A. Ezquerra

Porcine myelomonocytic markers and cell populations.

This review focuses in what is currently known about swine myeloid markers, the expression and function of these receptors in the biology of porcine myelomonocytic cells, the regulation of their expression along the different developmental stages of these cells and their utility to investigate the heterogeneity of monocyte and macrophage populations. Although the number of monoclonal antibodies recognizing surface antigens expressed on either swine granulocytes or monocytes is low compared with those available for human or mouse, they have contributed significantly to study the members of myeloid lineages in this species, allowing to discriminate different maturation stages of these cells in bone marrow and to reveal the heterogeneity of blood monocytes and tissue macrophages. Porcine myeloid cells share many similarities with humans, highlighting the relevance of the pig as a biomedical model.

Phenotypic and functional heterogeneity of porcine blood monocytes and its relation with maturation

Swine monocytes constitute a heterogeneous population of cells which can be divided into four subsets based on the expression of SWC3, CD14, CD163 and swine leucocyte antigen (SLA) DR markers. These subsets appear to represent different maturation stages in a pathway along which these cells up‐regulate the expression of SLA DR and CD163 antigens and reduce that of CD14. Differences in the expression of adhesion and costimulatory molecules are also patent, with a progressive increase in the expression of CD11a, wCD11R1, CD29, CD49d, CD61, CD1a and CD80/86, and a concomitant decrease in that of wCD11R2. Besides, these subsets differ in their capacity for tumour necrosis factor‐α (TNF‐α) production in response to lipopolysaccharide + interferon‐γ. The CD163+ CD14− SLA DR+ subset produces higher amounts of TNF‐α than the CD163− CD14+ SLA DR− subset, whereas CD163+ CD14+ SLA DR+ and CD163− CD14+ SLA DR+ subsets show intermediate values. CD163+ monocytes also display a higher ability to present soluble antigens to T cells than CD163− monocytes.

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.

Summary of workshop findings for porcine myelomonocytic markers

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

Summary of workshop findings for 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, Peyers 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.

Phenotypic characterization of porcine IFN-γ-producing lymphocytes by flow cytometry

Abstract We have developed a three-colour flow cytometric assay for phenotypic characterization of porcine IFN-γ-producing lymphocytes. Analyses of activated swine peripheral blood mononuclear cells (PBMC) showed a significant difference in the proportion of IFN-γ producing cells between young and adult animals (13.2±5.8% versus 34.2±5.7%). The majority of IFN-γ producing cells were αβ T lymphocytes, although there was also an important proportion of γδ T cells particularly in young animals. Within the αβ T lymphocytes, the double positive CD4 + CD8 lo subset, that contains memory T cells, produced high levels of IFN-γ, whereas the CD8 hi T cells ranged from low to high levels of IFN-γ. Also, consistent with a higher production by memory T cells, the CD45RA − subset of both CD4 + and CD8 + cells contained higher numbers of IFN-γ producing cells than the CD45RA + subset. Finally, no production of IFN-γ by either B cells (CD21 + ) or monocytes (SWC3 + ) was detected. This assay may be useful for the assessment of cell-mediated immunity in vaccine trials and may contribute to our understanding of the role of IFN-γ in protective immunity against important viral diseases of the pig.

Phenotypic characterization of monocyte subpopulations in the pig.

We have recently described the existence of two subsets of porcine monocytes based on the expression of CD163. In this study we compare the expression of a number of cell surface antigens in CD163+ and CD163- monocyte subsets using three-color flow cytometry. These monocyte subsets show differences with respect to the expression of MHC class II antigens (SLA-DR and DQ) and a variety of adhesion molecules (CD11a, wCD11c, wCD29, CD49d) that are expressed at higher levels on CD163+ monocytes, and of CD14 that is higher expressed by CD 163- cells. These differences on phenotype could reflect differences in the ability of these two subsets to migrate to tissues and may account for the higher allostimulatory capacity of CD163+ cells. In some aspects, the phenotype of CD163+ monocytes resembles that of mature macrophages. In vitro serum-induced maturation of monocytes into macrophages lead to the expression of SWC9 together with an increase in the expression of CD163 and a reduction in that of CD14. These results delineate a maturation pathway where CD14hiCD163-SWC9- monocytes develop into CD14loCD163+SWC9- monocytes and these cells into CD14loCD163+SWC9+ macrophages.

Monoclonal antibody 2F411 recognizes the α chain of a porcine β2 integrin involved in adhesion and complement mediated phagocytosis

Abstract The characterization of a new mAb, named 2F4 11 , specific for porcine myelomonocytic cells is described. This mAb immunoprecipitates a non-covalently linked heterodimer of 155000 95000 , which is expressed by granulocytes, monocytes and tissue macrophages but not by lymphocytes, erythrocytes or platelets. Immunoblot analysis localizes the 2F4 11 epitope on the largest subunit of the heterodimer. Mab 2F4 11 is able to block phagocytosis of complement-opsonized zymosan particles by PMN granulocytes and alveolar macrophages, as well as adherence to plastic surfaces of PMA-activated PMN. Together, these results suggest that mAb 2F4 11 recognizes the CD11b or α chain of the porcine complement type 3 receptor (CR3).

Targeting to porcine sialoadhesin receptor improves antigen presentation to T cells

Antibody-mediated targeting of antigen to specific antigen presenting cells (APC) receptors is an attractive strategy to enhance T cell immune responses to weak immunogenic antigens. Here, we describe the characterization of two monoclonal antibodies (mAb) against different epitopes of porcine sialoadhesin (Sn) and evaluate in vitro the potential of targeting this receptor for delivery of antigens to APC for T cell stimulation. The specificity of these mAb was determined by amino acid sequence analysis of peptides derived from the affinity purified antigen. Porcine Sn is expressed by macrophages present in the border between white and red pulp of the spleen and in the subcapsular sinus of lymph nodes, an appropriate location for trapping blood and lymph-borne antigens. It is also expressed by alveolar macrophages and monocyte-derived dendritic cells (MoDC). Blood monocytes are negative for this molecule, but its expression can be induced by treatment with IFN-a. MAb bound to Sn is rapidly endocytosed. MAb to sialoadhesin induced in vitro T cell proliferation at concentrations 100-fold lower than the non-targeting control mAb when using T lymphocytes from pigs immunized with mouse immunoglobulins as responder cells and IFN-a treated monocytes or MoDC as APC, suggesting a role of sialoadhesin in antigen uptake and/or delivery into the presentation pathway in APC.

Characterization of five monoclonal antibodies specific for swine class II major histocompatibility antigens and crossreactivity studies with leukocytes of domestic animals

A set of five monoclonal antibodies (mAb) against porcine major histocompatibility complex (MHC), or swine leukocyte antigens (SLA), class II molecules has been characterized. These mAbs appear to recognize monomorphic determinants on SLA-DR (2F4, 1F12 and 2E9/13) and SLA-DQ (BL2H5 and BL4H2) molecules, as assessed by flow cytometry and immunoprecipitation. By Western blot, the 2F4, 1F12, BL2H5 and BL4H2 epitopes were located on the beta-chains of these molecules. mAbs 2F4 and 1F12 crossreact with leucocytes of dog, cattle, horse and human; mAbs 2E9/13, BL2H5 and BL4H2 bind leucocytes of cattle but not those of human, dog and horse. These mAbs effectively blocked the mixed lymphocyte reaction and the proliferative response to viral antigens (African swine fever virus) and to staphylococcal enterotoxin B. Therefore, these mAbs can be useful reagents for studying MHC class II molecules of pig and crossreactive species, and the immunological processes where they are involved.