Richard M. Binns

Analysis of monoclonal antibodies that recognize γδ T/null cells

Abstract 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.

The immune system of the respiratory tract in pigs

Although the lung is not a lymphoid organ it contains large numbers of lymphocytes. These can be found in different compartments: (1) the pulmonary intravascular pool, which is organ-specific and shows a unique migration pattern; (2) the interstitial lymphocyte pool, which is equivalent in size to the whole blood pool; (3) the bronchus-associated lymphoid tissue (BALT) which develops as a result of microbial stimulation; (4) the intraepithelial and lamina propria lymphocytes of the bronchi, with their typical subset composition; (5) the lymphocytes in the bronchoalveolar space, which can be sampled by bronchoalveolar lavage. The size and kinetics of the lymphocyte pools have been studied in the pig in more detail than in most other species. Despite this organotypic compartmentalisation of the pulmonary lymphoid cells in the pig, the lung is part of the integrated mucosal immune system, as shown by protective oral immunisation against the lung-pathogenic bacteria Actinobacillus pleuropneumoniae. The lung immune system in the pig is not only of veterinary interest, but also a relevant model for the human respiratory tract.

Lymphoid tissue structure and lymphocyte trafficking in the pig

The organised lymphoid tissues of the pig, though conventionally mammalian in most respects, show several distinctive properties in their structure and physiology. Specialised antigen-presenting lymphoid organs function at three compartmental levels: the body surfaces, their draining lymph nodes and the spleen in the bloodstream. Other organs act as lymphocytic depots and sites of phagocytosis of debris. Pig lymphocytes recirculate continuously through these organs and through different forms of inflammatory change, experimentally induced by mitogens and cytokines, using a spectrum of distinctively different physiological mechanisms. These tissues in the young pig differ both in the resting and activated cell subsets and the molecules involved, many of which remain to be completely defined. Intriguing insights are evident in the subtlety of regulation of the specificity, level and foetal ontogeny of trafficking mechanisms in these different tissues, though they are as yet poorly explained. This subtle molecular physiology is only now emerging because appropriate monoclonal antibody reagents are being developed and rigorous attention is being paid to the use of gentle physiological methods in experiments in vivo.

Improved protocol for colorimetric detection of complement-mediated cytotoxicity based on the measurement of cytoplasmic lactate dehydrogenase activity.

The conventional cytotoxicity detection protocols based on the lactate dehydrogenase (LDH) activity assays rely upon the quantitation of the enzyme activity released into the assay medium upon cell lysis. In the case of complement-mediated cytotoxicity this results in the need to take into account the high and variable background LDH activity from the serum added to the cells. Using primate-derived COS-7 and pig kidney PK15 cell lines we show that, in the case of adherent cells, it is possible to overcome this drawback and to measure cell death caused by complement attack, by quantifying the amount of LDH activity retained by the undamaged cells. The modified assay is therefore quicker to carry out than the conventional procedure and cheaper because fewer control readings must be taken.

Analyses of monoclonal antibodies reactive with porcine CD44 and CD45

Twenty-six monoclonal antibodies (mAbs), assigned to the CD44/CD45 section of the First International Swine CD Workshop, were compared for their reactivity against a selected group of target cells by one- and two-color flow cytometric analysis. Based on staining and reactivity patterns the 26 mAbs were assigned to six groups, group F1 mAbs were designated CD44 mAbs; and groups F2 and F3 as CD45 mAbs. With the information available, a CD designation could not be given to the mAbs in groups F4, F5 or F6 consisting of four, three and four mAbs each, respectively. The reactivity of all six mAbs in group F1 (MAC35, 25-32, PORC24A, H22A, BAG40A, and BAT31A) was blocked by soluble porcine CD44. One mAb in this group (MAC325) reacted with a cell surface protein with a molecular weight of 80 kDa and was designated as CD44; the other five mAbs were designated as wCD44 because no molecular weight was known. Blocking experiments utilizing a cross reactive anti-human CD44 (mAb Z062) allowed the definition of the wCD44a epitope recognized by mAbs PORC24A and H22A. The group F2 mAbs (74-9-3; MAC323; K252.1E4; and 2A5) were designated as CD45 based on their broad reactivity pattern with lymphoid and myeloid cells and their ability to immunoprecipitate three polypeptides with an apparent molecular weight of 226, 210 and 190 kDa. The F3 mAbs (MAC327; MAC326; 3a56 and -a2) were designated as CD45R based on their restricted reactivity against lymphoid and myeloid target cells, and their ability to immunoprecipitate either two polypeptides with an apparent molecular weight of 226 and 210 kDa (mAbs MAC327 and MAC326) or a single polypeptide with an apparent molecular weight of 210 kDa (mAbs-a2 and 3a56). Sequential immunoprecipitation analyses confirmed the relatedness of the F2 and F3 group mAbs. The work conducted for this first workshop led to the definition of six mAbs specific for CD44, four mAbs specific for CD45, and four mAbs specific for CD45R which should prove to be very valuable reagents for the study of the porcine immune system.

Cellular xenoresponses: Observation of significant primary indirect human T cell anti-pig xenoresponses using co-stimulator-deficient or SLA class II-negative porcine stimulators

Abstract: We have documented the strength and characteristics of the in vitro human anti‐porcine indirect T cell response using porcine stimulators that were unable to provoke a direct xenoresponse. Our results indicate that HLA‐DR‐restricted and HLA‐DQ‐restricted CD4+ T cells recognise porcine xenoantigens presented by conventional APC, rather than xenospecific B cells. Limiting dilution analysis assays indicate a high precursor frequency of T cells with indirect xenospecificity in the peripheral blood of human responders. These data demonstrate that the indirect response requires no priming, but instead reflects the large number of pig‐specific antigens.

Mechanisms for variability in a member of the scavenger-receptor cysteine-rich superfamily

Abstract This study reports a molecular analysis of pig WC1, a new member of the scavenger-receptor cysteine-rich (SRCR) superfamily. The pig WC1 contains up to six extra-cellular SRCR domains, highly homologous to other members of the family. However, the striking feature of the WC1 gene, as for its cattle and sheep homologues, is that it is present as a multigene family showing extensive sequence diversity, for both DNA and predicted protein sequence. The basis of this diversity was examined and was shown to be attributable to several different causes. These included single base-pair changes within SRCR domains, the optional usage of whole domains or exons, including a SRCR domain and the proximal “hinge” region, and alternative isoforms of the putative cytoplasmic tail. These results suggest that WC1 may code for a new, though more primitive type of antigen recognition structure specific for γ/δ T cells.

Biochemical analysis of molecules reactive with monoclonal antibodies specific for porcine CD45

The apparent molecular weight of molecules reactive with eight mAbs, which were identified during the First International Swine CD Workshop as putatively specific for porcine CD45, was determined by immunoprecipitation analysis of 125I surface-labeled peripheral blood mononuclear cells. Four putative CD45 specific mAbs (K252.1E4, MAC323, 74-9-3 and 2A5) precipitated three polypeptides of 226, 210 and 190 kDa. Two putative CD45R specific mAbs (MAC326 and MAC327) precipitated two polypeptides with an apparent molecular weight of 226 and 210 kDa, while another two (3a56 and -a2) precipitated a single polypeptide of 210 kDa. Sequential immunoprecipitations analyses indicated that the comigrating molecules precipitated by the CD45 and CD45R were the same. The results from this study confirmed the assignment of mAbs K252.1E4, MAC323, 74-9-3 and 2A5 as CD45, and mAbs MAC326, MAC327, 3a56 and -a2, as CD45R. These CD45 and CD45R specific mAbs should prove to be valuable reagents for the study of the porcine immune system.

Early ontogeny of immune cells and their functions in the fetal pig

The origin of immune cells and their products have been studied in the prenatal period in miniature pigs. Macrophages were first detected on day 25, and myelocytes and lymphoid cells by day 28. Membrane antigens SLA-DR and CD45 were found by day 22, membrane molecules MG-7, 8/1, CD1, CD2 and 74-22 by day 28, Gamma/delta T cells were found initially in extrathymic sites (in the liver). The first gamma/delta T cells were detected as early as 40 days of gestation. The expression of fibronectin, Thy-1 and its message, Ig isotypes and the first induction of IFN alpha were described.

Infiltrating γδ T-cells and selectin endothelial ligands in the cutaneous phytohaemagglutinin-induced inflammatory reaction

The 24 h phytohaemagglutinin-induced skin inflammatory site (intradermal and subcutaneous) was studied in inbred MHC-homozygous (SLAb/b) pigs and it was found, by immunohistology, that the predominant lymphocytes in the infiltrate are CD2-CD4-CD8-sIg-T-cells, the Null/gamma delta T-cell family, identified using the monoclonal antibodies (mAbs) MAC320 and MAC319 (which recognises a subset of MAC320+ cells). A large percentage of the infiltrating cells expressed the gamma delta T-cell receptor phenotype identified by binding of the mAb 86D. Fewer CD2+, CD8+ and CD4+ cells were present and surface immunoglobulin positive (sIg+) cells were virtually absent in the infiltrate. Areas of lymphocytic infiltration were associated with endothelial activation as determined by expression of the E-selectin and a ligand for the L-selectin.