the CD8 standard mAbs, these three mAbs reacted with two proteins with a MW of 33 and 35 kDa from lymphocyte lysates and were, thus, given the wCD8 designation. Because the mAb STH101 inhibited the binding of mAb 76-2-11 but not of 11/295/33, it was given the wCD8a designation. The reactivity of the other two new mAbs in the T9 cluster with the various subsets of CD8⁺ lymphocytes were distinct from that of the other members in this cluster including the standards. Although the characteristic porcine CD8 staining pattern consisting of CD8^low and CD8^high cells was obtained with the mAb UCP1H12-2, a wider gap between the fluorescence intensity of the CD8^low and CD8^high lymphocytes was observed. In contrast, the mAb PG164A, not only exclusively reacted with CD4⁻/CD8^high lymphocytes, but it also failed to recognize CD4/CD8 double positive lymphocytes. It was concluded that this mAb is specific for a previously unrecognized CD8 epitope, and was, thus, given the wCD8c designation. A very similar reactivity pattern to that of PG164A was observed for two other mAbs (STH106, #094; and SwNL554.1, #009). Although these two mAbs were not originally positioned in the T cell subgroup because of their reactivity and their ability to inhibit the binding of PG164A, they were given the wCD8c designation. Overall, five new wCD8 mAbs were identified. Although the molecular basis for the differences in PBL recognition by these mAbs is not yet understood, they will be important in defining the role of CD8⁺ lymphocyte subsets in health and disease.     

Reactivity of workshop monoclonal antibodies on paraformaldehyde-fixed porcine blood mononuclear cells

One hundred sixty-four monoclonal antibodies (mAbs) of the second international swine CD workshop were tested for their reactivity with porcine blood mononuclear cells before and after fixing the cells with varying concentrations of paraformaldehyde (PFA) (1, 5 and 10 g l⁻¹). A total of 38 (out of 134) positive reacting mAbs were significantly affected in their binding behavior on fixed cells. Modulation was seen as reduction in binding (staining intensity and/or % positive cells, n=18) or in elevated values (n=20). Modified mAb binding occurred after fixing cells with 5 to 10 g l⁻¹ PFA.     

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.     

Analyses of monoclonal antibodies reacting with porcine CD3: results from the Second International Swine CD Workshop

Among the 57 monoclonal antibodies (mAb) analyzed within the T-cell group from the Second Swine CD Workshop, six mAb fell within clusters T10 and T11 (No. 088, STH164; No. 148, FY1A3; No. 149, FY2C1; No. 150, FY1H2; No. 151, FY2A11; No. 169, BB23-8E6). The mAb within these two groups gave a similar appearance on flow cytometry and stained all peripheral blood T-cells as defined by CD4 and wCD8 staining. All six mAb precipitated a 24 kDa protein. On the basis of inhibition analyses performed as part of the workshop and from published data, the mAb define at least three epitopes. There is only minimal stimulation of resting peripheral lymphocytes, but four of the mAb produce strong stimulation in the presence of PMA. With the exception of STH164, all have been shown to react with CD3-transfected COS cells. The new mAb, therefore, react with three epitopes on porcine CD3 designated CD3a (BB23-8E6, FY2A11), CD3b (FY1A3, FY2C1), and CD3c (FY1H2). mAb STH164 appears to be reactive with another epitope, however, since its reactivity with CD3 has not been confirmed it is designated as wCD3.     

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.     

Vaccination of calves with Mycobacteria bovis Bacilli Calmete Guerin (BCG) induced rapid increase in the proportion of peripheral blood γδ T cells

Changes in the proportion of peripheral blood T cell subsets after subcutaneous inoculation of cattle with Mycobacterium bovis Bacille Calmette-Guerin (BCG) were studied. Calves were injected with approximately 8 × 10⁶ BCG bacillus and blood samples collected at weekly intervals for flow-cytometric analyses to determine the proportion of CD4⁺, CD8⁺ and γδ T cells. In addition, whole blood samples were stimulated in vitro with M. bovis purified protein derivative (PPD) and the secreted IFN-γ quantified by ELISA. Results showed cellular and cytokine changes which could be categorized into three phases. The first phase occurred within the first 2 weeks after vaccination involving an increase in proportion of WC1⁺ γδ T cells and a concomitant increase in the secretion of IFN-γ. These two responses peaked at 2 weeks and waned thereafter. The second phase involved an increase in the CD4/CD8 ratio as a result of an increase in the proportion of CD4⁺ T cells between 4 and 6 weeks. The third phase involved a decrease in the CD4/CD8 ratio due to an increase in the proportion of CD8⁺ T cells between 8 and 10 weeks. Surprisingly, the IFN-γ response was associated with changes in the γδ rather than the CD4⁺ or CD8⁺ T cells, suggesting that this cytokine was secreted by γδ-T cells. These results are consistent with the reported ability of γδ T cells to act rapidly and bridging the innate and classically adaptive immune responses.     

Porcine SWC1 is CD52--final determination by the use of a retroviral cDNA expression library

During the last decades for several species--e.g. swine--many mAb to leukocyte-specific molecules have been developed and clusters of differentiation corresponding to human CD could be established. However, for a significant amount of the raised mAb the corresponding antigens were not characterized on the molecular level and therefore preliminary clusters--in swine so-called Swine workshop clusters (SWC)--were established. These clusters contain antigens with currently no obvious orthologs to human leukocyte differentiation antigens. In this study, we describe the generation of a eukaryotic cDNA expression library from in vitro activated porcine peripheral blood mononuclear cells. Screening of this library with an antibody recognizing SWC1 enabled isolation and sequencing of cDNAs coding for the porcine SWC1 molecule. A BLAST search of the obtained sequence revealed that SWC1 is the orthologous molecule of human CD52. Therefore, our study provides the basis for comparative studies on the role of CD52 in different mammalian species. In addition, the established cDNA library can be used for investigation of additional SWC-defined molecules.     

Molecular cloning and characterization of cDNA encoding CD11b of cattle

CD18, the common β subunit of β₂-integrins, associates with four distinct chains to give rise to four different β₂-integrins: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (CR4), and CD11d/CD18. Previously, we and others showed that CD18 of LFA-1 serves as a receptor for Mannheimia haemolytica leukotoxin (Lkt). Level of expression of Mac-1 is higher than that of LFA-1 and other β₂-integrins on polymorphonuclear leukocytes (PMNs), which constitute the leukocyte subset most susceptible to Lkt. Hence, it is likely that CD18 of Mac-1 also mediates Lkt-induced cytolysis. Co-expression of CD11b and CD18 of cattle on Lkt-resistant cells is necessary to irrefutably demonstrate the role of Mac-1 in Lkt-induced cytolysis. This approach is hindered by lack of availability of complete sequence of cattle CD11b. Therefore, in this study, we cloned and sequenced the full length cDNA encoding cattle CD11b. The 3459 bp cDNA of cattle CD11b encodes a polypeptide of 1152 amino acids. The deduced amino acid sequence of CD11b of cattle exhibits 75% identity to that of humans and chimpanzees, 74% identity to that of dogs, and 70% identity to that of mice and rats. Availability of cattle CD11b cDNA should facilitate the elucidation of Lkt-receptor interactions in cattle and other species.     

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).     

Polymorphism of the CD4 and CD5 differentiation antigens in cattle.

Monoclonal antibodies (mAbs) specific for bovine CD4 and CD5 antigens have been found to identify polymorphic determinants on these molecules. In the case of CD5, mAb IL-A67 recognises one allotypic form of the antigen while four other CD5-specific mAbs in the workshop (CC17, CC29, BLT-1 and 8C11) recognise a second allotype. The CD4-specific mAbs submitted to the workshop reacted with the cells of all animals tested. However, a further two mAbs (CC26 and IL-A18) specific for CD4 were found to react with cells only from about 85% of animals tested. Sequential immuno-precipitation experiments together with family studies showed that the allotypes of CD4 and CD5 are both inherited in a simple Mendelian manner and are co-dominantly expressed. One of the CD5 allotypes was not detected in Bos taurus animals while the gene frequency of the second allotype was only about 10% in the B. indicus animals tested. The gene frequency of the CD4 allotype detected by CC26 and IL-A18 was similar in the two sub-species.     

Individual antigens of cattle. Differentiation antigens expressed predominantly on CD4- CD8- T lymphocytes (WC1, WC2).

The 14 mAbs representing workshop cluster 1 recognise a 215/300 kDa antigen expressed on a subpopulation of lymphocytes which express low levels of CD5 but are negative for other B and T cell markers defined by workshop antibodies. Separate studies with cDNA probes for bovine CD3 and T cell receptor indicate that these lymphocytes are gamma/delta T cells. It is of note that the different mAbs react with varying proportions of this cell population, suggesting that the antigen undergoes considerable post-translational modification. A further two mAbs, designated workshop cluster 2, react with a 37/47 kDa heterodimeric molecule expressed in a subpopulation of the WC1+ cells and on an additional small population of T lymphocytes. The cell populations recognised by the two mAbs are different although they overlap in some animals. It is suggested that these mAbs may be specific for T cell receptor molecules.     

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.     

Opsonization of yeast cells with equine iC3b, C3b, and IgG

The main opsonins in serum are antibodies and complement factor C3. The opsonization mechanisms including complement activation and deposition are important in studies of phagocytosis and of mechanisms of microbial immune evasion. The objective of the present study was to monitor the deposition of complement C3 and IgG from equine serum on yeast cells (Saccharomyces cerevisiae) using a flow cytometric immunoassay. Correlations were made between the opsonic coating and phagocytic capacity using equine blood neutrophils. In addition, the bound C3 fragments were characterized by SDS–PAGE and Western blot analyses.

Opsonic coating of yeast with equine C3 and IgG occurred rapidly with detectable levels with as little as 0.75% serum. C3 deposition was a result of complement activation and no passive adsorption was observed. When complement was inactivated, the fluorescence indicating IgG deposition increased 3–6-fold, indicating spatial competition between C3 and IgG at binding.

Opsonization with 1.5% serum led to suboptimal equine neutrophil phagocytosis of yeast cells which was dependent on complement activation by the classical pathway. With ≥6.25% serum, IgG contributed to opsonization and phagocytosis. With 50% serum and more, C3 was deposited also by the alternative pathway. Phagocytosis rates became optimal with 3% serum, and did not increase further with higher serum concentrations. The main form of C3 on the yeast cells was iC3b and the rest was C3b without any detectable breakdown products (C3c or C3dg). The equine complement components are similar in size to the human equivalents.

It may be concluded that opsonization of yeast particles leading to phagocytosis, occurs at very low serum concentrations (1.5%) and that it is dependent on activation of the classical complement pathway at this low opsonic level. This is an important finding for efficient host defense, e.g. extravascular phagocytosis at infection sites.     

Workshop studies on monoclonal antibodies in the myeloid panel with CD11 specificity.

Several putative anti-human and swine CD11-specific monoclonal antibodies (mAbs) were included in the myeloid section of the Third International Swine CD Workshop. Failure of clustering analysis to group these mAbs together prompted additional analyses to define the specificities of these mAb. Combination of one and two-color flow cytometry (FCM) and immunoprecipitation (IP) allowed the definition of the mAb into three CD11 groups. Cellular distribution of the molecules recognized by anti-human CD11b and c mAbs on swine cells proved to be significantly different from that found in humans.