Karin Haverson

Analysis of the immunological cross reactivities of 213 well characterized monoclonal antibodies with specificities against various leucocyte surface antigens of human and 11 animal species.

213 Monoclonal antibodies (mAbs) raised against leucocyte surface antigens from human and 11 animal species were analyzed for reactivities against leucocytes from human and 15 different animal species. We found 77 mAbs (36%) to cross-react. Altogether, 217 cross reactions were registered out of 3195 possible combinations (7%). Most of the cross reacting mAbs had integrin or MHC class II specificities. This study defined cross reactions on the following markers: CD1a, 1c, 2, 4, 5, 8, 9, 11a, 11b, 14, 18, 20, 21, 23, 29, 31, 41, 43, 44, 45, 45R, 46, 49, 61, 62L, TCR gamma/delta, BCR, Thy-1, MHC class I and MHC class II, Swine-WC7 and Cattle-WC1. In order to characterize the molecular weight (MW) of the corresponding cross reacting antigens, selected mAbs were used to immunoprecipitate the antigens. The MWs of the analyzed precipitated antigens were in good agreement with the MWs of the homologous antigens. The followed strategy was found to be efficient and economical in defining new leucocyte antigen reactive mAbs.

Characterization of monoclonal antibodies specific for monocytes, macrophages and granulocytes from porcine peripheral blood and mucosal tissues.

A panel of four monoclonal antibodies produced in our laboratory, MIL1, MIL2, MIL3, MIL4, and the type-specific monocyte/granulocyte marker 74-22-15 were used to isolate and to discriminate between monocytes, macrophages and granulocytes derived from porcine peripheral blood, lung and gut lamina propria. Two-colour flow cytometry and cell sorting showed that while no monoclonal antibody was specific for just a single cell population, each cell type had a unique and characteristic combination of surface antigens. These differences could be used to identify and purify monocytes, macrophages, neutrophils, eosinophils and basophils from the three different sites. The study also demonstrated similarities and differences within cell types from the same site and from different sites: polymorphonuclear neutrophils (PMN) from peripheral blood were subdivided into two subpopulations by the presence or absence of the surface antigen recognized by MIL4, while PMN from alveolar lavage did not express this antigen. Peripheral blood eosinophils were also divided into subpopulations by the presence or absence of the same surface antigen. Lamina propria eosinophils strongly expressed the MIL4 marker and differed morphologically from blood eosinophils. Peripheral blood basophils and lamina propria mast cells were morphologically similar and expressed similar antigens. Monocytes and alveolar macrophages also expressed the same surface antigens.

Regulation of mucosal immune responses in effector sites

In human disease and rodent models, immune responses in the intestinal mucosa can be damaging. Damage is characterised by villus atrophy, crypt hyperplasia and reduced ability to digest and absorb nutrients. In normal individuals active responses to harmless environmental antigens associated with food and commensal bacteria are controlled by the development of immunological tolerance. Similar pathological changes occur in piglets weaned early from their mothers. Active immune responses to food antigens are observed in these piglets, and we and others have hypothesised that the changes occur as a result of transient allergic immune responses to novel food or bacteria antigens. The normal mechanism for producing tolerance to food antigens may operate at induction (Peyers patches and mesenteric lymph nodes) or at the effector stage (intestinal lamina propria). In our piglet studies immunological tolerance occurs despite the initial active response. Together with evidence from rodents, this observation suggests that active responses are likely to be controlled at the effector stage, within the intestinal lamina propria. Support for this mechanism comes from the observation that human and pig intestinal T-cells are susceptible to apoptosis, and that this process is accelerated by antigen. We suggest that the role of the normal mature intestinal lamina propria is a balance between immunological effector and regulatory function. In neonatal animals this balance develops slowly and is dependant on contact with antigen. Immunological insults such as weaning may tip the balance of the developing mucosal immune system into excessive effector or regulatory function resulting in transient or chronic allergy or disease susceptibility.

Professional and non-professional antigen-presenting cells in the porcine small intestine

We have previously presented evidence of a highly organized and compartmentalized structure of the small intestinal lamina propria of the pig. In this work, we have identified at least two major populations of cells in this site expressing high levels of major histocompatibility complex (MHC) class II antigens. One is CD45 positive and is a potent initiator of a primary immune response, this is a function usually associated with dendritic cells. These cells have characteristic dendritic morphology, but show evidence of phagocytosis as well as other phenotypic markers of immature dendritic cells. Some cells show evidence of ongoing immune maturation. We have also isolated CD45 negative endothelial cells bearing significant amounts of MHC class II, which do not trigger a mixed lymphocyte reaction. These findings have implications for the functional role of healthy gut lamina propria and clearly implicate this site as capable of differential antigen presentation by a heterogeneous population of antigen‐presenting cells.

Analysis of monoclonal antibodies reacting with molecules expressed on gammadelta T-cells.

gamma delta T cells in ruminants can be subdivided in two or more subpopulations on the basis of the expression of surface antigen WC1, which can exist in different isoforms. In this study, 18 monoclonal antibodies (mAbs) submitted to the Third International Workshop that were predicted to react with gamma delta TcR molecules were analysed and expression of their antigens was investigated on the different gamma delta T cell subpopulations. A set of control mAbs positive for TcR1 (86D), BoCD3 (MM1A), WC1 (B7A1, BAQ4A, CACTB32A, and BAQ89A) was included for comparative studies. Previous investigations demonstrated eight of the mAbs immunoprecipitated peptides with apparent M(r)s of 37 and/or 47 kDa, indicating they recognized determinants on the T cell receptor, TcR1. Two color flow cytometric analyses in the present study demonstrated the mAbs formed three groups; group 1, a set of mAbs that recognize TcR1 determinants expressed on all gamma delta T cells and groups 2 and 3, sets of mAbs that recognize TcR1 determinants on some gamma delta T cells: TcR1-N6 and TcR1-N7 respectively. mAbs from the latter groups define families of TcR1 molecules that express either one or both of the determinants. These antigenically distinct forms of TcR1 are expressed in equal proportion on the two gamma delta T cell populations that express one of the mutually exclusive isoforms of WC1, WC1-N3 and WC1-N4. The data indicate usage of the mAb-defined families of the gamma delta TcR is primarily restricted to the WC1+ subpopulation of gamma delta T cells. However, a small subpopulation of CD2+, WC1- gamma delta T cells expresses a form of TcR1 positive for the determinant TcR1-N6.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.

Expression of major histocompatibility complex class II antigens on normal porcine intestinal endothelium.

A novel monoclonal antibody (MIL 11) specific for an antigen expressed on porcine endothelial cells is described. The antigen recognized by MIL 11 is most strongly expressed in the intestine but is also expressed on the capillary endothelium of a wide range of tissues. Using two‐ and three‐colour immunofluorescence microscopy we demonstrated the extensive coexpression of MIL 11 and major histocompatibility complex (MHC) class II antigens on normal porcine capillary endothelium in the intestine, trachea, thymus and small veins, while endothelium of large vessels and the heart were negative for MHC class II. In contrast to humans and rodents, available reagents do not detect MHC class II on the intestinal epithlium of pigs. However, porcine intestinal endothelium expressed both DR and DQ antigens. A population of strongly class II‐positive cells was also detected immediately adjacent to the endothelium in the lamina propria. Three‐colour immunofluorescence microscopy highlighted the close association between endothelium and intestinal CD4+ T cells. Lamina propria T cells were mainly MHC class II positive, whereas those in the epithelial compartment were MHC class II negative.

A Preclinical Model For Laryngeal Transplantation: Anatomy And Mucosal Immunology Of The Porcine Larynx

BACKGROUND A major step in translating work on laryngeal transplantation into clinical practice is the establishment of a preclinical model. We have investigated the anatomy and mucosal immunology of the porcine larynx in eight Minnesota Minipigs (12-37 weeks). METHODS Neck dissections were carried out and the vascular tree was mapped. Snap-frozen biopsies from epiglottis, supraglottis, glottis, and subglottis were prepared for conventional histology, immunohistochemistry (CD45), and single and two-color immunofluorescence (CD3, MHC-II, CD45). RESULTS The anatomy of the laryngeal skeleton was broadly similar to that of the human larynx. The blood supply is predominantly via the caudal thyroid vessels, with assistance from the cranial laryngeal artery. The porcine larynx is clearly highly immunologically active. Structured collections of leukocytes were found in the mucosal epithelium, around tubuloacinar glands, and occasionally in the submucosa. MHC-II and CD 3 cells were predominantly found within the epithelium. The highest densities of all cell types were observed in the epiglottis, tailing off caudally. The lowest densities were seen in the vocal cords. CONCLUSIONS The porcine larynx is both anatomically and immunologically similar to the human larynx and contains a high level of immunological organization. It presents an ideal preclinical model for laryngeal transplantation.

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.

The larynx as an immunological organ: immunological architecture in the pig as a large animal model

The larynx is a mucosal organ positioned at the divergence of the respiratory and digestive tracts. It is exposed to a wide variety of environmental components, including foreign antigens, tobacco smoke, laryngopharyngeal reflux and pollutants. The mucosal immune system generates either active immune responses or tolerance, depending on the nature of the antigen and we hypothesize that the larynx is important organ for immunological decision‐making in the airway. Because the pig is an ideal large animal model in which to explore laryngological research questions, such as those relating to laryngeal transplantation, we investigated the normal mucosal immunology of the porcine larynx. Pig larynges and tracheae were processed and prepared for bright‐field microscopy and quantitative, multiple‐colour immunofluorescence histology using pig‐specific monoclonal antibodies. There was an abundance of immunologically active cells within the mucosa of the larynx and trachea of both the newborn and adult animal. Specifically, major histocompatibility complex class II (MHC class II+) cells, CD4+ and CD8+ cells were identified, although regional differences in numbers were apparent: specifically, the supraglottis contained fewer immunologically relevant cells than other sites sampled. There was a significant correlation between the numbers of MHC class II+ and CD4+ cells indicating co‐ordinate regulation and therefore functional local interactions. The presence of such an immunological structure suggests that the larynx may have important functions in respiratory immunology and that it may trigger strong alloresponses after laryngeal transplantation.

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.