II. Detection of an antigen on resting T cells down-regulated after activation

The expression of an antigen on porcine T lymphocytes detected by murine monoclonal antibody (mAb) 8/1 was investigated by functional studies and dual-parameter immunofluorescence. mAb 8/1 reacts with greater than 95% of thymocytes and in peripheral blood with all T lymphocytes and with cells of the monocyte/macrophage lineage, but not with B cells, erythrocytes, and platelets. Pretreatment of peripheral blood lymphocytes with mAb 8/1 plus complement abrogated the proliferative response in vitro to mitogen, soluble antigen, and MHC determinants. Dual-parameter immunofluorescence revealed that resting porcine T8+ as well as T4+ lymphocytes express the 8/1 antigen, whereas after in vitro activation, cell surface expression of the antigen was low or absent in both T cell subsets. Thus, the 8/1 antigen represents a marker that discriminates between resting and activated T lymphocytes. Distribution and functional criteria indicate that 8/1 represents a novel marker not described before for any other mammalian species

Ueber die fetten Säuren des Ricinusöls

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Simultaneous expression of CD4 and CD8 antigens by a substantial proportion of resting porcine T lymphocytes

The existence of four subpopulations in resting porcine T lymphocytes is documented. In addition to the two known subpopulations which are typified by a mutually exclusive expression of either the CD8 or the CD4 differentiation antigen, CD4-CD8+ and CD4+CD8- T lymphocytes, respectively, two unusual subpopulations were prominent not only in peripheral blood, but also in lymphoid tissues: CD4-CD8- T lymphocytes expressing neither of these antigens and CD4+CD8+ T lymphocytes coexpressing both antigens. While CD4+CD8+ lymphoblasts have been found also in other species, resting T lymphocytes with that phenotype are without precedent among all species analyzed to date. This unique composition of the porcine T lymphocyte population has to be taken into consideration when the swine is used as a large animal model in experimental medicine

Selective contribution of Tyk2 to cell activation by lipopolysaccharide

AbstractTyk2 deficient mice show a markedly reduced susceptibility to lipopolysaccharide (LPS) induced shock and a partial impairment of IL-12 and interferon (IFN) signals. To examine the underlying mechanisms, we analysed the activation of peritoneal macrophages (PMΦs) and spleen cells after LPS challenge. In PMΦs and spleen cells the contribution of Tyk2 to the induction of the T-cell co-stimulatory molecules CD86, CD40 and MHC II was small or insignificant. By contrast, induced expression of the early activation marker CD69 on PMΦs and splenic cell populations required type I interferons (IFN-I) and Tyk2. The data suggest a selective contribution of Tyk2 to the activation of inflammation-relevant cell types by LPS

Immunohistochemical characterization of type II pneumocyte proliferation after PRRSV (Type I) challenge

The study aimed to histologically and immunohistochemically characterize lung lesions after a challenge with a recently isolated PRRSV field strain in growing pigs 10 and 21 days post infection (DPI). In the first phase of the study lung lesions were evaluated microscopically on routine haematoxylin and eosin stained slides. The evaluation was performed as a blinded analysis and the lesions were scored based on the following criteria: (1) pneumocyte hypertrophy and hyperplasia, (2) septal mononuclear infiltration, (3) intraalveolar necrotic debris, (4) intraalveolar inflammatory cell accumulation and (5) perivascular inflammatory cell accumulation. For further characterization of the lung lesions, immunohistochemical stainings were performed using anti-cytokeratin, anti-Ki67, anti-TTF-1 (Thyroid Transcription Factor-1), anti-myeloid receptor (MAC387), and anti-PRRSV antibodies to identify alveolar epithelial cells, proliferating cells, type II pneumocytes, macrophages, and PRRSV antigen, respectively. The evaluation of the immunohistochemical stainings revealed that humanized anti TTF-1 antibodies can successfully identify type II pneumocytes in porcine lung tissue. Marked proliferation of these cells was confirmed by a significant (p<0.05) increase of TTF-1 positive cells in acute cases compared to the lungs of control pigs. Cytokeratin labeling marked the type I, and type II pneumocytes as well as bronchial epithelial cells, however this staining was not suitable for cell counting purposes. When the routine histological scores were compared to the number of immunohistochemically positive cells, Ki67 cell counts were found to show positive correlation (p<0.05) with the overall severity of the lesions

Porcine CD8αdim/-NKp46high NK cells are in a highly activated state

Natural Killer (NK) cells play a crucial role in the early phase of immune responses against various pathogens. In swine so far only little information about this lymphocyte population exists. Phenotypical analyses with newly developed monoclonal antibodies (mAbs) against porcine NKp46 recently revealed that in blood NKp46(-) and NKp46(+) cells with NK phenotype exist with comparable cytotoxic properties. In spleen a third NKp46-defined population with NK phenotype was observed that was characterised by a low to negative CD8α and increased NKp46 expression. In the current study it is shown that this NKp46(high) phenotype was correlated with an increased expression of CD16 and CD27 compared to the CD8α(+)NKp46(-) and NKp46(+) NK-cell subsets in spleen and blood. Additionally NKp46(high) NK cells expressed elevated levels of the chemokine receptor CXCR3 on mRNA level. Functional analyses revealed that splenic NKp46(high) NK cells produced much higher levels of Interferon-γ and Tumor Necrosis Factor-α upon stimulation with cytokines or phorbol-12-myristate-13-acetate/Ionomycin compared to the other two subsets. Furthermore, cross-linking of NKp46 by NKp46-specific mAbs led to a superior CD107a expression in the NKp46(high) NK cells, thus indicating a higher cytolytic capacity of this subset. Therefore porcine splenic NKp46(high) NK cells represent a highly activated subset of NK cells and may play a profound role in the immune surveillance of this organ

Pathogen-reactive T helper cell analysis in the pig

There is growing interest in studying host-pathogen interactions in human-relevant large animal models such as the pig. Despite the progress in developing immunological reagents for porcine T cell research, there is an urgent need to directly assess pathogen-specific T cells-an extremely rare population of cells, but of upmost importance in orchestrating the host immune response to a given pathogen. Here, we established that the activation marker CD154 (CD40L), known from human and mouse studies, identifies also porcine antigen-reactive CD4(+) T lymphocytes. CD154 expression was upregulated early after antigen encounter and CD4(+)CD154(+) antigen-reactive T cells coexpressed cytokines. Antigen-induced expansion and autologous restimulation enabled a time-and dose-resolved analysis of CD154 regulation and a significantly increased resolution in phenotypic profiling of antigen-responsive cells. CD154 expression identified T cells responding to staphylococcal Enterotoxin B superantigen stimulation as well as T cells responding to the fungus Candida albicans and T cells specific for a highly prevalent intestinal parasite, the nematode Ascaris suum during acute and trickle infection. Antigen-reactive T cells were further detected after immunization of pigs with a single recombinant bacterial antigen of Streptococcus suis only. Thus, our study offers new ways to study antigen-specific T lymphocytes in the pig and their contribution to host-pathogen interactions

MONOCLONAL ANTIBODIES REACTIVE WITH SWINE LYMPHOCYTES II. Detection of an Antigen on Resting T Cells Down-Regulated After Activation

The expression of an antigen on porcine T lymphocytes detected by murine monoclonal antibody (mAb) 8/1 was investigated by functional studies and dual-parameter immunofluorescence. mAb 8/1 reacts with greater than 95% of thymocytes and in peripheral blood with all T lymphocytes and with cells of the monocyte/macrophage lineage, but not with B cells, erythrocytes, and platelets. Pretreatment of peripheral blood lymphocytes with mAb 8/1 plus complement abrogated the proliferative response in vitro to mitogen, soluble antigen, and MHC determinants. Dual-parameter immunofluorescence revealed that resting porcine T8+ as well as T4+ lymphocytes express the 8/1 antigen, whereas after in vitro activation, cell surface expression of the antigen was low or absent in both T cell subsets. Thus, the 8/1 antigen represents a marker that discriminates between resting and activated T lymphocytes. Distribution and functional criteria indicate that 8/1 represents a novel marker not described before for any other mammalian species

Characterisation of monoclonal antibodies specific for hamster leukocyte differentiation molecules

Flow cytometry was used to identify mAbs that recognize conserved epitopes on hamster leukocyte differentiation molecules (hLDM) and also to characterize mAbs developed against hLDM. Initial screening of mAbs developed against LDMs in other species yielded mAbs specific for the major histocompatibility (MHC) II molecule, CD4 and CD18. Screening of sets of mAbs developed against hLDM yielded 22 new mAbs, including additional mAbs to MHC II molecules and mAbs that recognize LDMs expressed on all leukocytes, granulocytes, all lymphocytes, all T cells, a subset of T cells, or on all B cells. Based on comparison of the pattern of expression of LDMs expressed on all hamster leukocytes with the patterns of expression of known LDMs in other species, as detected by flow cytometry (FC), four mAbs are predicted to recognize CD11a, CD44, and CD45. Cross comparison of mAbs specific for a subset of hamster T cells with a cross reactive mAb known to recognize CD4 in mice and one recognising CD8 revealed they recognize CD4. The characterization of these mAbs expands opportunities to use hamsters as an additional model species to investigate the mechanisms of immunopathogenesis of infectious diseases