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).

Silent memory induction in maternal immune young animals.

Maternal immunity was shown to be an effector mechanism which does not include transfer of memory. 'Boosting' of maternal immunity by vaccination was not effective. Transferred maternal immunity negatively interfered with the induction of optimal protection by vaccination. Antibody formation was not observed after vaccination of maternally immune piglets. In contrast, induction of memory had occurred in animals under maternal immune suppression. Vaccination in young animals negatively interfered with or abrogated, effective maternal immune protection. There was no correlation between specific serum antibody titres in piglets and protection to PRV. Thus apart from protection provided by antibodies contributions of other soluble factors and the cellular immune compartment as represented in colostrum and/or milk were important for protection.

A mouse model to study immunity against pseudorabies virus infection: significance of CD4+ and CD8+ cells in protective immunity.

In this study we firstly established a vaccination/challenge model to study pseudorabies virus infection in mice. The mouse model was used to investigate the significance of CD4+ and CD8+ cells and of IFN gamma production in protective immunity. Functional depletion of CD4+ and CD8+ and IFN gamma was obtained in vivo by intraperitoneal injection of alginate-encapsulated anti-CD4, -CD8 or -IFN gamma producing hybridoma's before and at the moment of vaccination. The observed protective immunity was correlated with underlying immunologic responses such as PRV-specific DTH reactivity, lymphoproliferation and cytotoxicity. The significance of CD4+ and CD8+ cells and of IFN gamma production was also investigated for these immunological responses by the same in vivo depletion technique. The results demonstrated that protective vaccination of mice, that could be induced by immunization with 10(7) plaque forming units of the avirulent PRV mutant NIA3 TK-, was characterized by a typical anti-viral Th1 type immune response. A clear PRV-specific, CD4-dependent DTH reactivity and a classical CD8-dependent, MHC-restricted cytotoxicity was induced after protective immunization and the humoral immune response had a bias towards PRV-specific IgG2a formation. In vivo treatment with anti-CD8 and anti-IFN gamma demonstrated that the cytotoxic response and humoral IgG2a response, respectively, were strongly reduced, whereas protection against lethal challenge was unaffected. On the other hand anti-CD4 treatment reduced the induced protection so that 30% of the mice died after lethal challenge. The results of our study demonstrated that CD4+, DTH like effector cells are a crucial effector mechanism for protective immunity against PRV.

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.

Monoclonal antibodies putatively identifying porcine B cells.

Comparison was made of the binding of 38 test and three standard monoclonal antibodies (mAbs) to B cells from various pig lymphoid tissues by flow cytometry (FCM) and immunohistochemistry. Some mAbs were also tested on B cells from foetal pig tissues. Twenty of the new mAbs bound, though to variable degrees, to porcine B cells but only three were given cluster assignations: C35 (#147) and BB6-11C9 (#167) were assigned to wCD21 and 2F6/8 (#057) was assigned to SWC7.

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.

Jejunal and ileal Peyer's patches in pigs differ in their postnatal development.

The postnatal development of the jejunal and ileal Peyer's patches was studied before and after weaning in 1-, 1.5- and 2-month-old pigs. The follicles of the jejunal Peyer's patches grew with age and were two times longer and wider in specified pathogen-free and conventional pigs than in germ-free animals, thus indicating an influence of the living microbial antigens from the gut lumen. In germ-free pigs the size of the ileal Peyer's patch follicles increased between the 1st and 2nd month, whereas in the specified pathogen-free and conventional animals these follicles were comparable in size in all three age groups. In 1- to 1.5-month-old pigs the interfollicular area of jejunal Peyer's patches was wider (0.1 +/- 0.04 mm) than that of the ileal Peyer's patch (0.04 +/- 0.03 mm). Immunohistological studies showed that in germ-free pigs preferentially surface IgM+ but few IgA+ B cells were present in the follicles, domes and dome epithelia. In specified pathogen-free and conventional pigs the B cells expressed different levels of surface or cytoplasmic IgM or IgA. In all groups studied, more T cells were observed in the jejunal than in the ileal Peyer's patch. Here, few T lymphocytes were found because of the small interfollicular areas. Small numbers of Null cells were distributed in the interfollicular regions of all animals. The results show that living microbial antigens have a major influence on the jejunal and ileal Peyer's patches in pigs. The morphological differences between the two types of Peyer's patches are an indication that they develop differently during postnatal life. So far it remains unclear whether these morphological differences reflect a specific function of the pig's ileal Peyer's patch, such as the expansion of the genetically determined B cell repertoire as has been reported for sheep.

Immunohistological characterization of the local cellular response directed against pseudorabies virus in pigs.

The aim of this study was to investigate the kinetics of a primary and secondary immune response against pseudorabies virus (PRV). Pigs vaccinated with strain 783 and unvaccinated pigs were challenged with wild-type PRV by either intranasal or subcutaneous infection. Non-challenged pigs were used as controls. On days 1, 3 and 7 after challenge, tissues from the site of infection, and the tonsils of intranasally and the draining lymph nodes of subcutaneously challenged pigs were sampled. Immunohistological staining was used to characterize the various cell populations at the primary site of virus replication and in the lymphoid tissue. Tissue sections were stained for the T-cell markers CD2, CD3 gamma delta, CD4 and CD8, for the B-cell markers IgM, IgA and IgG, for a macrophage marker, and for PRV antigen. After challenge, PRV was detected during a shorter period in vaccinated pigs, and was less disseminated than in unvaccinated pigs. Cellular infiltrates were detected both in the nasal mucosa and the subcutaneous tissue of both unvaccinated and vaccinated pigs. Cell infiltrates, however, appeared earlier in vaccinated than in unvaccinated pigs, indicating a difference in kinetics of the primary and secondary immune response. The appearance of T-cells preceded the appearance of B-cells, but the proportion of the various subsets did not differ between unvaccinated and vaccinated pigs. These findings suggest that the early immune response in vaccinated pigs may contribute to the rapid clearance of virus at the primary site of infection. In addition, T-cells appear to have a more important role in the clearance of PRV than B-cells.

Monoclonal antibodies recognising differentiation antigens on porcine B cells.

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

Distribution of lymphocyte subpopulations in thymus, spleen, and peripheral blood of specific pathogen free pigs from 1 to 40 weeks of age.

Using flow-microfluorometry analysis and cluster determinant (CD) markers, we studied how lymphocyte subpopulations in lymphoid organs of specific-pathogen-free pigs developed in pigs from birth to young adulthood. Cell suspensions of the thymus and spleen were prepared and peripheral blood cells were collected at 1, 4, 10, and 40 weeks of age. Tissue sections of the thymus and spleen were stained with monoclonal antibodies directed against CD2 and immunoglobulin to localize the CD2-Ig- lymphocyte subpopulation. In the thymus, only limited changes were observed in the lymphocyte subpopulations with time. Most thymocytes expressed CD4 or CD8 or both. Most CD2-Ig- cells or, 'null cells', (5-13%) were observed in the medulla of the thymus and probably represented a recirculating cell type. In the spleen and blood the percentage of CD2+ and Ig+ cells increased significantly with time, the former increasing from about 30-60% owing to an increase of CD8+ cells. Therefore, the selective increase of the CD8+ population also caused the CD4/CD8 ratio to change. Although CD2+ cells in the spleen and blood are positive for CD4 or CD8, but not for both, quantities of CD4+ CD8+ cells were also observed. Half of the lymphocytes in the spleen and blood were typed as null cells at 1 week of age and decreased in proportion to the increase of the CD8+ and Ig+ cells. Nevertheless, quantities of null cells were still present in the spleen blood at 40 weeks of age. Almost all these were located in the red pulp of the spleen. This study indicates an effect of age and housing conditions on the distribution of the lymphocyte subpopulations, and especially on the CD8+ subset. Quantities of CD4+CD8+ cells as well as CD4-CD8- were observed in blood, but also in spleen of pigs. The function of high numbers of null cells directly after birth are discussed.

Development of the B- and T-cell compartments in porcine lymphoid organs from birth to adult life: an immunohistological approach.

Using immunohistological techniques, we studied the development over time of B- and T-cell compartments in the lymphoid organs of specific-pathogen-free pigs. Tissue samples were collected at various time-points, starting 2 days before the pigs were born until the pigs were 10 months old. The samples were collected from the spleen, thymus, peripheral lymph node, mesenteric lymph node, duodenum, jejunum, ileum, jejunal Peyer's patch and ileal Peyer's patch. Monoclonal antibodies specific to B- and T-cells were used to identify where the following cells were localized: IgM-B cells (cells positive to surface immunoglobulin), IgM-, IgG- and IgA-containing cells (cells positive to cytoplasmic immunoglobulin), and CD2-, CD4- and CD8-positive cells. The development of the B- and T-cell subpopulations in each organ was analysed. Two days before birth, most organs contained quantities of IgM-B cells. The spleen, lymph nodes, Peyer's patches and, notably, the thymus, contained some immunoglobulin-containing cells (Ig-CC); this finding indicates that pigs have cells that secrete immunoglobulins before birth. Just after birth, the incidence of Ig-CC increased in most organs; first IgM-CC increased, then either IgG- or IgA-CC increased, depending on the organ. T-cell development was observed clearly in spleen and in the lamina propria of the small intestine, in contrast to other organs, in which the T-cell compartments containing various T-cell subpopulations were well developed before birth. Comparison of the incidence of CD4+ and CD8+ cells showed that the CD4:CD8 ratio of these cells in the spleen, lymph nodes, Peyer's patches and small intestine is low, especially in adult pigs, compared with the CD4:CD8 ratio in other species. Weaning had little influence on the incidence of B- and T-cells in lymphoid organs. This study is the first immunohistological survey to describe the development of the major B- and T-cell subpopulations in various lymphoid organs of pigs, and it should be useful for future immunopathological and comparative immunological studies in pigs.

Induction of an enteric Ig-response against ovalbumin and stimulation of the response by cholera toxin and its B-subunit in mice.

We induced ovalbumin (OA)-specific antibody responses in the murine small intestine and quantitated the responses by counting ovalbumin-specific antibody-containing cells (OACC) in the intestinal lamina propria, and by measuring anti-OA Ig titers in intestinal secretions. OA is a "weak" mucosal antigen and we could only induce intestinal anti-OA responses by intraperitoneal, primary injection of OA in water-in-oil emulsion and a mucosal booster with OA. Double staining of OACC for OA- and isotype-specificity demonstrated that 95% of all mucosal (intestinal) OACC produced IgA, whereas 95% of all systemic (splenic) OACC produced IgG. The intestinal anti-OA response could be stimulated by addition of cholera toxin (CT) or cholera toxin B-subunit (CTB) during the mucosal booster immunization. The stimulatory effect of CT did not depend on covalent coupling of CT and OA, while the stimulatory effect of CTB required covalent coupling of CTB with OA. Mucosal (intraduodenal) application of OA as such does not prime for a mucosal and systemic OACC response but induces tolerance. We demonstrated that coupling of OA with CT or CTB could overcome the inability of mucosal application of OA to prime for a mucosal OACC response. Although CT proved to be much more effective than CTB in stimulation of mucosal immune responses and possibly in prevention of tolerance induction, our results indicate that CTB is a useful (and safer) alternative.