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Utilizing a 'sandwich' ELISA assay we have been able to demonstrate that mAb W6/32, B1G6 and IL-A19 are reactive with three different monomorphic determinants on bovine class I major histocompatibility complex (MHC) molecules. Sequential immunoprecipitations performed with the mAb revealed that class I molecules on PBM comprise a single population with respect to reactivity with the mAb in that the beta 2m-associated proteins bear all three epitopes. By contrast, TCGF-driven lymphoblasts and cells transformed by Theileria parva (Tp) additionally express molecules of Mr 45000 bound to beta 2m which are recognized by mAb B1G6 and IL-A19 but not by W6/32. These two subclasses of molecules were further distinguished on the basis that, when tunicamycin was added to cultures in the preparation of cells for analysis, mAb W6/32 precipitated class I heavy chains of Mr 39000 while the extra molecules detected only by mAb B1G6 and IL-A19 were of Mr 37000 and 39000. On thymocytes, the mAb W6/32-non-reactive class I molecules are present in low amounts and are expressed by cells in the medulla area, unlike BoT1 (analogous to human CD1) molecules which are expressed by the cortical cells. Our studies also revealed that the supposed beta 2m-specific mAb B1G6 does not recognize the beta 2m-associated molecules (BoT1) precipitated by mAb TH97A and thus the specificity of mAb B1G6 in cattle is for an epitope on bovine beta 2m which is strongly influenced by the nature of the heavy chain with which the beta 2m is associated.

Monoclonal antibodies have been derived which detect the bovine equivalents of the human pan-T cell marker CD2 and the T lymphocyte subpopulation markers CD4 and CD8. We refer to the bovine analogues as BoT2, BoT4 and BoT8. Monoclonal antibodies have also been derived which detect an antigen(s) with similarities to CD3, although the precise nature of the target molecule(s) in this instance remains to be elucidated. In general there is close similarity between the tissue distributions and, where these have been determined, the molecular masses of the BoT2, BoT4, BoT8 and putative BoT3 entities and their counterparts in other species. BoT2 is expressed on a majority of peripheral blood T lymphocytes and thymocytes and BoT2+ cells are found in both thymic cortex and medulla. In contrast, the putative BoT3 marker is expressed by a minority of thymocytes which are moreover, largely restricted to medulla. Monoclonal antibodies detecting BoT2 determinants have been shown to precipitate 55 kDa molecules. Antibodies to the BoT2 and BoT3 entities have been shown to induce proliferation in peripheral blood mononuclear cells of some cattle, and to be capable of inhibition of antigen-driven proliferative responses and cytolytic function. The BoT4 and BoT8 markers are expressed in a mutually exclusive manner by bovine peripheral blood mononuclear cells but they are coexpressed on a large population of thymocytes. Monoclonal antibodies have been used to precipitate molecules of 52 and 55 kDa in the case of those detecting BoT4 and 34 and 35 kDa in the case of an antibody reactive with a BoT8 determinant. The BoT4 and BoT8 markers have been associated with specificity for, and restriction by, MHC class II and class I molecules respectively.

In this study we report on the tissue distribution and functional characteristics of bovine T-cell differentiation antigens recognized by the monoclonal antibodies (mAbs) IL-A26, IL-A27, and IL-A28. All three mAbs are able to stimulate proliferation of peripheral blood mononuclear cells (PBM) and inhibit proliferation of responder cells in mixed leucocyte cultures (MLC). MAbs IL-A27 and IL-A28 are believed to react with the same molecule, which is different to that recognized by IL-A26 as determined by a number of criteria. MAbs IL-A27 and IL-A28 inhibit binding of one another, but not of IL-A26. MAbs IL-A27 and IL-A28 react with 25% of thymocytes confined to the medulla, whereas IL-A26 reacts with approximately 80% of thymocytes, including medullary and cortical populations. MAbs IL-A27 and IL-A28 react with thymocytes which express BoT4 or BoT8 singularly, whereas IL-A26 reacts with all cells which express BoT4 or BoT8, either singularly or dually, in addition to all thymocytes which react with IL-A27/28. Only IL-A26 inhibits spontaneous sheep erythrocyte (E)-rosette formation by bovine T cells. Based on tissue distribution and functional characteristics, IL-A26 is believed to recognize the bovine homologue of CD2, designated BoT2, whereas IL-A27/28 reacts with a mature T-cell antigen. Cells reactive with the mAbs constitute approximately 60% of bovine PBM. Using these mAbs in dual immunofluorescence analyses, at least three populations of bovine T cells are demonstrable in PBM. The majority of T cells are BoT4+ or BoT8+ and also react with IL-A26/27/28. A second small population of PBM is negative for BoT4 and BoT8 but is IL-A26/27/28+. A third population (less than 5%) is BoT4-/BoT8-/ILA27/28- but reacts with IL-A26.

Three monoclonal antibodies (MoAb), TH97A, CC13, and CC14, define a thymic differentiation antigen in cattle. The antigen is expressed on 50-60% of bovine thymocytes, located mainly in the cortical areas, but is not expressed on peripheral blood mononuclear cells (PBMC). In cryostat sections of lymph node, the antibodies react with large dendritic-like cells in the paracortical regions. They also react with a proportion of the large 'frilly' cells in afferent lymph and with dendritic-like cells in the dermis. The antibodies apparently do not react with cells in the epidermis. Biochemical analysis of the antigen recognized by MoAb TH97A reveals two bands of 44 kDa and 12 kDa under reducing conditions. These polypeptides are distinct from bovine class I major histocompatibility complex molecules reactive with the MoAb w6/32. The tissue distribution of positive cells together with results of biochemical analyses indicate that the antigen recognized by these MoAb is the bovine analogue of the human CD1.

A panel of monoclonal antibodies (MoAb) with specificities for bovine leucocyte subsets were used in conjunction with routine haematological procedures to analyse sequential changes in peripheral blood leucocyte populations during the course of tsetse fly-transmitted Trypanosoma congolense infection in trypanotolerant N'Dama and trypanosusceptible Boran cattle. Subsequent to the first parasitaemic wave, the N'Dama cattle maintained packed cell volumes (PCV) above 22 and lower levels of parasitaemia than Boran throughout the 160 days of the experiment. In contrast, the Borans developed severe anaemia and required curative drug therapy (i.e., PCV dropped to less than 15) by 55 days (range: 22-55 days) post infection. There were significant (P less than 0.05) decreases in total white blood cells and total lymphocytes from pre-infection levels to the first peak of parasitaemia (day 16 post-infection) in both groups. Flow cytometric analyses using MoABs revealed that this change was due to an absolute decrease in T cells expressing BoT2 and either BoT4 or BoT8, surface immunoglobulin M-positive (sIgM+) B cells, and null cells which did not express T cell, B cell or monocyte markers. During this period there was significant variation over time, but no overall increase or decrease, in the number of cells expressing class II major histocompatibility (MHC) molecules or monocyte markers, or in the number of circulating neutrophils or eosinophils. The BoT4/BoT8 ratios were significantly (P less than 0.01) increased in both groups of infected animals at the first peak of parasitaemia. After day 22 in the infected N'Damas and in the Borans which required drug therapy, there was a leucocytotic response characterized by an increase in the total number of B cells, T cells, and null cells. Prior to infection and throughout the course of the experiment N'Dama cattle had significantly (P less than 0.01) higher numbers of B cells and null cells than Boran.

Monoclonal antibody (mAb) IL-A17 characterizes a subset of bovine T lymphocytes. IL-A17 recognizes a 34,000-35,000 MW doublet, designated BoT8, which is expressed on the surface of approximately 20% of peripheral blood mononuclear leucocytes (PBM), a subpopulation of lymphocytes in T-dependent areas of lymph nodes and spleen, and about 70% of thymocytes. This molecule is not expressed on B lymphocytes, monocytes/macrophages or granulocytes. Double labelling of PBM showed that the BoT8+ population is distinct from the T lymphocyte subset expressing BoT4. BoT8+ lymphocytes purified with a fluorescence-activated cell sorter (FACS) proliferated poorly in response to mitogenic and alloantigenic stimulation in the absence of exogenous growth factors. IL-A17 had no inhibitory effect on proliferation of PBM to mitogens (Con A and PHA) or alloantigens and no measurable effect on the in vitro generation of cytolytic effector cells. However, in some experiments IL-A17 was found to block partially allospecific cytolytic function mediated by bulk effector cell populations when included in 51Cr-release assays. Fractionation of effector cells generated in an allogeneic mixed leucocyte culture (MLC) demonstrated that cytotoxic cells specific for class I major histocompatibility complex (MHC) antigens reside within the BoT8+ population. Based on these data, and information reported elsewhere on alloreactive bovine T-cell clones, BoT8 is considered to be analogous to CD8 in humans and equivalent molecules in other species.

We report the identification of a subset of bovine T cells by two monoclonal antibodies (mAb), IL-A11 and IL-A12, and some functional analyses of these cells. Both mAb precipitate two polypeptides, called BoT4, with apparent molecular mass of 52 and 55 kilodaltons. The epitopes recognized by these two mAb are different, however. BoT4 is found on approximately 70% of thymocytes and 30% of peripheral blood mononuclear cells (PBM), is not expressed by monocytes or B cells, and is found on cells in the T-dependent areas of lymph nodes. BoT4+ lymphocytes purified by a fluorescence-activated cell sorter proliferate in response to mitogenic and alloantigenic stimulation without addition of exogenous growth factors, whereas BoT4- lymphocytes do not. Monoclonal antibodies IL-A11 and IL-A12 have no effect on mitogen- (PHA and Con A) or alloantigen-induced proliferation of PBM. Monoclonal antibody IL-A12 has no inhibitory effect on the cytolytic activity of bulk populations of alloreactive T lymphocytes, and most of the cytolytic activity generated in mixed leukocyte culture is ascribable to the BoT4- population. Using cloned alloantigen-specific lymphocytes, we found that the ability of BoT4+ clones to proliferate to alloantigenic stimuli without exogenous growth factors is a characteristic of some clones, as is susceptibility to inhibition of proliferation by mAb IL-A12. These results implicate the BoT4 molecule in antigen recognition but indicate that the requirement for BoT4 is variable among clones. Our findings, together with those in the companion paper, which provides evidence that BoT4+ lymphocytes are class II restricted, indicate that BoT4+ lymphocytes are the bovine equivalent of Leu3/T4+ lymphocytes of humans and analogous lymphocytes of other species.

Following prescapular lymphadenectomy, the cellular composition of afferent (peripheral) lymph from eight out of 13 calves in which the afferent and efferent lymphatic ducts had anastomosed was examined using monoclonal antibodies (mAb) specific for bovine lymphoid populations. Afferent lymph leucocytes (ALL) consisted of around 50% T lymphocytes, of which 35% and 14% expressed BoT4 and BoT8, respectively, 20-25% B lymphocytes and 23% non-lymphoid cells. When populations of non-lymphoid cells were prepared from ALL by adherence to gelatin-plasma-coated flasks, 81% contained alpha-napthyl acetate esterase (ANAE) and 70% were phagocytic, whereas the non-adherent population contained around 5% of these cell types, the cells being largely non-phagocytic and negative for ANAE. Following purification of non-lymphoid cells on the fluorescence-activated cell sorter (FACS) using IL-A22 (mAb detecting macrophages and polymorphonuclear leucocytes, PMN), only 72% of the positive cells contained ANAE, 19% were phagocytic, and 84% expressed Fc1 receptors. While the ALL depleted of IL-A22+ cells could not proliferate in the presence of specific antigen in culture, the addition of purified monocytes from peripheral blood leucocytes (PBL) or IL-A22-positive ALL to macrophage-depleted PBL or IL-A22-negative ALL promoted proliferative responses to specific antigens. Similar populations of non-lymphoid cells stimulated mixed leucocyte reactions (MLR) in allogeneic combinations. When these non-lymphoid cells were pulsed with specific antigens prior to addition to autologous PBL or ALL in culture, normal or gamma-irradiated but not glutaraldehyde-fixed cells from PBL or ALL induced proliferation and immunoglobulin (Ig) synthesis in responder cells. Proliferation was depressed in cultures containing mAb detecting BoT4, MHC class II antigens and a mAb which detected non-lymphoid cells in PBL; Ig synthesis was inhibited only by anti-class II mAb. Monocyte-depleted PBL or IL-A22-negative ALL responded specifically to autologous antigen-presenting cells which had been pulsed with the original immunogen and not other antigens.

Five monoclonal antibodies (MAb) produced against cell surface antigens on bovine mononuclear phagocytes (MPh) were characterized. None of the MAb recognized erythrocytes, thrombocytes, B lymphocytes or resting or activated T lymphocytes. Two MAb (IL-A22 and IL-A24) reacted with the majority of monocytes and granulocytes in peripheral blood, with 20-40% bone marrow cells comprising myelo-monocytic cells, and with a proportion of mature macrophages. Reactivity of the remaining three MAb was restricted to MPh: one of these (IL-A25) was apparently specific for pulmonary macrophages, whereas the molecules recognized by the other two (IL-A23 and CH16A) were expressed on subpopulations of blood monocytes and tissue macrophages. None of the MAb inhibited adherence of MPh to plasma-coated gelating surfaces or Fc-mediated rosette formation. One of the MAb, IL-A24, which reacts with MPh and granulocytes, inhibited antigen-specific proliferative response or peripheral blood mononuclear leukocytes (PBM) to the soluble antigen, keyhole limpet hemocyanin (KLH) but did not inhibit responses to concanavalin A or allogeneic leukocytes. This MAb was shown to react with two polypeptides of approximately 75 kD and 110 kD on the surface of peripheral blood monocytes.

During the course of a lethal infection with Theileria parva (Muguga), the surface phenotypes of efferent lymphatic lymphocytes (ELL) were analysed to determine whether the parasite preferentially infected any particular subpopulation of cells. In the second week of infection, when the proportion of lymphoblasts and parasitized cells increased to 50% of the total ELL, greater than 99% of infected cells expressed T-lymphocyte markers including both BoT4 and BoT8. From day 10, a population of T-lymphocytes coexpressing BoT4 and BoT8 appeared in ELL, reaching 33% by day 14. Similar changes were observed in peripheral blood mononuclear cells (PBM) and lymph node cells (LNC). Analysis of ELL sorted into populations differing on the basis of expression of BoT4 and BoT8, revealed a higher level of parasitosis in the BoT4+ and BoT8+ lymphocytes than in the BoT4+ BoT8- or BoT4- BoT8+ populations. For comparison, the phenotypes of 28 cloned cell lines, obtained by infection of PBM with sporozoites in vitro, were examined. All of these clones exhibited T-cell markers. Nine of the clones expressed both BoT4 and BoT8; within each of these lines, BoT4 was expressed on all cells, whereas BoT8 was expressed at variable concentrations on 20-70% of cells. That BoT4+ cells were induced by T.parva (Muguga) to coexpress BoT8 was demonstrated directly by the finding that a BoT4+ BoT8- T-cell clone expressed BoT8 following infection with the parasite.

Monoclonal antibodies (mAb) CH128A and CH61A react with molecules of 50,000-60,000 MW. They are expressed by all T cells in cattle, comprising 44-69% of peripheral blood mononuclear leucocytes (PBM), the majority of lymphocytes in T-dependent areas of lymph node, and 75-80% of cells derived from the thymus including both cortical and medullary thymocytes. The molecule recognized by these mAbs is not expressed on B lymphocytes, monocytes/macrophages, or granulocytes. Both mAb inhibit spontaneous rosette formation by sheep erythrocytes and bovine lymphocytes. We postulate that these mAb see the bovine homologue of the human sheep red blood cell receptor CD2 and has been named BoT2.