W.I. Morrison

Improved methods for purification and depletion of monocytes from bovine peripheral blood mononuclear cells: Functional evaluation of monocytes in responses to lectins

We have compared different techniques for the enrichment and depletion of monocytes from bovine peripheral blood mononuclear cells. Adherence to plasma-coated gelatin was the most efficient and reproducible method for enrichment of monocytes (80% monocytes), whereas depletion of peripheral blood mononuclear cells of monocytes (0.3% monocytes and less) was best achieved by defibrination of the blood from which the PBM were separated. In both instances, purity of the cell population could be improved further by an additional step, namely, FACS sorting with a monocyte-specific monoclonal antibody to purify monocytes (97% monocytes and more), and adherence to polystyrene to remove residual monocytes from defibrinated PBM (0.1% monocytes and less). Depletion of monocytes abolished the response of PBM to concanavalin A and phytohaemagglutinin. The lectin-induced response could be restored by adding gelatin/plasma purified monocytes. This activity of monocytes could be replaced by 2-mercaptoethanol.

Monoclonal antibodies identify phenotypically and functionally distinct cell types in the bovine lymphoid system

Monoclonal antibodies were produced against bovine lymphoid cells. The reactivities of the antibodies for membrane determinants were examined on both cell suspensions and cryostat tissue sections prepared from bovine blood, thymus, spleen and lymph nodes. The antibodies were putatively grouped into sets which reacted with monomorphic and polymorphic determinants associated with bovine class I and class II major histocompatibility complex (MHC) antigens (MAbs P12 and P3, and R1 and P2 respectively), or associated with differentiation antigens expressed on T cells and monocytes (MAb P5) or exclusively on monocytes (MAb P8). The antibodies were used to identify the surface phenotypes of cells which stimulate (R1+ P5+ P8+) and proliferate (R1- P5+ P8-) in the bovine mixed leukocyte cultures, and cells which proliferate in response to the mitogen, concanavalin A (R1- P5+).

Differentiation antigens on bovine mononuclear phagocytes identified by monoclonal antibodies

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.

Bovine helper T‐cell clones specific for lymphocytes infected with Theileria parva (Muguga)

Summary T‐cell clones specific for lymphocytes infected with Theileria parva were derived from animals immunized by infection with T. parva (Muguga). These clones were non‐cytolytic and had the BoT4+ BoT8‐ surface phenotype, BoT4 and BoT8 being the bovine analogues of human CD4 and CD8 molecules. The clones proliferated in response to irradiated autologous lymphoblasts infected with T. parva (Muguga) but not to autologous uninfected lymphoblasts or monocytes. They were parasite strain‐specific, in that they did not respond to autologous lymphoblasts infected with another parasite stock, T. parva (Marikebuni). The clones proliferated in the absence of exogenous T‐cell growth factor (TCGF) and produced TCGF when stimulated with concanavalin A. Induction of proliferation of the cloned T‐cells was genetically restricted, and evidence was obtained which indicated that they were restricted by determinants on class II major histocompatibility complex (MHC) molecules. These findings demonstrate that infections with T. parva stimulate antigen‐specific MHC‐restricted T‐cells with the properties of T‐helper cells. The results also provide further evidence for the expression of a parasite strain‐specific antigen on the surface of T. parva‐infected lymphocytes.

Characterization of a polymorphic immunodominant molecule in sporozoites and schizonts of Theileria parva

Summary This study examines several aspects of a polymorphic, immunodominant molecule (P!M) found in the protozoan parasite. Theileria parva. The antigen is present in all T. p. parva stocks examined, and in the related subspecies, T. p. bovis and T. p. lawrencei. It is the predominant antigen recognized by antisera from immune cattle on Western blot analysis of schizont‐infected lymphocytes, and is the only antigen which has been shown to react with anti‐schizont monoclonal antibodies (MoAbs) on Western blots or in immunoprecipitations. The antigen shows polymorphism in both size and expression of antibody epitopes among the different stocks of T. parva. The antigen is present in sporozoites as well as schizonts.

Protective immunity and specificity of antibody responses elicited in cattle by irradiated Trypanosoma brucei

The level and specificity of antibody responses elicited in cattle by irradiated non‐infective Trypanosoma brucei were examined and related to the development of protective immunity. These responses were compared with those induced by infection and by inoculation with purified variable surface glycoprotein (VSG) in adjuvant. It was found that 107 or more irradiated trypanosomes inoculated intravenously into cattle conferred complete protection against challenge with 103 homologous trypanosomes 14 days later. Animals immunized with 106 organisms showed partial protection. Of the assays used for detection of antibody, neutralization of infectivity was slightly more sensitive than either the Farr assay or the immunofluorescence test which were both more sensitive than solid‐phase radioimmunoassay (RIA). Detection of specific antibody correlated with immunity, in that all animals inoculated intravenously with 106 or more trypanosomes developed neutralizing activity in their sera. The antibody responses after intravenous inoculation were consistently superior to those induced by the subcutaneous route. By carrying out blocking assays, most of the antibody elicited by irradiated trypanosomes was found to be specific for antigenic determinants on the VSG exposed on the surface of live trypanosomes. A similar specificity was found for the antibody induced during infection with T. brucei. Conversely, a large component of the antibody induced by purified VSG in adjuvant appeared to be directed against determinants on the VSG which are not exposed on live trypanosomes and are probably not involved in protective immunity.

Theileria parva (Muguga) infects bovine T‐lymphocytes in vivo and induces coexpression of BoT4 and BoT8

Summary 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, > 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.

Regulation of the growth and differentiation of Trypanosoma (Trypanozoon) brucei brucei in resistant (C57B1/6) and susceptible (C3H/He) mice

Summary While Trypanosoma brucei brucei GUTat 3 were equally infective for C3H/Heand for C57B1/6 mice at doses ranging from 5 to 5 × 103 organisms and had similar prepatent periods in both strains of mice, infected C57B1/6 mice displayed lower parasitaemia, shorter times to parasite wave remission and survived for a longer time than infected C3H/He mice. Parasite growth and differentiation rates and host immune responses were similar for the first 5 days in both strains of mice after infection with 103 T.b.brucei GUTat 3 but, thereafter, parasite differentiation proceeded more rapidly and specific antibodies reached higher titres in C57B1/6 than in C3H/He mice. In contrast, parasite growth and differentiation rates were similar in irradiated mice of both strains. Furthermore, following inoculation of intact mice with irradiated T.b.brucei GUTat 3, C3H/Hemice actually mounted higher titred antibody responses than C57B1/6 mice showing that they were not intrinsically defective in their capacity to respond to GUTat 3 antigens. Parasite differentiation occurred at the same rate in irradiated (650r) C57B1/6 mice and in irradiated C57B1/6 mice reconstituted with syngeneic spleen cells although T.b.brucei GUTat 3 specific antibody was detected in the latter mice prior to peak parasitaemia. Furthermore, it was shown directly in C57B1/6 mice that there was no selective destruction of slender form T.b.brucei GUTat 3 parasites during the phase of accumulation of stumpy form parasites. These studies indicate that the more rapid differentiation of T.b.brucei GUTat 3 parasites in infected C57B1/6 mice as compared to infected C3H/Hemice was unlikely to be directly related to the more efficient antibody response in the infected C57B1/6 mice. The observations suggest that there might be an association between host mechanisms which regulate differentiation of T.b.brucei parasites and those which regulate antibody responses.

Cell-mediated immune responses of cattle to Theileria parva.

Theileria parva is a protozoan parasite that infects lymphocytes of cattle and African buffalo. As is the case with certain viruses, the parasite causes antigenic changes on the cell surface against which the host mounts cytotoxic T-cell. Precise definition of the cells participating in these response and their specificity has been facilitated by the recent identification of markers for bovine T-cell subpopulations and functional analyses of bovine lymphocytes at the clonal level. In this paper Ivan Morrison and his colleagues discuss current information on the parasite specificity and MHC restriction of anti-Theileria cytotoxic T cell, in relation to their role in protective immunity.

BOVINE MONONUCLEAR PHAGOCYTIC-CELLS - IDENTIFICATION BY MONOCLONAL-ANTIBODIES AND ANALYSIS OF FUNCTIONAL-PROPERTIES

Monoclonal antibodies (mAb) which react with bovine monocytes have been produced. These include three mAb (P8, IL-A22 and IL-A24) that recognize the majority of monocytes and granulocytes in peripheral blood; two of these mAb were also shown to react with 30-40% of cells in bone marrow, including both monocytic and granulocytic cells, and with variable percentages of tissue macrophages. Thus these mAb can act as markers for myeloid cells in haemopoietic tissues and for monocytes in cell populations devoid of granulocytes. A further two mAb (IL-A23 and IL-A25) recognize monocytes and/or macrophages. The reactivity of one of these mAb (IL-A25) appears to be mainly restricted to pulmonary macrophages. The other mAb reacts with a variable proportion of blood monocytes and generally with a higher percentage of tissue macrophages, suggesting that its expression may relate to activation or maturation of monocytes. In order to study the functional properties of peripheral blood monocytes, techniques were developed for obtaining populations of peripheral blood mononuclear cells (PBM) depleted of monocytes to less than 0.2% and monocyte populations of greater than 97% purity. Removal of monocytes from PBM abrogated the capacity of the cells to proliferate in response to Con A and PBS, although addition of 2-mercaptoethanol to the cultures restored proliferation. In both allogeneic and autologous mixed leukocyte cultures (MLC), monocytes were required in the stimulator cell populations for induction of the proliferative responses, and both responses could be elicited with purified monocytes. However, proliferation in the autologous MLC occurred only with responder cell populations that were depleted of monocytes. Moreover, it was shown that addition of more than 5% unirradiated monocytes to the autologous MLC suppressed proliferation. These findings indicate that monocytes play an important role in the induction and regulation of cellular immune responses in cattle. Two of the mAb that react with monocytes and granulocytes were tested for their capacity to inhibit proliferative responses of PBM to mitogens, alloantigens or the soluble antigen, KLH.(ABSTRACT TRUNCATED AT 400 WORDS)