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A rat monoclonal antibody which recognizes a product of the HLA-DR locus is described. The determinant recognized by this antibody is monomorphic in man but polymorphic in mice. Mapping studies with inbred and recombinant strains of mice show that this antigen is associated with H-2 haplotypes b, d and q and is coded for by genes within the I-A subregion. [ABSTRACT FROM AUTHOR]

Efferent lymph from the popliteal node of sheep challenged with antigen was found to suppress the in vitro transformation of sheep peripheral blood lymphocytes to a variety of antigens. The suppressor factor appeared 6-20 hr after challenge of the node and was shown to bc prostaglandin E2, probably complexed to albumin. [ABSTRACT FROM AUTHOR]

Prostaglandin E2 (PGE2) can mediate changes in lymphocyte traffic through lymph nodes. By measuring PGE2 levels in efferent lymph from cannulated sheep lymph nodes it was shown that increased synthesis of PGE2 always correlated with cell shutdown; infusion of PGE2 alone induced cell shutdown and inhibition of PGE2 synthesis by the node abolished cell shutdown induced by antigen. [ABSTRACT FROM AUTHOR]

Complement activation within lymph nodes has been shown to alter lymphocyte traffic through the node. Challenge of cannulated sheep lymph nodes with either immune complexes or complement activators was found to initiate the phenomenon of cell shutdown associated with antigenic challenge of lymph nodes in primed animals. [ABSTRACT FROM AUTHOR]

An antigen-dependent factor showing migration inhibition (MIF) and gold uptake stimulation (GUS) activities which has been previously described (Lowe & Lachmann, 1974) has been further purified from efferent lymph collected from cannulated nodes of BCG-sensitized sheep undergoing a delayed hypersensitivity response to PPD. During purification, fractions containing MIF activity also exhibited GUS activity. Initial purification by salt precipitation showed that antigen-dependent MIF activity was in the 40-90% ammonium sulphate precipitate. Non-specific activity and contaminating immunoglobulin were found in the 0-20% and 20-40% precipitates. Gel filtration on Sephadex G-200 and affinity chromatography on Concanavalin A-Sepharose have shown that antigen-dependent MIF is a glycoprotein of approximately 70,000 molecular weight (Lowe & Lachmann, 1974). Traces of contaminating antibody in the glycoprotein fraction were removed by immunoadsorption on monospecific anti-sheep IgG-Sepharose. Antigen-dependent MIF was also purified by affinity chromatography on PPD-Sepharose. The eluted fractions with all the antigen-specific activity, contained less than 1 % of the applied material. Analysis by polyacrylamide gel electrophoresis showed that the major protein component in the purified MIF preparation has a molecular weight and electro-phoretic mobility identical with that of sheep albumin. Although this represents a high degree of purification of antigen-dependent MIF it seems that albumin is still present as a contaminant and that the protein associated with MIF activity is present in trace quantities. [ABSTRACT FROM AUTHOR]

Antisera have been raised to the Fv fragment derived from a mouse α/λ myeloma. These had different reactivities for Fv and mouse immunoglobulin and two of them appeared to detect v region framework determinants on normal mouse immunoglobulins. A large proportion of mouse lymphocytes were shown to have Fv determinants on the surface which was part of the B-lymphocyte antigen receptor. Most of the reacting cells were B lymphocytes. Fv determinants were not detected on the majority of T lymphocytes including cells with known T-helper cell activity. It is concluded that in mice, T cells do not express immunoglobulin v region framework determinants on their surface. [ABSTRACT FROM AUTHOR]

Immunoglobulin (Ig) bearing, antigen-binding and antibody-secreting cells were investigated for the presence of membrane receptors for the Fc part of IgG and for C3b on their surface. Fc and C3b receptors were detected on the surface of most Ig-bearing and of most antigen-binding cells. Fc receptors were also detected on IgM antibody-secreting cells but not on IgG-secreting cells. C3b receptors were not detected on any antibody-secreting cells. These receptors are either lost from the B cell surface as they differentiate into antibody-secreting cells or are blocked in vivo by immune complexes or C3b. [ABSTRACT FROM AUTHOR]

We have investigated several aspects of γδ T cells in sheep. γδT cells of sheep express a unique transmembrane protein termed T19 but lack the expression of particular cell-surface molecules such as CD2, CD4 and CD8 which are typically associated with αβ T cells. The majority of γδ T cells isolated from animals of all ages examined lacked the expression of CD45RA. A faster rate of activation by γδ T cells compared to either CD4 or CDS T cells was seen in the time-course of IL-2 receptor a chain (CD25) cell-surface expression. All γδ T cells expressed the CD25 protein within 8 hr of activation whereas the majority of CD4 or CD8 T cells did not express CD25 until 24 hr post-concanavalin A (Con A) stimulation. This difference in the rate of expression of activation molecules was not restricted to CD25, as a similar trend was seen with cell-surface expression of major histacompatibility complex (MHC) class II molecules. We have used the distinct phenotypic profile of ovine γδ T cells to purify these cells by positive selection via the T cells molecule to assess their in vitro proliferative response to various antigens. Routinely, cell populations comprising more than 93% γδ T cells with yields of approximately 55% were obtained. Purified γδ T cells were capable of responding to Mycobacterium tuberculosis antigen in a primary and secondary in vitro proliferation assay and to ovalbumin in a secondary response. Ovine γδ T cells showed little, if any, proliferative response to allogeneic stimulator cells. [ABSTRACT FROM AUTHOR]

In order to identify and characterize the sheep major histocompatibility complex (MHC) class II molecules, a panel of 19 monoclonal antibodies (mAb) has been raised following immunization of mice with a variety of class II antigen preparations. Antibodies were selected by ELISA using immunopurified sheep class II as antigen and further screened for the ability to react on immunoblots. Nine mAb reacted with the β-chain and four reacted with the α-chain. The chain specificity of the remainder could not be determined as they did not blot. The anti-α and anti-β mAb all reacted with deglycosylated class II. With the exception of one anti-α and one anti-β antibody, the antibodies reacted with both efferent lymph cells and peripheral blood mononuclear cells (PBMC). Three of the anti-sheep class II mAb reacted with human PBMC. [ABSTRACT FROM AUTHOR]

The majority of studies that characterize lymphokines utilize in vitro activation of lymphocytes. In an attempt to identify and characterize lymphokines released from tissue sites, we have cannulated sheep lymphatic vessels and collected lymph that drains a site of in vivo antigen challenge. Lymph draining directly from a site of intradermal antigen challenge (afferent lymph) and lymph draining an antigen- stimulated lymph node (efferent lymph) were assayed for lymphokine activity by the ability of cell- free lymph fluid to stimulate the proliferation of sheep Con A-blasts. Afferent and efferent lymph, both collected at 24 and 48 hr following in vivo antigen challenge, with either ovalbumin or PPD in primed animals, stimulates the proliferation of sheep Con A-blast cells. This in vivo-derived lymphokine activity and in vitro-generated sheep Con A supernatant has an active component with properties similar to interleukin-2 (IL-2) that has been described in several other species. The IL-2- like material is precipitated by 40-80% ammonium sulphate saturation, has a molecular weight (MW) of 20,000 MW as judged by gel filtration chromatography, and is eluted from an anion- exchange HPLC column with 125 mM NaCl. HPLC ion-exchange fractionation of the 20,000 MW material from lymph fluid shows differences between afferent and efferent lymph material. The fractionation of afferent material is similar to that of in vitro generated Con A supernatant material with a single peak of activity eluted by 125 mM NaCI. In contrast, the 20,000 MW material from efferent lymph elutes with peaks of activity at 125 and 300 mM NaCl. [ABSTRACT FROM AUTHOR]

Antigen-specific sheep T-cell lines have been generated in vitro from peripheral blood mononuclear cells (PBMC). PBMC prepared from antigen-primed animals were cultured in the presence of ovalbumin (OVA) or purified protein derivative (PPD). After 5 days of culture, activated antigen- specific cells were expanded by further culture in the presence of recombinant human interleukin-2 (IL-2). Cell lines generated after two cycles of antigen stimulation followed by expansion with IL-2 show a proliferative response to antigen only in the presence of autologous antigen-presenting cells (APC) and recognize only the antigen used in the original stimulation. An OVA-specific cell line was found to be capable of recognizing a synthetic peptide corresponding to amino acid residues 323-338 of OVA. The cell lines also responded by proliferation in an allogeneic mixed leucocyte reaction (MLR). Cell-surface phenotyping shows that the cell lines comprise both CD4- and CD8-positive cells. [ABSTRACT FROM AUTHOR]

A rat monoclonal antibody is described that recognizes sheep MHC Class II antigen and appears to recognize a determinant that is non-polymorphic. This antibody precipitates polypeptides of apparent molecular weight 33,000 and 28,000/29,000 (under non-reducing conditions). The binding of this antibody was inhibited by an anti-human DP, DQ, DR β-chain monoclonal(DA6.231) and its reactivity on murine cells suggests specificity for the I-E gene product. Using this antibody we have studied the distribution of Class II molecules in sheep and also the variations in Class II expression by cells in both peripheral and efferent lymph, produced as a result of in vivo antigen inoculation. [ABSTRACT FROM AUTHOR]

Efferent lymph from the popliteal node of sheep challenged with antigen was found to suppress the in vitro transformation of sheep peripheral blood lymphocytes to a variety of antigens. The suppressor factor appeared 6-20 hr after challenge of the node and was shown to bc prostaglandin E2, probably complexed to albumin. [ABSTRACT FROM AUTHOR]

Using trypanosomes labelled with [75Se]- methionine a series of experiments was conducted to investigate the respective roles of antibody, macro- phage activation and complement in the removal of trypanosomes from the circulation of immune mice. It was found that clearance in such animals is largely accomplished by antibody-mediated hepatic phagocytosis, which, at least in passively immunized animals, is dependent on opsonization involving C3. No evidence was found to suggest that intravascular lysis or activated macroph ages are involved in immune clearance. [ABSTRACT FROM AUTHOR]

A proportion of the plasma cells in lymph nodes of allergized guinea-pigs and mice were found to possess membrane-bound receptors for antigen when tested for rosette-formation at 4° by the suspension-centrifugation technique. This was shown by staining rosetted cells with pyronin-methyl green. Rosette-forming cells of the guinea-pig were further examined by staining with fluorescein-conjugated antigen (rabbit Fab') and by electron microscopy. By these techniques it was found that many plasma cells which contain antigen-specific intracellular antibodies do not form rosettes, and that cells of the plasmacytic series represented in the rosetted population are limited to plasmablasts and immature plasma cells. The lack of rosette-forming mature plasma cells suggests that a loss of receptors from the cell-membrane occurs as an accompaniment to maturation. [ABSTRACT FROM AUTHOR]

Scrapie is a transmissible spongiform encephalopathy in which there is an accumulation of the abnormal form of the prion protein, PrPsc, in the lymphoreticular system and nervous system. There is a particular accumulation of PrPsc on follicular dendritic cells within the germinal centre of B-cell follicles. Because accumulation of PrPsc in the nervous system leads to neuronal cell loss we have examined PrPsc accumulation in the prescapular and mesenteric lymph nodes in relation to lymph node architecture of scrapie-challenged sheep. We demonstrate that an accumulation of PrPsc in the lymph node fails to result in gross defects in the microanatomy and phenotype of T- and B-cell areas in the lymph nodes.

Dendritic cells (DCs) are professional antigen-presenting cells with a highly immunostimulatory function and the capacity to activate naïve T cells. In recent years the rapid progress in mouse and human DC research can be mainly attributed to the generation of DCs from precursor cells in vitro, although a lack of reagents has hampered DC research in many large animal models. Here we describe the generation and characterization of ovine monocyte-derived DCs in vitro. In addition to the characteristic morphology and non-adherence of DCs, peripheral blood mononuclear cell monocytes cultured with ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) expressed CD11c and major histocompatibility complex (MHC) class II, but did not express CD14. High levels of endocytosis and an ability to stimulate antigen-specific proliferation of CD4 T lymphocytes were also demonstrated.

We have investigated several aspects of gamma delta T cells in sheep. gamma delta T cells of sheep express a unique transmembrane protein termed T19 but lack the expression of particular cell-surface molecules such as CD2, CD4 and CD8 which are typically associated with alpha beta T cells. The majority of gamma delta T cells isolated from animals of all ages examined lacked the expression of CD45RA. A faster rate of activation by gamma delta T cells compared to either CD4 or CD8 T cells was seen in the time-course of IL-2 receptor alpha chain (CD25) cell-surface expression. All gamma delta T cells expressed the CD25 protein within 8 hr of activation whereas the majority of CD4 or CD8 T cells did not express CD25 until 24 hr post-concanavalin A (Con A) stimulation. This difference in the rate of expression of activation molecules was not restricted to CD25, as a similar trend was seen with cell-surface expression of major histocompatibility complex (MHC) class II molecules. We have used the distinct phenotypic profile of ovine gamma delta T cells to purify these cells by positive selection via the T19 molecule to assess their in vitro proliferative response to various antigens. Routinely, cell populations comprising more than 93% gamma delta T cells with yields of approximately 55% were obtained. Purified gamma delta T cells were capable of responding to Mycobacterium tuberculosis antigen in a primary and secondary in vitro proliferation assay and to ovalbumin in a secondary response. Ovine gamma delta T cells showed little, if any, proliferative response to allogeneic stimulator cells.

In order to identify and characterize the sheep major histocompatibility complex (MHC) class II molecules, a panel of 19 monoclonal antibodies (mAb) has been raised following immunization of mice with a variety of class II antigen preparations. Antibodies were selected by ELISA using immunopurified sheep class II as antigen and further screened for the ability to react on immunoblots. Nine mAb reacted with the beta-chain and four reacted with the alpha-chain. The chain specificity of the remainder could not be determined as they did not blot. The anti-alpha and anti-beta mAb all reacted with deglycosylated class II. With the exception of one anti-alpha and one anti-beta antibody, the antibodies reacted with both efferent lymph cells and peripheral blood mononuclear cells (PBMC). Three of the anti-sheep class II mAb reacted with human PBMC.

The majority of studies that characterize lymphokines utilize in vitro activation of lymphocytes. In an attempt to identify and characterize lymphokines released from tissue sites, we have cannulated sheep lymphatic vessels and collected lymph that drains a site of in vivo antigen challenge. Lymph draining directly from a site of intradermal antigen challenge (afferent lymph) and lymph draining an antigen-stimulated lymph node (efferent lymph) were assayed for lymphokine activity by the ability of cell-free lymph fluid to stimulate the proliferation of sheep Con A-blasts. Afferent and efferent lymph, both collected at 24 and 48 hr following in vivo antigen challenge, with either ovalbumin or PPD in primed animals, stimulates the proliferation of sheep Con A-blast cells. This in vivo-derived lymphokine activity and in vitro-generated sheep Con A supernatant has an active component with properties similar to interleukin-2 (IL-2) that has been described in several other species. The IL-2-like material is precipitated by 40-80% ammonium sulphate saturation, has a molecular weight (MW) of 20,000 MW as judged by gel filtration chromatography, and is eluted from an anion-exchange HPLC column with 125 mM NaCl. HPLC ion-exchange fractionation of the 20,000 MW material from lymph fluid shows differences between afferent and efferent lymph material. The fractionation of afferent material is similar to that of in vitro generated Con A supernatant material with a single peak of activity eluted by 125 mM NaCl. In contrast, the 20,000 MW material from efferent lymph elutes with peaks of activity at 125 and 300 mM NaCl.

Prostaglandin E2 (PGE2) can mediate changes in lymphocyte traffic through lymph nodes. By measuring PGE2 levels in efferent lymph from cannulated sheep lymph nodes it was shown that increased synthesis of PGE2 always correlated with cell shutdown; infusion of PGE2 alone induced cell shutdown and inhibition of PGE2 synthesis by the node abolished cell shutdown induced by antigen.

Efferent lymph from the popliteal node of sheep challenged with antigen was found to suppress the in vitro transformation of sheep peripheral blood lymphocytes to a variety of antigens. The suppressor factor appeared 6-20 hr after challenged of the node and was shown to be prostaglandin E2, probably complexed to albumin.

A rat monoclonal antibody is described that recognizes sheep MHC Class II antigen and appears to recognize a determinant that is non-polymorphic. This antibody precipitates polypeptides of apparent molecular weight 33,000 and 28,000/29,000 (under non-reducing conditions). The binding of this antibody was inhibited by an anti-human DP, DQ, DR beta-chain monoclonal (DA6.231) and its reactivity on murine cells suggests specificity for the I-E gene product. Using this antibody we have studied the distribution of Class II molecules in sheep and also the variations in Class II expression by cells in both peripheral and efferent lymph, produced as a result of in vivo antigen inoculation.

An antigen-dependent factor showing migration inhibition (MIF) and gold uptake stimulation (GUS) activities which has been previously described (Lowe & Lachmann, 1974) has been further purified from efferent lymph collected from cannulated nodes of BCG-sensitized sheep undergoing a delayed hypersensitivity response to PPD. During purification, fractions containing MIF activity also exhibited GUS activity. Initial purification by salt precipitation showed that antigen-dependent MIF activity was in the 40-90% ammonium sulphate precipitate. Non-specific activity and contaminating immunoglobulin were found in the 0--20% and 20--40% precipitates. Gel filtration on Sephadex G-200 and affinity chromatography on Concanvalin A-Sepharose have shown that antigen-dependent MIF is a glycoprotein of approximately 70,000 molecular weight (Lowe & Lachmann, 1974). Traces of contaminating antibody in the glycoprotein fraction were removed by immuno-adsorption on monospecific anti-sheep IgG-Sepharose. Antigen-dependent MIF was also purified by affinity chromatography on PPD-Sepharose. The eluted fractions with all the antigen-specific activity, contained less than 1% of the applied material. Analysis by polyacrylamide gel electrophoresis showed that the major protein component in the purified MIF preparation has a molecular weight and electrophoretic mobility identical with that of sheep albumin. Although this represents a high degree of purification of antigen-dependent MIF it seems that albumin is still present as a contaminant and that the protein associated with MIF activity is present in trace quantities.

Human peripheral blood lymphocytes were found to give an opsonic adherence reaction with IgM-coated erythrocytes. This reactivity could only be detected after incubating the lymphocytes at 37 degrees, was confined to the T-lymphocyte population and could be inhibited by IgM but not IgG. It is concluded that human T lymphocytes possess a receptor for IgM.

Using trypanosomes labelled with [75Se]-methionine a series of experiments was conducted to investigate the respective roles of antibody, macrophage activtion and complement in the removal of trypanosomes from the circulation of immune mice. It was found that clearance in such animals is largely accomplished by antibody-mediated hepatic phagocytosis, which, at least in passively immunized animals, is dependent on opsonization involving C3. No evidence was found to suggest that intravascular lysis or activated macrophages are involved in immune clearance.