Analysis of the reactivity of anti-bovine CD8 monoclonal antibodies with cloned T cell lines and mouse L-cells transfected with bovine CD8

Mouse L-cells transfected with bovine CD8 and two Theileria parva-infected cloned T cell lines expressing bovine CD8 were used to screen the panel of ten monoclonal antibodies (mAbs) submitted to the workshop. Eight of the ten mAbs reacted with the transfectant and both the cloned T cell lines. However, two mAbs CC58 and BAT82A did not recognise the transfectant and only reacted with one of the T cell lines. Further biochemical studies indicated that the eight mAbs react with both homo- and heterodimeric forms of bovine CD8 whilst the two mAbs CC58 and BAT82A react with only heterodimeric forms. These data suggest that bovine DC8 is encoded by two genes as is the case in mouse and man.     

Analysis of pokeweed mitogen-induced in vitro proliferative and antibody responses of bovine lymphocytes.

The functional role of subpopulations of bovine cells in the regulation of pokeweed mitogen (PWM)-induced proliferative and antibody responses of peripheral blood mononuclear cells (PBM) was analysed. Subpopulations of bovine PBM identified by specific monoclonal antibodies (mAbs) were isolated by sorting them through the fluorescence activated cells sorter (FACS). The depletion from PBM of T cells bearing the CD4 marker, recognised by mAb IL-A12, resulted in a reduction of PWM-induced responses, which could be partly reversed by the addition of CD4 positive T cells. The depletion of cells belonging to the macrophage/monocyte lineage also resulted in a reduction of PWM-induced proliferative responses. PBM depleted of CD8 positive T cells, recognised by mAb IL-A51, showed increased PWM-induced responses, which in turn were reduced by the addition of mAb IL-A51 positive cells. Proliferative and antibody responses were also obtained by PWM stimulation of FACS-purified B cells, in the presence of bovine T cell growth factor.     

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.     

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.     

The role of CD4(+) or CD8(+) T cells in the protective immune response of BALB/c mice to Neospora caninum infection

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.     

Cellular interactions regulating the in vitro response of bovine lymphocytes to ovalbumin.

We examined the contribution of MHC class II-restricted T cells (CD4+), MHC class I-restricted T cells (CD8+), gamma/delta T cell receptor (TCR)+ T cells, B cells and macrophages to the development and control of in vitro proliferative responses of bovine lymphocytes to ovalbumin (OA). Cell populations for in vitro assay were obtained from peripheral blood (peripheral blood leukocytes, PBL) of OA-primed cattle. Specific cell populations were depleted or purified from PBL by staining with monoclonal antibodies (MAbs) against the appropriate differentiation antigens and sorting on a Fluorescence Activated Cell Sorter (FACS). OA-specific in vitro responses of in vivo primed PBL were dependent on the presence of CD4+ T cells. Their presence could not be replaced by the inclusion of T cell growth factor (TCGF) in the culture system, indicating that CD4+ T cells probably actively proliferate in response to antigenic stimulation. Bovine CD8+ T cells and gamma/delta TCR+ T cells appeared to exert a suppressive effect on proliferative responses. No proliferation was observed in PBL after the depletion of MHC class II+ cells. In this case, the response could be restored by the addition of macrophages or LPS-activated B cells to the MHC class II- population.     

Individual antigens of cattle. Bovine CD2 (BoCD2).

The data obtained in the workshop provide further evidence that CH128A and IL-A26 and the 12 new mAbs that form a cluster recognise the bovine orthologue of CD2. The mAbs inhibit rosetting with SRBC, stain cells in primary and secondary lymphoid organs in patterns consistent with those obtained in humans with anti-CD2 mAbs, and the 11 IgG mAbs all immunoprecipitate a peptide with a Mr of 58-62 kDa. It is not clear from the studies whether the epitopes defined by the mAbs correspond with the region I and II epitopes present on CD2. None of the data suggest that any of the mAbs recognise the region III (CDD2R) epitope (Peterson and Seed, 1987; Knapp et al., 1989). Further studies are now needed to define the physical and functional relation of the epitopes and establish whether antibody-mediated activation corresponds with that noted in humans. Data reported in one study (Baldwin et al., 1988) with IL-A26 suggest possible differences in the requirements for activation. In addition, further studies are needed to demonstrate how many cell types express BoCD2. In mice, evidence has been presented which shows the mouse orthologue is expressed on some B cells (Yagitta et al., 1989). Studies in cattle have clearly shown CD2 is present on the majority of CD4+ and CD8+ T-cells and a small population of CD4-/CD8- cells (Baldwin et al., 1988; Davis, unpublished observations). Evidence presented in this workshop has shown that some CD2+ cells express a WC2 molecule (Sopp et al., 1991).(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of monoclonal antibodies that recognize γδ T/null cells

Thirty two monoclonal antibodies (mAbs) from the first round of analysis in the Second International Swine CD Workshop were placed together with additional mAb derived from the first workshop in the null cell panel for further evaluation. Preparations of peripheral blood leukocytes, concanavalin A stimulated peripheral blood mononuclear cells, and spleen cells were used in flow cytometric analyses. Nineteen mAbs identified molecules that were not expressed on null cells, not lineage specific, or recognized activation molecules. Sixteen mAbs including control mAbs were identified that were specific for null cells. One of the latter mAbs, 041 (PGBL22A), that recognizes a determinant on a constant region of porcine γδ TcR established the majority of null cells are γδ T cells. Use of this mAb in further comparisons demonstrated the γδ T cell population is comprised of two major subpopulations, one negative and one positive for CD2. Two color analyses demonstrated that 11 of the mAbs formed a broad cluster that included control mAbs 188 (MAC320) that defined the CD2 negative SWC6 cluster in the first workshop and mAb 122 (CC101) that might recognize an orthologue of bovine WC1 and nine mAbs that recognize determinants on one or more molecules with overlapping patterns of expression on subsets of CD2⁻ γδ T cells. Two groups of mAbs formed the previously identified subset clusters SWC4 and SWC5. Two new mAbs formed a third subcluster. Three mAbs did not form clusters. Three mAbs predicted to recognize TcR in the first workshop (020 [PT14A], 021 [PT79A], and 022 [MUC127A]) and mAb PGBL22A were shown to immunoprecipitate a 37, 40 kDa heterodimer.     

Characterization of new monoclonal antibodies against porcine lymphocytes: molecular characterization of clone 7G3, an antibody reactive with the constant region of the T-cell receptor delta-chains

A battery of mouse monoclonal antibodies (mAbs) reactive with porcine peripheral blood (PB) leukocytes was generated. Among the mAbs, 6F10 was found to react probably with cluster of differentiation (CD)8 alpha-chain, while 7G3 and 3E12 were found to recognize gammadelta T-cells, as revealed by two-color flow cytometric and immunoprecipitation studies. 7G3 was shown to react with the constant (C) region of the T-cell receptor (TCR) delta-chain by the following facts: (1) 7G3 immunoprecipitated full-length TCR delta-chain protein fused with glutathione S-transferase (GST) produced by Esherichia coli and (2) 7G3 reacted with TCR delta-chain expressing Cos-7 cells transfected with either full-length or N-terminal deleted mutant cDNA, but did not react with Cos-7 cells transfected with C-terminal deleted mutant TCR delta-chain cDNA. All three mAbs produced high-quality immunostaining results on frozen sections, revealing a distinct distribution of gammadelta T-cells and CD8(+) cells. This report precisely characterizes mAbs against porcine TCR for the first time, facilitating molecular biological investigations of the porcine immune system.     

Characterisation of lymphocyte populations in African buffalo (Syncerus caffer) and waterbuck (Kobus defassa) with workshop monoclonal antibodies

In order to measure different lymphocyte populations in buffalo (Syncerus caffer) and waterbuck (Kobus defassa), we analysed the monoclonal antibodies from the 1st International Workshop on Leukocyte Antigens in Cattle, Sheep and Goats for suitable cross-reactive reagents. Peripheral blood mononuclear cells from three buffalo and three waterbuck were tested with the whole panel of monoclonal antibodies (mAbs) together with some additional antibodies against MHC and Ig. In some clusters almost all antibodies cross-reacted (CD2, CD8), in others almost none cross-reacted (CD4, CD5) and in cluster CD6, mAbs only reacted with buffalo but not waterbuck. Double staining experiments were performed on buffalo PBM with the cross-reacting antibodies, to confirm that they detected similar cell populations as in bovine PBM. This was shown with reagents against CD2, CD4, CD6, CD8, CD11, WC1, WC3 and Ig. The molecular weights of the buffalo antigens correlated well with those of the homologous cattle antigens. In the CD5 cluster, only one mAb reacted with the two wild species, and defined an unusual CD2+ CD5- cell population in buffalo. Also mAbs cross-reacting with buffalo MHC class II detected unusual expression on resting T cells. From the results presented, it is clear that the workshop panel contains mAbs against the most important T and B cell antigens of buffalo and probably waterbuck, which will allow us to compare functional lymphocyte populations in cattle and wild ruminants.     

Two monoclonal antibodies (CC17, CC29) recognizing an antigen (Bo5) on bovine T lymphocytes, analogous to human CD5

Two new monoclonal antibodies (CC17 and CC29) raised against bovine thymocytes are described. The antibodies, both of which were IgG1, recognize a molecule of approximately 67,000 molecular weight on bovine T cells. They react T cells in peripheral blood, the lymph node paracortex and the periateriolar lymphoid sheath in the spleen. Both the cortex and medulla of the thymus are stained but the medulla reacts more intensely. They do not stain B cells in peripheral blood, the ileal Peyer's patch, the cortex or the primary follicles in lymph nodes. No activity was found on cells outside the lymphoid system, i.e. monocytes, alveolar macrophages or endothelial and epithelial tissue. The antigen recognized is considered to be the bovine homologue of CD5 (T1) in humans and Lyt1 in mice. The mAbs appear to be particularly useful for detecting cells in the peripheral blood of young calves which are of the T cell lineage but do not express BoT2 or the mature pan T cell antigen recognized by mAb IL-A27 and may thus allow identification of a population of bovine lymphocytes previously described as null cells.     

The detection of CD2+, CD4+, CD8+, and WC1+ T lymphocytes, B cells and macrophages in fixed and paraffin embedded bovine tissue using a range of antigen recovery and signal amplification techniques

In order to develop procedures to label the main bovine leucocyte populations in paraffin embedded sections, the immunoreactivity of 25 monoclonal antibodies (mAbs) to different leucocyte antigens was assessed with formal dichromate (FD5) and 10% formalin fixation, a battery of antigen retrieval (AR) methods, and the biotin-tyramide amplification system. All the leucocyte populations investigated (CD2+, CD4+, CD8+, WC1+ T lymphocytes, B cells and macrophages) were strongly and specifically detectable under an appropriate combination of mAb, AR method and signal amplification system. CD4 and CD8 required the most stringent conditions and could only be demonstrated in FD5 fixed sections. For detection of CD2, WC1+ T lymphocytes, B cells and macrophages, all the mAbs produced immunoreactivity in FD5 or formalin fixed tissues. The need to check a range of different AR methods is stressed, as the method of choice varied for each individual mAb. The incorporation of the signal amplification system was necessary to observe a strong signal and the complete distribution of CD4, CD8 and B cells. Fixation by FD5 proved to be better than formalin for the preservation of surface antigens but it was inferior for the detection of markers which were found to show cytoplasmic immunoreactivity, such as the macrophage marker MAC387 or the B cell markers BAQ155 or IL-A59.     

Polymorphism of the CD4 and CD5 differentiation antigens in cattle.

Monoclonal antibodies (mAbs) specific for bovine CD4 and CD5 antigens have been found to identify polymorphic determinants on these molecules. In the case of CD5, mAb IL-A67 recognises one allotypic form of the antigen while four other CD5-specific mAbs in the workshop (CC17, CC29, BLT-1 and 8C11) recognise a second allotype. The CD4-specific mAbs submitted to the workshop reacted with the cells of all animals tested. However, a further two mAbs (CC26 and IL-A18) specific for CD4 were found to react with cells only from about 85% of animals tested. Sequential immuno-precipitation experiments together with family studies showed that the allotypes of CD4 and CD5 are both inherited in a simple Mendelian manner and are co-dominantly expressed. One of the CD5 allotypes was not detected in Bos taurus animals while the gene frequency of the second allotype was only about 10% in the B. indicus animals tested. The gene frequency of the CD4 allotype detected by CC26 and IL-A18 was similar in the two sub-species.     

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.     

Monoclonal antibody specific for bovine CD 5 antigen which enhances mitogen-induced blastogenesis and IL-2 production.

This study reports on the functional characteristics of a bovine T-cell differentiation antigen recognized by the monoclonal antibody (mAb) 8C11. This mAb has previously been found to react with a 67-kD molecule shared by thymocytes and peripheral blood T cells and to be undetectable on the B cells of healthy animals. This antigen is also largely expressed on the B cells from bovine leukemia virus-infected animals. Molecules with a similar cell distribution have been described in other species (mouse, human, rat and sheep), and were termed CD5 molecules. In order to confirm the CD5-like nature of the target molecule recognized by 8C11, functional T-cell assays were carried out. We report here that this mAb, like its human and murine homologues, enhances the proliferative responses of T cells to mitogens or alloantigens but does not directly stimulate T-cell division. Moreover, we have shown an enhancing effect of this 8C11 mAb on bovine interleukin-2 production by concanavalin A-stimulated bovine peripheral blood mononuclear cells.     

Detection of bovine herpesvirus 4 in CD11b+ leukocytes of experimentally infected rabbits

The presence and numbers of bovine herpesvirus 4 (BoHV-4) infected CD11b+ leukocytes were investigated during experimental infections of New Zealand White rabbits by Fluorescence Activated Cell Sorter (FACS) analysis. Peripheral blood leukocytes (PBL) were collected every second day, and the cells were stained with phycoerythrin-labelled CD11b-specific mouse monoclonal antibody and fluorescein-conjugated bovine herpesvirus 4-specific mouse monoclonal antibody. The numbers of double-stained cells from PBLs of the control and inoculated groups were measured and compared in FACSTREK analyser. Double-stained cells were detected in the virus-inoculated group on postinoculation days (PID) 2-5 and 9-12. The results indicated that CD11b+ PBLs were permissive for BoHV-4 infection, and are probably the main reservoir of the virus during the latent period. The data did not indicate production of infectious viral particles, but virus-specific proteins were expressed on the surface of CD11b+ cells. The two waves of double-stained cells gave similar results to the PCR assays from serum samples, which showed the presence of viral DNA in the serum on the same days when virus-infected CD11b cells were also present. Productive BoHV-4 infection of mast cells or undifferentiated leukocytes in the bone marrow and the antiviral immune response might be responsible for this periodic appearance of the virus in CD11b+ PBLs and in the serum. The paper provides evidence that CD11b+ PBLs are the main target cell populations in the blood for BoHV-4.     

Generation and characterization of monoclonal antibodies to equine NKp46

The immunoreceptor NKp46 is considered to be the most consistent marker of NK cells across mammalian species. Here, we use a recombinant NKp46 protein to generate a panel of monoclonal antibodies that recognize equine NKp46. The extracellular region of equine NKp46 was expressed with equine IL-4 as a recombinant fusion protein (rIL-4/NKp46) and used as an immunogen to generate mouse monoclonal antibodies (mAbs). MAbs were first screened by ELISA for an ability to recognize NKp46, but not IL-4, or the structurally related immunoreceptor CD16. Nine mAbs were selected and were shown to recognize full-length NKp46 expressed on the surface of transfected CHO cells as a GFP fusion protein. The mAbs recognized a population of lymphocytes by flow cytometric analysis that was morphologically similar to NKp46+ cells in humans and cattle. In a study using nine horses, representative mAb 4F2 labeled 0.8-2.1% PBL with a mean fluorescence intensity consistent with gene expression data. MAb 4F2+ PBL were enriched by magnetic cell sorting and were found to express higher levels of NKP46 mRNA than 4F2- cells by quantitative RT-PCR. CD3-depleted PBL from five horses contained a higher percentage of 4F2+ cells than unsorted PBL. Using ELISA, we determined that the nine mAbs recognize three different epitopes. These mAbs will be useful tools in better understanding the largely uncharacterized equine NK cell population.     

FACS analysis of bovine leukemia virus (BLV)-infected cell lines with monoclonal antibodies (mAbs) to B cells and to monocytes/macrophages.

The eighteen monoclonal antibodies (mAbs) to B cells and the fourteen mAbs to accessory cells submitted to the workshop were analysed by FACS on three established, bovine leukemia virus (BLV)-infected bovine cell lines. Several mAbs of previously defined specificity were run in parallel. This analysis allowed us to gain further insight on the precise phenotype of those peculiar cells and to cluster the submitted mAbs according to their staining patterns. The BLV-infected cell lines seemed to belong to the B cell type though some of them lack detectable surface immunoglobulins. Moreover, all lines express the CD5 T cell marker and several myeloid markers.     

Loss of naïve (CD45RA+) CD4+ lymphocytes during pediatric infection with feline immunodeficiency virus

Feline immunodeficiency virus (FIV) infection of cats is an animal model for the pathogenesis of CD4+ lymphopenia and thymus dysfunction in HIV-infected humans. Recently, a monoclonal antibody (755) was reported to recognize the feline homologue to CD45RA, allowing the enumeration of na?ve T cells in cats. We tested the hypothesis that pediatric FIV infection would be associated with a selective loss of na?ve CD4+ lymphocytes by inoculating newborn cats with a pathogenic clone of FIV (JSY3) or a related clone with an inactive ORF-A gene (JSY3-DeltaORFA), and compared the data to age-matched uninfected control cats. Both FIV inocula were associated with a reduction in the CD4-CD8 ratio (p=0.01), which was attributable to a disproportionate loss of na?ve CD4+ cells (p=0.01) vs. na?ve CD8+ cells. Therefore, the reduced CD4:CD8 ratio in FIV-infected juvenile cats is associated with a selective depletion of na?ve CD4+ cells from the blood.     

Equine herpesvirus-1 infection induces IFN-gamma production by equine T lymphocyte subsets

A commercial bovine IFN-gamma-specific monoclonal antibody was used to measure antigen-specific IFN-gamma production by equine lymphocytes. Paired PBMC samples were collected from six ponies prior to and 10 days after challenge infection with equine herpesvirus-1 (EHV-1). Each sample was stimulated in vitro with EHV-1, virus-free medium, or PMA and ionomycin, and labelled with monoclonal antibodies specific for various equine lymphocyte subset markers. Following fixation, intracellular IFN-gamma was detected using a FITC-conjugated bovine IFN-gamma-specific monoclonal antibody. In vitro restimulation of PBMC with EHV-1 induced IFN-gamma production by a significantly higher percentage of total (CD5(+)) T lymphocytes, and CD4(+) and CD8(+) T lymphocyte subsets among post-EHV-1 infection PBMC samples compared to pre-infection samples. This response was associated with an increase in virus-specific CTL activity, a critical immune effector for the control of EHV-1 infection and disease. No significant increase in IFN-gamma production by B lymphocytes was observed. These data demonstrate that EHV-1 challenge infection of ponies results in increased production of IFN-gamma by virus-specific T lymphocytes, and that this response can be quantitated using flow cytometry.