The influence of age and Rhodococcus equi infection on CD1 expression by equine antigen presenting cells

There is a distinct age-associated susceptibility of horses to Rhodococcus equi infection. Initial infection is thought to occur in the neonatal and perinatal period, and only foals less than 6 months of age are typically affected. R. equi is closely related and structurally similar to Mycobacterium tuberculosis, and causes similar pathologic lesions. Protective immune responses to M. tuberculosis involve classical major histocompatibility complex (MHC)-restricted T cells that recognize peptide antigen, as well as MHC-independent T cells that recognize mycobacterial lipid antigen presented by CD1 molecules. Given the structural similarity between these two pathogens and our previous observations regarding R. equi-specific, MHC-unrestricted cytotoxic T lymphocytes (CTL), we developed 3 related hypotheses: (1) CD1 molecules are expressed on equine antigen presenting cells (APC), (2) CD1 expression on APC is less in foals compared to adults and (3) infection with live virulent R. equi induces up-regulation of CD1 on both adult and perinatal APC. CD1 expression was examined by flow cytometric analysis using a panel of monoclonal CD1 antibodies with different species and isoform specificities.

Results

Three CD1 antibodies specific for CD1b showed consistent cross reactivity with both foal and adult monocyte-derived macrophages (MDM). CD1b and MHC class II expression were significantly higher on adult MDM compared with foals. R. equi infected MDM showed significantly lower expression of CD1b, suggesting that infection with this bacterium induces down-regulation of CD1b on the cell surface. Histograms from dual antibody staining of peripheral blood mononuclear cells also revealed that 45–71% of the monocyte population stained positive for CD1b, and that the majority of these also co-expressed MHC II molecules, indicating that they were APC. The anti-CD1 antibodies showed no binding or minimal binding to bronchoalveolar lavage (BAL)-derived macrophages.

Conclusion

The CD1b isoform is evolutionarily conserved, and is present on equine MDM, as well as on circulating blood monocytes. The unique susceptibility of foals to R. equi infection may be due in part to lower expression of CD1 and MHC class II, as observed in this study. The data also suggests that infection with R. equi induces down-regulation of CD1b on equine MDM. This may represent a novel mechanism by R. equi to avoid detection and killing of infected cells by the immune system, similar to that observed when human APC are infected with M. tuberculosis.     

Cloning, expression and characterization of monkey (Macaca fascicularis) CD137

CD137 plays an important role as a co-stimulatory molecule in activated T cells. Agonistic CD137 specific antibodies have been investigated as therapeutic agents to promote tumor-specific immune responses by direct activation of T cells. As part of the pre-clinical pharmacological evaluation of cynomolgus monkeys, monkey CD137 was cloned and characterized. The deduced amino acid sequence encoded a full-length gene of 254 amino acids 95% identical to human CD137. Sequence variants identified in monkey CD137 include four splice variants lacking the transmembrane domain. These variants were detectable in human including two previously unreported variants. Two missense single nucleotide polymorphisms were detected present in 42 and 50% of 36 monkeys tested. In both monkey and human, mRNA expression of full-length CD137 and splice variants were significantly increased in peripheral blood mononuclear cells (PBMCs) upon stimulation by anti-CD3 antibodies. Recombinant monkey CD137 protein was bound with high affinity by an agonistic anti-human CD137 antibody but not by an anti-mouse CD137 antibody. In summary, compared to human, monkey CD137 showed distinct extracellular domain amino acid sequence and sequence polymorphisms. Thus, antibodies directed against epitopes in this extracellular domain could have differences in pharmacologic activity between cynomolgus monkeys and human or across individual cynomolgus monkeys.     

Human intravenous immunoglobulin (hIVIG) inhibits anti-CD32 antibody binding to canine DH82 cells and canine monocytes in vitro

The IgG receptors CD16 and CD32 (Fc_γRIII and Fc_γRII) link the humoral immune response to effector cell immune responses by binding immune complexes. Human intravenous immunoglobulin (hIVIG) consisting of immunoglobulin from pooled donors is reported to block Fc_γRs and has been used to treat a variety of canine autoimmune disorders. Fc_γRs have been poorly described for canine monocytes; therefore, the objectives of this study were to: (1) identify canine monocyte/macrophage Fc_γR (CD16 and CD32) expression and (2) demonstrate in vitro hIVIG binding to these receptors. The canine monocyte/macrophage-like cell line (DH82) and monocytes isolated from peripheral blood of healthy dogs were evaluated by flow cytometry (FACS) for CD16 and CD32 expression using commercially available anti-CD16 and anti-CD32 antibodies directed against the human isoforms. The mean percentage of cells expressing CD16 was 55% of DH82 cells and 13% of blood monocytes and the mean percentage of cells expressing CD32 was 85% of DH82 cells and 73% of blood monocytes. Immunoprecipitation of canine DH82 cells lysate using the same anti-CD16 or anti-CD32 antibodies suggested that these anti-human antibodies recognize the canine homologues. To demonstrate Fc_γR blockade, cells were incubated with increasing concentrations of hIVIG and then incubated with anti-CD16 or anti-CD32 antibodies. The percentage of CD32 expression decreased in a concentration dependent fashion in DH82 cells and blood monocytes after incubation with increasing concentrations of IVIG, suggesting that hIVIG was binding to CD32 and inhibiting anti-CD32 antibody binding. The same results were not demonstrated with anti-CD16 antibody. We believe this is the first report to demonstrate Fc_γ receptors CD16 and CD32 expression on canine monocytes and in vitro CD32 binding by human IgG, which may represent one of the immunomodulatory mechanisms of hIVIG.     

CD20 expression in normal canine B cells and in canine non-Hodgkin lymphoma

We examined the expression of CD20 in normal canine peripheral blood mononuclear cells, normal canine spleen, and canine non-Hodgkin lymphoma (NHL) to determine the feasibility of using this antigen as a diagnostic aid and as a possible target for therapy. An antibody generated against a C-terminal (intracytoplasmic) epitope of human CD20 recognized proteins of 32-36 kd in normal and malignant canine lymphocytes. This antibody showed restricted membrane binding in a subset of lymphocytes in peripheral blood, in the B-cell regions from a normal canine spleen and lymph node, and in malignant cells from 19 dogs with B-cell NHL, but not from 15 dogs with T-cell NHL. The patterns of CD20 reactivity in these samples overlapped those seen using an antibody that recognizes canine CD79a. This anti-CD20 antibody is therefore suitable as an aid to phenotype canine NHL. In contrast, normal canine B cells were not recognized by any of 28 antibodies directed against the extracellular domains of human CD20 (including the chimeric mouse-human antibody Rituximab) or by any of 12 antibodies directed against the extracellular domains of mouse CD20. Thus, the use of CD20 as a therapeutic target will require the generation of specific antibodies against the extracellular domains of canine CD20.     

Monoclonal antibodies to equine CD23 identify the low-affinity receptor for IgE on subpopulations of IgM+ and IgG1+ B-cells in horses

CD23, also called FcεRII, is the low-affinity receptor for IgE and has first been described as a major receptor regulating IgE responses. In addition, CD23 also binds to CD21, integrins and MHC class II molecules and thus has a much wider functional role in immune regulation ranging from involvement in antigen-presentation to multiple cytokine-like functions of soluble CD23. The role of CD23 during immune responses of the horse is less well understood. Here, we expressed equine CD23 in mammalian cells using a novel IL-4 expression system. Expression resulted in high yield of recombinant IL-4/CD23 fusion protein which was purified and used for the generation of monoclonal antibodies (mAbs) to equine CD23. Seven anti-CD23 mAbs were further characterized. The expression of the low-affinity IgE receptor on equine peripheral blood mononuclear cells was analyzed by flow cytometric analysis. Cell surface staining showed that CD23 is mainly expressed by a subpopulation of equine B-cells. Only a very few equine T-cells or monocytes expressed CD23. CD23(+) B-cells were either IgM(+) or IgG1(+) cells. All CD23(+) cells were also positive for cell surface IgE staining suggesting in vivo IgE binding by the receptor. Two of the CD23 mAbs detected either the complete extracellular region of CD23 or a 22kDa cleavage product of CD23 by Western blotting. The new anti-CD23 mAbs provide valuable reagents to further analyze the roles of CD23 during immune responses of the horse in health and disease.     

Analysis of T lymphocyte subsets proliferating in response to infective and UV-inactivated African swine fever viruses.

The proliferative response to infective and UV-inactivated African swine fever virus was analyzed in cells from pigs surviving an experimental infection with attenuated virus. All the pigs showed strong dose-dependent proliferative responses to both infective and UV-inactivated virus. This response was also observed when nitrocellulose-bound solubilized virus proteins were used in the assay. Heterologous isolates also induced proliferation, however it was significantly lower than that induced by the isolate used to infect the animals. The response to infective virus was blocked equally by anti-CD4 and anti-CD8 monoclonal antibodies (mAb); the response to UV-inactivated virus was almost abolished by anti-CD4 and 60% inhibited by anti-CD8 mAb. FACS analysis of 28-day T cell lines derived from peripheral blood mononuclear cells demonstrated the progressive increase of the CD8+ subset when the cells were stimulated with infective virus, whereas the stimulation with UV-inactivated virus induced the increase of both CD4+ and CD8+ subsets. In this case, the sum of CD4+ and CD8+ percentages was higher than the total percentage of T cells, suggesting the presence of cells positive for both CD4+ and CD8+.     

Cross-species reactivity of seven monoclonal antibodies with equine lymphocytes by flow cytometry

The recognition of equine lymphocyte antigens by monoclonal antibodies (mAbs) directed against human CD11a, CD18, CD21, CD23, CD29 and DR, as well as mouse CD23 was studied by flow cytometry. Unlike anti-CD11a, -CD21, -CD23 and DR mAbs, anti-CD18 and CD29 mAbs labelled the same percentage of horse peripheral blood lymphocytes (PBL) as human PBL. Double-staining with anti-horse immunoglobulin antibodies showed that anti-CD21 and -CD23 mAbs are mainly bound to peripheral blood B lymphocytes. The seven mAbs were also tested on the lymph node and thymus cells. The molecular targets of anti-CD11a, CD18 and CD29 mAbs were confirmed by immunoprecipitation of the membrane proteins. Our results suggest that anti-CD18, -CD29 and -DR mAbs recognise similarly expressed molecular homologues on equine cells, but that anti-CD11a, -CD21 and -CD23 mAbs recognise either different molecules or homologues that are expressed at different levels on horse cells.     

Phenotypic analysis and effects of sequential administration of activated canine lymphocytes on healthy beagles

We attempted to accumulate the basic data for evaluation of activated lymphocyte therapy for small animal medicine. The peripheral blood mononuclear cells (PBMCs) collected from healthy dogs were activated using anti-CD3 antibody and human recombinant (hr) interleukin (IL)-2 and reactivated using hr interferon (IFN)-alpha and hr IL-2. The property of obtained cells was compared with PBMCs. The number of cells was shown to have increased approximately>50 -fold by cultivation. The proportion of CD8+ cells was significantly increased, the cytotoxicity of the cultured cells was revealed to have been reinforced. Additionally, CD56 mRNA levels tended to have increased. The cells obtained by this method were confirmed to be activated lymphocytes. Furthermore, we investigated the effects of sequential administration of the obtained cells to healthy dogs. By sequential administration of the activated lymphocytes, the cell proliferative activity, proportion of CD4+ cells and CD8+ cells, and serum IFN-gamma concentration were shown to have increased, and no severe adverse effects were observed. Consequently, activated lymphocytes could be induced using anti-CD3 antibody and IL-2 in healthy dogs, and sequential administration of activated lymphocytes reinforced the recipient's immunity.     

Immunophenotyping of chicken peripheral blood lymphocyte subpopulations: individual variability and repeatability

T-cell lymphocyte populations can be delineated into subsets based on expression of cell surface proteins that can be measured in peripheral blood by monoclonal antibodies and flow cytometry percentages of the lymphocyte subpopulations. In order to accurately assess immunocompetence in birds, natural variability in both avian immune function and the methodology must be understood. Our objectives were to (1) further develop flow cytometry for estimating subpopulations of lymphocytes in peripheral blood from poultry, (2) estimate repeatability and variability in the methodology with respect to poultry in a free-range and environmentally diverse situation, and (3) estimate the best antibody and cell marker combination for estimating lymphocyte subpopulations. This work demonstrated the repeatability of using flow cytometry for measurements of peripheral blood in chickens using anti-chicken antibodies for lymphocyte subpopulations. Immunofluorescence staining of cells isolated from peripheral blood revealed that the CD3(+) antibodies reacted with an average of approximately 12-24% of the lymphoid cells in the blood, depending on the fluorescence type. The CD4(+) and CD8(+) molecules were expressed in a range of 4-31% and 1-10% of the lymphoid cells in the blood, respectively. Both fluorescence label and antibody company contribute to the variability of results and should be considered in future flow cytometry studies in poultry.     

Enhancement of interleukin-2 production by bovine peripheral blood mononuclear cells treated with the combination of anti-programmed death-ligand 1 and cytotoxic T lymphocyte antigen 4 chimeric monoclonal antibodies

Our previous studies demonstrate the therapeutic efficacy against bovine diseases of an anti-bovine programmed death-ligand 1 (PD-L1) chimeric antibody. In humans, PD-1 and PD-L1 antibodies are more effective when combined with an antibody targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and these combination therapies are therefore clinically used. Here we generated an anti-bovine CTLA-4 chimeric antibody (chAb) to enhance the therapeutic efficacy of the PD-L1 antibody. We further analyzed the effects of dual blockade of CTLA-4 and PD-1 pathways on T-cell responses. The established anti-bovine CTLA-4 chAb showed comparable blocking activity on the binding of bovine CTLA-4 to CD80 and CD86 as the anti-bovine CTLA-4 mouse monoclonal antibody. Anti-bovine CTLA-4 chAb also significantly increased IL-2 production from bovine peripheral blood mononuclear cells (PBMCs). Further, the combination of anti-CTLA-4 chAb with anti-PD-L1 chAb significantly upregulated IL-2 production by PBMCs. These results suggest that the combination of antibodies have higher potential to enhance immune responses against pathogens compared with single administration.     

Evaluation of elutriation and magnetic microbead purification of canine monocytes

An elutriation technique was developed to obtain large quantities of pure canine monocytes. Firstly, peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll gradient. Then, the PBMC were separated by an elutriation procedure. We demonstrated that these techniques allow the isolation of canine peripheral blood monocytes with a purity of 64% +/- 7.9 when labelled with anti-CD14 antibody. This purity increased to 83% +/- 2.2 after separation by magnetic anti-CD14 microbeads. The cell viability was more than 95% and apoptotic cells were less than 10%. The monocytes purified by these methods were functionally active in a mixed leukocyte reaction (MLR). A lymphocyte fraction was obtained directly only by elutriation with an average of 79.9% +/- 10.7 of CD5+, 7.9% +/- 3.5 of CD21+ and 1.78% +/- 2.53 of CD14+. Our results indicate that this elutriation procedure is a safe method to purify monocytes as well as lymphocytes, useful in MLR.     

Peripheral blood mononuclear cell subsets during Trichinella spiralis infection in pigs

The immune response of 'Yugoslav meat breed' pigs inoculated with low doses of Trichinella spiralis muscle larvae was followed over two to nine weeks of primary infection, by analysing changes in peripheral blood mononuclear cell subsets, the development of a humoral antibody response and muscle larvae burden. During the course of the infection, infected animals showed a persistent elevation of both CD4+ and CD8+ T cell subsets from days 15 to 60 after the parasite exposure. During this time, the number of peripheral blood mononuclear cells expressing major histocompatibility complex class II antigens was also increased, while no significant differences were found in the number of circulating monocytes/macrophages and B cells over time. Humoral antibody responses to muscle larvae excretory-secretory products were evident as early as 41 days after infection, while the muscle larvae were recovered as early as 27 days after infection. The increased levels of CD4+ and CD8+ T cell subsets, as well as cells expressing major histocompatibility complex class II antigens in pigs exposed to T spiralis, may be indicative of some considerable alterations in cell subsets that are involved in the regulation of the swine immune response to this parasite.     

Response of bovine gammadelta T cells to activation through CD3

Since the T cell receptor of γδ T cells is associated with CD3 molecules, it is a reasonable postulate that signal transduction through CD3 would occur in γδ T cells as it does in β T cells. However, while a small percentage of bovine γδ T cells divided in cultures of peripheral blood mononuclear cells (PBMCs) in response to stimulation by anti-CD3 monoclonal antibody (mAb) the majority of viable γδ T cells at the end of the culture period had not. This was assessed by carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis here and previously [Res. Vet. Sci. 69 (2000) 275]. When intracytoplasmic staining for interferon-γ (IFN-γ) was also used here to assess activation through CD3, a small proportion of γδ T cells (approximately 14%) produced IFN-γ during the first 4 h of culture and by 72 h of culture that number had doubled. By comparison, a much larger proportion of CD4 and CD8 T cells stimulated with anti-CD3 mAb divided and although the percentage of CD4 and CD8 T cells that produced IFN-γ at 4 h was similar to that of γδ T cells, by 72 h the majority of CD4 and CD8 T cells were IFN-γ⁺. Addition of IL-2 did not increase the proportion of γδ T cells that responded to anti-CD3 stimulation by cell division. To test the hypothesis that γδ T cells were inhibited from responding by other mononuclear cell populations within PBMC, monocytes were removed from the PBMC or γδ T cells were purified by magnetic-bead sorting. Only a small distinct population of the sorted cells underwent multiple cell divisions in response to anti-CD3 mAb and removal of monocytes resulted in only a moderate increase in γδ T cell replication. The anti-CD3 mAb stimulation system may provide a useful system to evaluate the difference in the requirements for activation and clonal expansion for γδ T cells versus β T cells.     

Enhanced protocol for CD14+ cell enrichment from equine peripheral blood via anti‐human CD14 mAb and automated magnetic activated cell sorting

Reasons for performing study: CD14 positive (CD14+) cells are the precursor cells of monocyte‐derived dendritic cells (DCs). In horses their potent antigen‐presenting capacity and ability to induce an effective immune response classify these cells suitable for several therapeutic approaches such as for equine sarcoid. However, in horses, the generation efficiency of DCs from adherent peripheral blood mononuclear cells (PBMCs) is currently still poor. Objectives: Establishment of a simple short protocol to enhance DC generation in horses by using a human CD14 monoclonal antibody (mAb) and an automated magnetic activated cell sorting (MACS) system. Methods: Peripheral blood mononuclear cells were isolated from fresh heparinised blood samples of 3 horses and primarily stained for flow cytometric analysis (FACS) with a mAb against human CD14 as well as a secondary phycoerythrin (PE) conjugated antibody to determine the initial percentage of CD14 cells in the sample. Peripheral blood mononuclear cells were used for automated MACS using the same primary and secondary antibodies and analysed by FACS. CD14+ selected cells were cultured for 4 days adding granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4) to the culture media. Dendritic cell generation was assessed analysing cell morphology and surface marker expression (hCD83, hCD86, eqMHCII). Results: Prior to selection, the mean percentage of CD14+ cells in the total cell population was 5.5%, further gaiting of this cell population resulted in 78.46% CD14+ monocytes. After our positive selection the mean percentage of CD14+ cells in the population was 98% without affecting viability. After culture, DC yield was 2‐fold higher than in previous published outcomes. Conclusions: The additional CD14 cell separation step after PBMC isolation significantly amplified the number of CD14+ cells, increasing the number of generated DCs. Potential relevance: The number of DCs available is critical for further use of these cells and the herein described protocol will therefore help to improved DC generation for therapeutic approaches in horses.     

Phenotypic and functional characteristics of bovine T lymphocytes

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.     

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.     

Immunophenotyping of Leukocyte Subsets in Peripheral Blood and Palatine Tonsils of Prefattening Pigs

The quantitative and distribution patterns of porcine peripheral blood and tonsillar lymphoid/myeloid cell subsets were assessed in order to establish the immune status of farm pigs prior to their transfer to fattening units. Peripheral blood and tonsillar samples were taken from clinically healthy, nonvaccinated, 12-week-old pigs, either ex vivo or following euthanasia. Single-colour flow cytometry, using monoclonal antibodies (mAbs) reactive with the swine leukocyte cluster of differentiation (CD) antigens, gave the proportions of lymphoid (9.7% CD4⁺, 8.0% CD8⁺, 36.9% CD5a⁺, 20.3% CD16⁺, 6.9% CD21⁺, 86.3% CD45⁺, 41.8% CD45RA⁺, 48.3% CD45RC⁺), null cells (6.9%) and myeloid cells (23.7% CD11b⁺ and 5.4% SWC3a⁺) in peripheral blood. In situ identification and distribution of lymphoid cells in the tonsils (CD3a⁺, CD21⁺, CD45RA⁺, CD45RC⁺) was performed with anti-CD mAbs using the avidin–biotin complex method. Most CD3a⁺ cells were in the parafollicular areas, with many cells in the follicles. CD21⁺ cells were scattered throughout the parafollicular area, with only a few cells inside lymphoid follicles. CD45RA⁺ cells were mostly concentrated in the follicles but many positive cells were present in the parafollicular area. Many CD45RC⁺ cells were visible in the parafollicular area, a few positive cells were in the crypt epithelium, and single cells were inside the follicles.     

Flow cytometric analysis of blood lymphocyte phenotypes in horses infected with the equine infectious anemia virus

Lymphocyte phenotypes were evaluated in bloodsamples taken from horses in the persistent phase EIA virus infection (n=10), from diseased controls (n=5) and from normal controls (n=10). A single animal in the acute phase of EIA was also studied. Cells were identified using flow cytometry after labelling with polyclonal antibodies to horses immunoglobulins for B-lymphocytes, or monoclonal antibodies (MAbs) to CD4, CD5, CD8 and MHC Class-II antigens. In horses persistently infected with EIA virus, the percentage of CD4+T-lymphocytes is systematically reduced and the percentage of CD8+T-lymphocytes is irregular, ranging from normal to severely reduced. Most of them have low values for cells expressing class-II antigens, but high B-cell percentages. Total CD5+ cell percentages are low in all diseased horses examined compared to normal controls. The acutely-infected foal differed from the persistently infected animals in having an elevated percentage of CD8+ T-lymphocytes, and a severely reduced percentage of B-cells.BSA, bovine serum albumin; EIA, equine infectiousanemia; FITC, fluorescein isothiocyanate; MAb, monoclonal antibody; PBL, peripheral blood lymphocytes; PBMC, peripheral blood mononuclear cells, PBS, phosphate buffered saline.     

Bulk cultures of canine peripheral blood lymphocytes with solid phase anti-CD3 antibody and recombinant interleukin-2 for use in immunotherapy

Interleukin (IL)-2 can induce large numbers of lymphokine-activated killer cells in peripheral blood lymphocytes (PBL), but IL-2 alone cannot induce proliferation of a large number of canine (c) PBL. We used the solid phase anti-CD3 antibody and soluble recombinant (r) IL-2 in order to establish a large scale culture method for cPBL. The number of lymphocytes seeded (3 x 10 (7)) increased to 1 x 10(9) after incubation for 10 days. The phenotype of cultured cPBL cells (after 2 weeks) showed a CD4(+) or CD8(+) predominant cell population. The cultured cell solutions were administered with physiological saline intravenously to each dog. After transfusion of the cultured cells, the cPBL counts, especially the number of CD4(+), CD8(+) and CD4(-)CD8 (-)(DN) cells increased significantly in the recipient dogs. Natural killer (NK) cells, gammadeltaT cells and B cells were considered to be present in the DN cell population. The NK cells and gammadeltaT cells showed no adverse reaction to the transfusion of the activated cPBL. Therefore, it is necessary to recognize the B cells present in the DN cell population by detecting CD21(+) cells. In conclusion, the bulk culture system of cPBL with rIL-2 and solid phase anti-CD3 antibody may be useful for the development of novel immunotherapy in dogs.     

The bovine p150/95 molecule (CD11c/CD18) functions in primary cell-cell interaction.

The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.