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.     

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.     

Summary of the first round analyses of the Third International Workshop on Swine Leukocyte Differentiation Antigens.

The reactivity of 155 monoclonal antibodies submitted to the Third International Workshop on Swine Leukocyte Differentiation Antigens, together with 41 internal standards, was analysed by flow cytometry on 29 different pig cell targets as well as two human cell targets as a means of establishing suitable panels of monoclonal antibodies for more detailed clustering analyses by the various subsections of the workshop. Results were collected either without further gating, with gating based on FS/SS characteristics or with gating based on the co-expression of a reference antibody in two-colour flow cytometry. The CD or SWC reactivity of the internal standards had been established in previous workshops. Data sets were subsequently analysed by statistical clustering using the Leucocyte Typing Database IV software. The resulting 18 cluster groups were allocated to the appropriate second round sections of the workshop, after reviewing the overall cellular reactivity of each cluster as well as the specificity of known standards which clustered in a group.     

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.     

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.     

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).     

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 workshop findings for porcine adhesion molecule subgroup

Fifty-nine monoclonal antibodies (mAb) were assigned to the adhesion section of the Second International Swine CD Workshop. They were analysed for their reactivity to selected lymphoid cell populations, as well as to non-lymphoid cell lines. Cell lysate ELISAS and Western Blot analyses were also carried out. As a result, thirteen separate cluster groups emerged (p>0.95). Workshop assignments for adhesion molecules were made: wCD29/49 for mAbs UCP1D2 (#133) and FW4-101 (#165), and PNK-I (#194) and MUC76A (#025) could be assigned to wCD18. For one cluster (FQ1D7, #161 and 2F4, #069) the cellular distribution and MW were characteristic for MHC Class II, and another cluster comprising several antibodies which appeared to recognise MHC Class I. Other clusters could not be assigned to cell surface structures known to be linked to cellular adhesion, however, two further antibodies, 335-2 (#112) and FG1F6 (#156), could be added to SWC1, and the new SWC8 was defined by MIL3 (#077) and MUC20A (#029), binding a ligand of 29–32 kDa. Clustering for these two antibodies was confirmed by blocking studies. The cellular distribution is known for MIL3, recognising an epitope present on granulocytes, B cells, and a subset of T cells expressing CD8 at high intensity.     

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.     

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.     

Analyses of monoclonal antibodies reacting with porcine wCD6: Results from the Second International Swine CD workshop

Among the 57 monoclonal antibodies analyzed within the T-cell group of the Second International Swine CD Workshop, one mAb fell within cluster T14a that included the CD6 standard a38b2 (No. 175). The new mAb MIL8 (No. 082) and a38b2 both precipitated from activated T-cells a 150 kDa monomeric protein. Staining patterns on the various cell types were similar. There was no inhibition of binding of either mAb to peripheral blood T-cells with the opposite mAb. The new mAb, MIL8, reacts with a separate epitope on porcine wCD6.     

Workshop studies with monoclonal antibodies identifying a novel porcine differentiation antigen, SWC9

Two monoclonal antibodies (mAb) within cluster M4 of the myeloid section of the Second International Swine CD Workshop, C4 (No. 144) and PM18-7 (No. 192), showed reactivity with thymocytes and among cells of myelomonocytic origin with mature macrophages but not with monocytes and granulocytes. Both mAb recognize a protein showing two bands of 205 kDa and 130 kDa under both reducing and non-reducing conditions. Although epitope mapping with these mAb could not be performed, this cluster received the SWC9 designation.     

Cross-reaction of anti-human CD monoclonal antibodies on guinea pig cells: a summary of the guinea pig section of the HLDA8 animal homologues data

A panel of 377 commercially available monoclonal antibodies (mAbs) specific for a total of 144 CD antigens was submitted to the animal homologue section of the Eighth International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies in a range of vertebrate species. Each of the mAbs in this study was screened for positive reactivity with guinea pig splenocytes by flow cytometry. In the first phase of this study 36 of the total 367 mAbs (9.81%) cross-reacted with splenocyte surface molecules. The majority (26 of 36) of these cross-reactive mAbs were analysed further to confirm appropriate cell subset expression by two-color immunofluorescence. Our results indicate that 15 anti-human CD9, CD10, CD14, CD20 (two clones), CD22, CD25, CD29 (two clones), CD32, CD47 (two clones), CD49d, CD49e, and CD86 mAbs exhibit clear cross-reactivity with guinea pig splenocytes. These mAb can potentially be added to the limited repertoire of reagents available for studies in this model system. This data clearly indicates that mouse anti-human CD mAb guinea pig cross-reactions have been defined and that an aim of this HLDA8 section has been fulfilled, i.e., to identify mAbs which recognize conserved, species-independent CD epitopes. These results will contribute to the availability of mAbs and tools in veterinary medicine and immunology.     

Summary of workshop findings for porcine B-cell markers.

Based on cluster groups from the first-round analyses of the Third International Swine CD Workshop, 38 monoclonal antibodies (MAbs) including eight internal controls were analysed by flow cytometry (FCM) and immunohistochemistry (IH) in the second-round analysis of the B-cell section of this workshop. Targets in this section included peripheral blood lymphocytes and cells isolated from ileal Peyer's patches (PP), mesenteric lymph nodes (MLN) of adult animals, bone marrow cells from newborn piglets and thymus cells isolated from foetuses at day 105 of gestation.Immunohistochemistry of these 38 MAbs identified four sets, whose ligands were co-expressed with CD21, which showed a tissue distribution compatible with specificity for cells including those of the B-cell lineage. Another group of miscellaneous antibodies appeared to identify other cells, several antibodies were negative. Two-colour flow cytometry (2C-FCM) was carried out by pairing each antibody of interest with antibodies to SWC7, CD21, sIgM and a polyclonal rabbit anti-swine immunoglobulin antiserum (RaSwIg).The anti-CD21 MAb BB6-11C9 (no. 20) and IAH-CC51 (no. 19), established in previous workshops, as well as the cross-reactive anti-human CD21 B-1y4 (no. 146), clustered together in FCM analyses of the first round and showed similar cellular distribution in IH. A further cluster was formed by the standard CC55 (no. 55) and 2A10/8 (no. 102) submitted as SWC7 specific. The second SWC7 standard 2F6/8 (no. 100) clustered separately, but IH showed an identical pattern of reactivity to the other SWC7 MAb.Unfortunately, this work could not identify any other novel clusters with specificity for B-cells, as the statistical clustering of other MAbs could not be substantiated by IH or subsequent two-colour-FCM work. However, we could identify MAb with similar cellular distribution. The ligands for the cross-reactive anti-human CD40 G28.5 (no. 25) and STH224 (no. 153) were expressed on very similar targets, similarly the ligands for the MAb pair JM1H1 (no. 139) with BB6-10A10 (no. 142) and the MAb pair 3F7/11 (no. 115) with 1C2F10 (no. 187).     

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.     

Cross-reactivity of mAbs to human CD antigens with sheep leukocytes

A panel of 377 commercially available mAbs was submitted to the animal homologue section of the Eighth International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies in a range of vertebrate species. Eight commercial suppliers participated by providing isotype controls and mAbs specific for a total of 144 CD antigens. In this study, we describe the results of flow cytometric testing of the reactivity of these mAbs with leukocyte populations isolated from blood, bronchoalveolar lavage, and ileal Peyer's patches of sheep. A total of 52 mAbs were identified as potentially reacting with sheep blood leukocytes in the first round of screening with blood leukocytes. In the second phase, reactivity of selected mAbs was further analyzed by repeating the screening with blood leukocytes at an independent facility. Screening of selected mAbs for reactivity with myeloid antigens was completed with alveolar macrophages and screening for reactivity with B cell antigens was completed with ileal Peyer's patch B cells. This screening identified mAbs that consistently reacted with both putative myeloid (CD10, CD22, CD23, CD27, CD29, CD32, CD49d, CD81, CD86, CD88, CD163, CD165) and B cell (CD10, CD22, CD23, CD27, CD29, CD32, CD49d, CD81, CD86, CD88, CD165) activation or differentiation antigens. Further studies will be required to determine if each mAb cross-reacts with an orthologous leukocyte antigen.     

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.     

Cross-reactivity of mAbs to human CD antigens with cells from cattle

A panel of 377 commercially available mAbs were submitted to the animal homologue section of the 8th International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies on different animal species. In this study we describe the results of testing the mAbs on cattle cells by flow cytometry and Western blot. Eight commercial suppliers participated, providing mAbs to a total of 144 CD antigens plus controls. Fifty-two mAbs were identified as potentially staining cattle cells in the first round screen. In the second phase, 38 mAbs were confirmed as staining cattle cells. This included some that may recognise polymorphic determinants and others with atypical distribution patterns compared to humans. mAb to human CD9, CD11a, CD14, CD18, CD21, CD23, CD29, CD44, CD45R, CD47, CD49d and CD172a cross-reacted with bovine cells and mAb to CD22, CD88, CD119 and CD163 stained CD antigens that have not previously been identified in cattle.     

Identification of anti-human CD antibodies reactive with rhesus macaque peripheral blood cells

Three hundred and seventy seven commercially available monoclonal antibodies (mAb) were tested for their cross-reactivity with rhesus macaque (Macaca mulatta) peripheral blood cells. These antibodies were collected by the animal homologue section of the HLDA8 Workshop in order to assign their potential applicability for in vitro assays. Reactivity of each mAb with lymphocyte, monocyte and granulocyte populations obtained from peripheral blood of adult rhesus macaques was evaluated. Single-colour flow cytometry and indirect labeling method was used in first-round screening. Based on their reactivity with rhesus macaque cells 57 positive mAb were selected for second-round testing. Multi-colour flow cytometry and combinations of direct and indirect labeling was used to compare the reactivity of the respective mAb. In addition, reference reagents known to react with rhesus macaque CD3, CD20 and CD56 were used to further characterization of the reactivity of the selected 57 mAb on peripheral blood cells.     

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.