Development of detection methods for ruminant interleukin (IL)-4.

Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314. A pair of mAb (CC313 and CC314) was identified that together could be used to detect both recombinant and native bovine IL-4 by ELISA and a luminometric detection method was applied to the ELISA. Using this method native bovine IL-4 was detected in supernatants of PBMC stimulated with mitogens. In addition, high level secretion of IL-4 by Fasciola hepatica specific Th2 clones, but not by a Babesia bovis specific Th1 clone, was confirmed. The ELISA was also able to detect recombinant ovine IL-4. The pair of mAb used for ELISA could also be used for the detection of IL-4 spot forming cells by ELISPOT. In addition intracytoplasmic expression of IL-4 could be detected. The ability to detect ruminant IL-4 by three methods: ELISA, ELISPOT and by flow cytometric analysis of intracytoplasmic expression will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

Differences in the induction of CD8+ T cell responses by subpopulations of dendritic cells from afferent lymph are related to IL-1 alpha secretion.

The major subset of dendritic cells (DC) from bovine afferent lymph expresses the SIRP alpha MyD-1 antigen, but not CD11a or the antigen recognized by mAb CC81, and potently stimulates CD4+ and CD8+ T lymphocyte proliferation. The minor subpopulation, that is CD11a+ CC81+ MyD-1-, effectively stimulates CD4+ but not CD8+ T lymphocyte proliferation. CD11a+ CC81+ MyD-1- DC did not induce anergy or death or secrete an inhibitory factor. However, supernatant from cultures of CD8+ T cells with CD11a- CC81- MyD-1+ DC significantly enhanced proliferation of CD8+ T cells in response to CD11a+ CC81+ MyD-1- DC, an effect that was blocked by interleukin (IL)-1alpha, but not IL-1beta, specific mAb. The proliferation of CD8+ T cells with CD11a+ CC81+ MyD-1- DC was also enhanced by adding IL-1alpha. IL-1beta slightly enhanced proliferation, whereas IL-2, IL-6, IL-12, and IL-15 had no effect. We conclude that the failure to stimulate CD8+ T cell proliferation results from the lack of IL-1alpha synthesis by this population, which may have important consequences in vivo.

Development of detection methods for ruminant interleukin (IL)-12.

Recombinant bovine IL-12 (rbo IL-12) was transiently expressed in COS-7 cells and shown to upregulate the synthesis of IFNgamma by bovine cells stimulated with a suboptimal concentration of mitogen in vitro. Mice were immunised with a plasmid encoding rbo IL-12 and boosted with rbo IL-12 and a number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-12 in an ELISA. Some of these mAb neutralised the ability of rbo IL-12 to induce IFNgamma synthesis by bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-12 by ELISA and a luminometric detection method was applied to the ELISA making it more sensitive. Using this method native bovine IL-12 was detected in supernatants of dendritic cells (DC) cultured in vitro with a synthetic lipopeptide known to stimulate secretion of IL-12 by human DC. The ELISA was also able to detect recombinant ovine IL-12 and, less effectively, recombinant human IL-12. In contrast, bovine IL-12 was not detected by a commercial human IL-12 ELISA kit. Intracytoplasmic IL-12 was detected in bovine DC using the antibodies described herein. The ability to detect ruminant IL-12 by three methods: ELISA, bioassay with neutralising mAb and cytoplasmic staining, will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

Systemic vaccination with inactivated bovine virus diarrhoea virus protects against respiratory challenge.

Inactivated bovine virus diarrhoea virus, strain 11249nc, inoculated subcutaneously three times with Quil-A into calves protected against intranasal challenge with the same strain. Virus was isolated from nasopharyngeal swabs taken 4 to 8 days post challenge and blood taken 4 to 6 days post challenge from control calves but not from vaccinated calves. A second strain of virus, Ky1203nc, was selected on the basis of previously established data on its antigenicity and the amount of viral antigen produced by five cell cultures compared using an ELISA. Cultures of one cell line, MDBK, yielded a greater amount of viral antigen than the others. Strain Ky1203nc grown in MDBK cells was inactivated with beta-propiolactone, mixed with adjuvant and used as a vaccine inoculated into calves subcutaneously three times. All of 5 calves were protected against intranasal challenge with a heterologous strain. In contrast virus was isolated from nasopharyngeal swabs taken from 5 control calves and from the blood of 4 controls. All 5 control calves, but none of the vaccinates, had a leukopenia after challenge. We conclude that the selected strain and system of vaccine preparation provide an effective means of protecting calves against respiratory infection and that live vaccines are not required to protect against challenge via the respiratory tract.

Cluster analysis.

Cluster analysis was performed on flow cytometry data generated from the reactivities of the 302 workshop monoclonal antibodies with 36 target cell preparations. The antibodies were assigned to 42 preliminary clusters that were subjected to further examination in subsequent stages of the workshop.

Initial statistical clustering: definition of temporary clusters of monoclonal antibodies for the duration of the workshop.

Statistical clustering was performed on flow cytometry data generated from the reactivities of 189 mAbs with 56 cell preparations or cell lines (targets). Six targets were excluded from the statistical clustering because of non-reproducibility of data from the internal controls. Thirty-six temporary clusters (TC) were formed which were subjected to further examination in the second phase of the workshop.

Final clustering.

Final statistical cluster analysis was performed on 51 target cells. These included three new complete datasets that were submitted to the workshop in time for the final cluster analysis. Two human cell lines that were used for the initial temporary clustering were omitted from the final targets. Most temporary clusters remained unchanged but several, previously loosely clustered, mAbs no longer formed clusters.

IFN gamma and IL-4 production by CD4, CD8 and WC1 gamma delta TCR(+) T cells from cattle lymph nodes and blood.

The synthesis of IFN gamma and IL-4 by CD4, CD8 and WC1 gamma delta TCR(+) T cell sub-populations, and T cells stained with activation/memory-sub-set markers has been examined by flow cytometric analysis. Cells from blood, prescapular, bronchial and mesenteric lymph nodes and Peyer's patches were incubated with phorbol 12-myristate 13-acetate (PMA), ionomycin and brefeldin-A before staining. Lymphocytes that stained for cytoplasmic IFN gamma were evident within the CD4 and CD8 populations from all tissues and also in the WC1 population from lymph nodes. IL-4 producing cells were primarily evident within the CD4 population. IFN gamma synthesis was evident within both CD45RO(+) and CD45RB(+) populations, but IL-4 synthesis was predominantly by cells that were CD45RO(+)/CD45RB(-). Expression of CD62L is not related to functional memory in CD4(+) T cells from cattle and CD62L(+) cells, particularly from the lymph nodes draining the skin and the lungs, stained with mAb to IFN gamma and IL-4. The findings indicate that at least for CD4(+) T cells, where CD45 isoform expression is related to functional memory, these two cytokines are produced predominantly by cells with a memory phenotype. The observation that some WC1(+) cells produce IFN gamma implies the presence of distinct sub-sets of this gamma delta TCR(+) population cattle and suggests a functional role.

Cross-reactivity of monoclonal antibodies to defined human leucocyte differentiation antigens with bovine cells.

Thirty-seven subpanels of monoclonal antibodies (mAbs) included within the Vth International Workshop on Human Leucocyte Differentiation Antigens (Vth Workshop) were assayed for reactivity with bovine peripheral blood leucocytes. Sixty-five of the 772 mAbs (8.4%) stained bovine cells. mAbs from each of the 27 different CD groups that contained a mAb reacting with cattle were further investigated to compare the cellular expression of the antigen in cattle with that reported for the different CD antigens in humans. Two-colour immunofluorescence staining of the Vth Workshop mAbs against characterized bovine leucocyte subpopulation markers that identified monocytes, B cells, CD4, CD8 and WC1 +T cells were used for these analyses. Eighteen of the mAbs to different human CD antigens (CD11a, CD14, CD18, CD21, CD27, CD29, CD49a, CD49b, CD49d, CD49e, CD51, CD61, CD62L, CD62P, CD63, CDw78, CD98, CD100) stained bovine antigens with an almost identical cellular distribution to that reported in humans. This implies that these mAb react with the homologous cattle molecules. Nine mAbs (CD35, CD37, CD49c, CD50, CD54, CD66, CD81, CD88, CD102) stained bovine cells but the cellular distribution of the bovine antigen was different to that reported in humans implying either a different cellular distribution for these antigens in cattle or a reaction with a different molecule. The investigation has allowed the identification of several bovine homologues of human CD antigens that have not been previously defined in cattle and the cross-reacting mAbs will be valuable reagents for future investigations of bovine immunology.

Cross-reactivity of human leucocyte differentiation antigen monoclonal antibodies on porcine cells.

Thirty-six subpanels of monoclonal antibodies (mAbs) supplied to the Fifth International Workshop on Human Leucocyte Differentiation Antigens were assayed on porcine peripheral blood leucocytes for cross-reactivity. Sixty-two of the 752 mAbs-stained porcine cells. These mAbs identified 30 different CD groups and will be valuable reagents in the field of porcine immunology.

Investigation of PC36 (BoCD45R).

Eight monoclonal antibodies (mAbs) clustered together in a statistical analysis of data submitted to the Third Workshop on Ruminant Leucocyte Antigens to form provisional cluster (PC) 36. PC36 included the CD45R workshop control mAb CC76. The mAbs were compared by two-colour immunofluorescence with mAbs against other leucocyte subpopulation antigens. The flow cytometry results indicated that all of the mAbs identified CD45R.

An investigation of temporary workshop clusters reacting with cells of the mononuclear phagocytic system.

Monoclonal antibodies (mAbs) within ten temporary workshop clusters (TC3, TC8, TC9, TC11, TC16, TC17, TC19, TC20, TC21 and TC31) which from preliminary evidence appeared to recognize molecules expressed by cells of the mononuclear phagocytic system were studied by immunohistology and flow cytometry. A number of mAbs were identified that were potentially useful for immunohistological studies of the mononuclear phagocytic system. The results confirmed the specificity of a number of CD11a, CD11b, CD11c and CD44 mAbs. The findings also indicated that for several TCs the mAbs could not be regarded as recognizing the same molecule.

Cross-reactivity with bovine cells of monoclonal antibodies submitted to the 6th International Workshop on Human Leukocyte Differentiation Antigens.

Twelve subpanels of monoclonal antibodies (MAb) included within the 6th International Workshop on Human Leukocyte Differentiation Antigens (6th HLDA) were assayed for reactivity with bovine peripheral blood leukocytes. Sixty-nine of the 807 MAb (8.6%) stained bovine cells. These MAb represented 30 different human CD groups. Nine of the MAb to different human CD antigens (CD19, CD23, CD39, CD47, CD86, CD117, CD120b, CDw149, CD165) potentially recognized antigens on cattle cells that had not previously been identified. These were investigated further by two-colour immunofluorescence to compare the cellular expression of the antigen on cattle cells with that reported for the different CD antigens in humans. Four of the MAb that belonged to CD23, CD39, CD47, and CDw149 stained bovine cells in a manner that indicated an almost identical cellular distribution of the antigen to that reported in humans. This implied that these MAb reacted with the homologous cattle molecules. Further work would be necessary to confirm specificity of CD19, CD86, CD117, CD120b and CD165 MAb. Other cross-reacting MAb either recognized antigens already defined in cattle or antigens not yet clustered in humans. The study has identified valuable new reagents for studies of cattle and confirmed that most common cross-reactive MAb are to epitopes on integrins.

A new non-lineage specific antigen with an M(r) of 115 kDa and 39 kDa present on bovine leukocytes identified by monoclonal antibodies within BoWC10.

Five mAbs out of a total of 189 submitted to the Second Workshop were shown to be similar when statistical analyses of flow cytometry data were performed. For the duration of the workshop the five mAbs were assigned to temporary cluster TC35. The mAbs precipitated two polypeptides by SDS-PAGE under reducing conditions. One had an M(r) of 115 kDa, the other an M(r) of 39 kDa. The antigen recognized was present on thymocytes, a subpopulation of CD2+ T cells and a major subpopulation of B cells. The antigen is upregulated after long-term activation with concanavalin A. It is proposed that the mAbs in TC35 should be regarded as a single cluster identifying a novel leukocyte differentiation antigen in cattle. These mAbs were accepted as a new workshop cluster BoWC10.

Competitive binding with putative Bo5 (CD5) cluster of monoclonal antibodies.

The relationship of seven monoclonal antibodies, putatively to the Bo5 (CD5) antigen, was tested. Five of the mAbs were confirmed to be directed against the Bo5 antigen. Three mAbs, CC29, BLT-1 and 8C11, effectively blocked binding to bovine PBM of mAb CC17, previously reported to be directed against this antigen. MAb 8-3F4 also blocked binding of mAb CC17, but less effectively than the others. MAbs IL-A67 and 79-5 did not inhibit binding of mAb CC17 because of antibody allelic specificity or technical reasons.

Immunohistology of workshop monoclonal antibodies to the bovine homologue of CD1.

Six monoclonal antibodies putatively to the BoCD1 antigen were compared by immunohistology on cryostat sections from a range of tissues. The different staining patterns observed allowed the mAbs to be placed in three groups (a) 20-27, (b) CC13, CC14, TH97A and (c) CC20, CC40. An ovine mAb VPM5 did not stain bovine tissues sufficiently strongly to enable a comparison with the other CD1 mAbs.

Investigating monoclonal antibodies to bovine "null" cell antigens using two-colour immunofluorescence.

Eighteen monoclonal antibodies (mAbs) putatively to non T4/T8 (null) cell antigens were tested by two-colour immunofluorescence and antibody binding inhibition (blocking), with one selected mAb (CC15) that previous studies had indicated to be specific for null cells. None of the other mAbs blocked binding of CC15 to lymphocytes. Three main patterns of reaction were observed in two-colour immunofluorescence studies: mAbs that stained the same cells as CC15, mAbs that only stained a sub-population of the cells that stained with CC15 and mAbs that stained a sub-population of the cells that stained with CC15 but also some cells that did not react with CC15.

Identification of bovine CD14.

Six monoclonal antibodies (mAbs) that clustered together in a statistical analysis of data submitted to the Third Workshop on Ruminant Leucocyte Antigens and mAb CC-G33 were tested for ability to stain COS-7 cells transfected with cDNA encoding human CD14. Only mAb CC-G33 recognised the human molecule. The six mAbs were compared with mAb CC-G33 by flow cytometry and three were shown to be directed against bovine CD14.

Identification of monoclonal antibodies specific for bovine leukocyte common antigen (CD45) together with a novel broadly expressed leukocyte differentiation antigen, BoWC11.

Ten mAbs that comprised temporary clusters TC5 and TC7, plus an additional mAb, CC171, were compared. All mAbs reacted broadly and stained antigen expressed on thymocytes as well as T and B cells in peripheral blood. The five mAbs CC1, CC171, 2E8, TD14 and TD15 precipitated molecules of 180, 205 and 220 kDa, and we conclude that they recognize the bovine leukocyte common antigen and are BoCD45 mAb. The three mAbs IL-A117, 1C10 and BAGB27A precipitated molecules of 86 and 63 kDa that were broadly expressed. It is proposed that these three mAbs should be regarded as a cluster (named BoWC11 within the Second Workshop) that is distinct from BoCD45.

Studies of monoclonal antibodies identifying two novel bovine lymphocyte antigen differentiation clusters: workshop clusters (WC) 6 and 7.

Analysis of flow cytometric staining of 50 target cells resulted in five monoclonal antibodies (mAbs) forming two adjacent clusters called temporary cluster (TC) 2. The five mAbs gave similar immunostaining of frozen sections, but flow cytometric analysis and the M(r) of molecules indicated that they comprised two distinct specificities. Mabs CC98, IL-A114 and IL-A53 all immunoprecipitated a molecule of 210 kDa that preclearing experiments with mAbs to the leukocyte common antigen (CD45) showed did not recognize isoforms within the CD45R family of molecules. The 210 kDa molecule was present on all CD2+ cells and B cells, but not expressed by WC1+ gamma/delta T cells. mAbs TH1A and TH18A precipitated molecules of 69 and 62 kDa which were present on cells expressing CD2, WC3 (B cells) and WC1 (gamma/delta T cells). It is proposed that these five mAbs should be considered as comprising two new bovine lymphocyte differentiation clusters, WC6 (mAbs CC 98, IL-A114 and IL-A53) and WC7 (TH18A and TH1A).