Swine CD Research Articles

Development of new immune reagents for swine health, vaccine and disease studies

Abstract Current research efforts require a broad range of immune reagents; those available for pigs are limited. The goal of the US-UK Collaborative Swine Immune Toolkit Initiative is to generate priority immune reagents, based on international input, and pipeline them for marketing. UK researchers focus on mucosal targets, including production of monoclonal antibodies (mAbs) to chemokine receptors and IgE. Plans for mAbs to each 7-transmembrane G-protein coupled receptor (CCR3, CCR9, CCR10, CXCR1, CXCR6, F4/80) are to target peptides from extracellular loop 2 (ECL-2) based on threaded protein structures. These peptides have been designed for CCR3 and CCR9 and mAb plans are underway. US efforts are aimed at expression of soluble proteins and swine CD molecules, and production of panels of mAbs. The team has set up collaborations with commercial partners for protein expression and mAb production, and updated protocols to evaluate reagent specificity. New panels of mAbs reactive with IL-6, IFNβ and IFNγ are currently being evaluated. Immunizations are underway to produce panels of mAbs for CXCL10, CX3CL1 (fractalkine), IL-13, IL-17A, IL-17F, IFNω1 and IFNω5. Plans for mAbs to porcine CD1d using 4 synthetic peptides are in progress using CD1d knockout (KO) mice and screening on CD1d KO pig cells. Plans continue to use reagents produced to develop new assays for swine immune markers, e.g., multiplex assays and intracellular staining to provide the veterinary community with new commercial reagents and techniques for their research efforts. Tools and reagents generated by this project will undoubtedly advance swine immune, disease and biomedical research efforts. Supported by USDA ARS, NIFA grant #2015-67015-23216 and BBSRC grant BB/M028232/1.

Dynamic Expression of the Signal Regulatory Protein α and CD18 on Porcine PBMC During Acute Endotoxaemia

Endotoxaemia elicits a massive inflammatory insult affecting the beta2 integrin CD18. Being an adhesion molecule, CD18 is pivotal in inflammation and, moreover, exiting data suggest that CD18 is a lipopolysaccharide (LPS) receptor. Early LPS-induced inflammation is regulated by the signal regulatory protein (SIRPalpha), which is identical to the porcine panmyelocytic marker swine CD workshop 3 (SWC3), and LPS-induced downregulation of SIRPalpha has been described in vitro. The dynamic SIRPalpha/SWC3 and CD18 expression on peripheral blood mononuclear cells (PBMC) in vivo during LPS-induced inflammation is the focus of this study. Pigs were randomized into LPS (n = 12) or control (n = 6) groups. At start 0 min, LPS infusion was stepwise (2.5-15 mug/kg/h, 30 min) followed by maintenance infusion (2.5 mug/kg/h, 330 min). PBMC were isolated at 0, 60, 240 and 360 min, and two-colour flow cytometry was performed using monoclonal antibodies identifying SWC3 and CD18. Viability was tested using 7-amino-actinomycin D. LPS dramatically changed the relative distribution of circulating myeloid cells. At 60 min monocytes disappeared. This was followed by reappearance of a distinct population with low CD18 and SIRPalpha/SWC3 expression. Cell sorting showed that the appearing population comprised band neutrophils and apoptotic/dead cells. The remaining monocytes expressed less CD18 at 360 min than the controls (P = 0.03). The appearance of a distinct cell population comprising apoptotic cells and band neutrophils consistent with LPS-induced apoptosis, and decreased CD18 expression on monocytes suggests that early CD18 downregulation is profitable for the host in a situation with an intense LPS stimulus.

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.

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.

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) γ/δ 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.

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).

Characterization of monoclonal antibodies assigned to the CD45 subgroup of the Third International Swine CD Workshop

As a result of the first-round cluster analysis, a panel of 16 novel monoclonal antibodies (mAbs) was assigned for detailed analysis to the CD45 subgroup of the Third International Swine CD Workshop. The specificity of the mAbs was initially determined by examining their reactivity with Chinese hamster ovary (CHO) cells engineered to express individual isoforms of porcine CD45. These analyses indicated that seven of the mAbs (PG77A, PG96A, PG167A, PGB78A, 3C/9, MIL13, NHT 101) recognized the portion of the CD45 molecule encoded by the A exon (CD45RA), while one (MIL15) was specific for that portion encoded by the C exon (CD45RC). In each case, the designation was supported by the demonstration that the molecular weight(s) of the recognized antigen(s) in porcine mononuclear cells, as determined by immunoprecipitation, corresponded to the predicted size(s) according to their specificity. As expected, a similar correlation was obtained for five standard mAbs whose specificity for either common or restricted epitopes of porcine CD45 had been established in previous workshops. Screening of the remaining 174 mAbs that comprised this workshop but were excluded from the CD45 subgroup by cluster analysis failed to detect any additional ones reactive with the porcine CD45-expressing cells.

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).

A porcine cell surface receptor identified by monoclonal antibodies to SWC3 is a member of the signal regulatory protein family and associates with protein-tyrosine phosphatase SHP-1.

SWC3 was defined at the First International Swine CD Workshop as a specific myelomonocytic antigen of 230 kDa with mAbs 74-22-15, 6F3 and DH59B. In this report, we describe two new mAbs (BL1H7 and BA1C11) that react selectively with granulocytes, monocytes and macrophages. These monoclonal antibodies (mAbs) recognize a molecule in the range of 90-115 kDa in immunoprecipitation and/or Western blotting analyses. Two-colour FACS analyses showed that the distribution of BL1H7 and BA1C11 antigens was identical to that of SWC3. Moreover, in this assay, mAb 74-22-15 appeared to partially block the binding of mAbs BL1H7 and BA1C11, suggesting that all these mAbs reacted with the same or spatially close epitopes. Cross-blocking analyses indicated that it was the case with mAbs 74-22-15 and BL1H7. Immunoprecipitation experiments with mAbs 74-22-15, BL1H7 and BA1C11, followed by immunoblotting with mAb BL1H7 confirmed that all three mAbs recognize the same molecule. Analysis of the N-terminal sequence carried out on the affinity purified protein revealed homology with members of signal regulatory protein (SIRP) family. Like other members of this family, after treatment with sodium pervanadate, SWC3 became phosphorylated in tyrosines, and associated with the protein-tyrosine phosphatase SHP-1.

Monoclonal antibodies 2F6/8 and 2A10/8 recognize a porcine antigen (SWC7) expressed on B cells and activated T cells

This report describes the production and characterization of two monoclonal antibodies (mAbs), 2F6/8 1 MAb 2F6/8 was assigned to SWC7 cluster in the Second International Swine CD Workshop. 1 and 2A10/8, that recognize a porcine antigen (SWC7) expressed on B cells in lymphoid tissues. The antigen was not detectable on resting PBMC but its expression could be induced after treatment with phorbol esters (PMA) but not by ConA, PWM, LPS or Ca ionophore. Kinetic studies showed that the antigen was expressed 24 h after PMA treatment, peaked at day 2 or 3 and slightly declined by day 6. Interestingly, the antigen was also found on a subset of CD3+ T cells, with levels of expression similar to those of B cells. By immunohistochemistry, the antigen was detected on follicular dendritic cells of germinal centers in tonsils, spleen, lymph nodes and Peyer's patches. MAb 2F6/8 precipitates a molecule of approximately 40 kDa under non-reducing conditions, and 24 kDa under reducing conditions. The restricted and tightly regulated expression of this antigen may reflect an important role in B cell differentiation within the germinal center. These mAbs will be useful reagents for phenotypic analysis of porcine lymphoid cell populations by flow cytometry and immunohistochemistry.

Immunoprecipitation studies of monoclonal antibodies submitted to the Second International Swine CD Workshop

Immunoprecipitation studies of monoclonal antibodies submitted to the Second International Swine CD Workshop

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.

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.

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.

Appendix: Flow cytometry data included in first round cluster analysis

Tables 1-16 contain the results of flow cytometry analyses of 18 different cell types labelled with all 176 monoclonal antibodies submitted to the Second International Swine CD workshop. The 19 standards (mAb #171-#189) characterized for their specificity in the first workshop served as controls. Data derived from 40 different analyses are presented as % positive cells.

Determination of the specificity of CD45 and CD45R monoclonal antibodies through the use of transfected hamster cells producing individual porcine CD45 isoforms

The exclusive presence of the tyrosine phosphatase, CD45, on the surface of hematopoietic cells coupled with the differential expression of its various isoforms has enabled the selection of lymphocyte subsets based on their reactivity with monoclonal antibodies (mAbs) specific for one or more CD45 species. As a prelude to defining the specificity of anti-porcine CD45 mAbs for this purpose, Chinese hamster ovary cells were transfected with constructs containing cDNAs encoding the extracellular and transmembrane domains of four pig CD45 isoforms. Cells expressing only one of the three predominant types (CD45RO, CD45RC, and CD45RAC) or the minor species (CD45RA) of porcine CD45 on their surface were sorted based on positive reactivity with the CD45 mAb K252.1E4. Initially, these CD45 + cells were used as a source of antigen when determining the specificities of nine mAbs, which had been identified during the First and Second International Swine CD Workshops as being reactive with porcine CD45. Later, cloned cell lines were established and phenotypically verified for the production of the correct CD45 transcript by RT-PCR. Binding of two more mAbs (74-9-3 and 10-14-1) in addition to the original mAb panel to these cell lines was assessed by using a cell ELISA in lieu of one-color flow cytometry. Despite differences in detection methodology, identical mAb binding results were obtained. As anticipated, CD45 mAbs K252.1E4, MAC 323, and 74-9-3 which recognize an epitope(s) in the common portion of porcine CD45, reacted with cells expressing any one of the four isoforms but not with the parental CHO cells. In contrast, none of the restricted (CD45R) mAbs bound to cells producing the CD45RO isoform which lacks any of the alternate extracellular regions. However, three of these mABs (6E3/7, FG2F9 and STH267) did react specifically with the CD45RA and CD45RAC isoforms, indicating their specificity for an epitope(s) encoded by the CD45 A exon. The other four CD45R mAbs (MAC326, 3a56, MIL5, and -a2) recognized the CD45RC isoform. Interestingly, only CD45R mAb MAC326 also bound to cells expressing the CD45RAC isoform, suggesting that the epitope(s) recognized by the other three may have arisen due to the juncture of the invariant 5′ leader sequence with the CD45C exon. The eleventh mAb (10-14-1) was unique in that it did not react with any of the expressed CD45 isoforms. This inability coupled with the previously demonstrated recognition of a 240 kDa protein suggests that it may be specific for CD45RABC. Overall, this panel of CD45R mAbs should prove useful for obtaining functionally distinct subpopulations of B and T lymphocytes.

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.

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 γ δ TcR established the majority of null cells are γ δ T cells. Use of this mAb in further comparisons demonstrated the γ δ 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 − γ δ 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.

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.

Summary of the first round analyses of the Second International Swine CD Workshop

The reactivity of 176 monoclonal antibodies (mAb) submitted to the Second International Swine CD Workshop, together with 19 internal standards, was analyzed by flow cytometry on 16 different cell types as a means of establishing the proper cell subset for later detailed clustering analyses. The exact CD subset reactivity of the 19 internal standard mAb had been characterized in the First International Swine CD Workshop. The flow cytometric analyses resulted in 40 data sets which were then subjected to statistical clustering using the Leukocyte Typing Database IV (LTDB4) software. As result of this work, 22 clusters were defined. After review of these results, panels of mAb from the defined first round clusters were assigned to cell subsets. The respective mAb in those first round clusters were then distributed to subset group researchers for further examination during the second round of the workshop.