Wheat germ agglutinin (WGA): a bovine T lymphocyte marker

Bovine peripheral blood lymphocytes (PBL) were examined for their ability to bind wheat germ agglutinin (WGA). This lectin labelled 43.8% +/- 11.95 of bovine PBL, whereas peanut agglutinin (PNA), a T cell marker, bound 59.4% +/- 8.67 cells, and surface immunoglobulin (SLG)-bearing cells constituted 24.15% +/- 8.47 of PBL. After panning fractionation of B (Slg+) and T (PNA+) lymphocytes. WGA labelled 89 to 97% of the enriched T cell population (80/87% PNA+; 2-4% Slg+) but only 6 to 8% of the enriched B cell population (85-91% Slg+; 5-7% PNA+).     

Enrichment of T lymphocytes from bovine peripheral blood mononuclear cells using an immuno-affinity depletion technique ("panning")

A simple and efficient method to enrich bovine T lymphocytes from peripheral blood mononuclear cells (PBMC) by immuno-affinity depletion ("panning") has been developed. The PBMC were initially separated by density gradient centrifugation on Histopaque of density 1.077 g/ml. The T lymphocyte subset was then separated from PBMC by depletion of membrane immunoglobulin (Ig) bearing cells which had an affinity for anti-Ig antibodies bound to polystyrene tissue culture flasks. An average of 95% of the nonadherent "panned" cells were identified as T lymphocytes using a label of peanut agglutinin conjugated with fluorescein isothiocyanate (PNA-FITC). Two percent of the PNA negative cells were Ig bearing cells. The average yield was 50% of the original T lymphocytes found in the PBMC population, and the cell viability as assessed by trypan blue exclusion was greater than 95%. The separation took approximately 2 hours, and the total number of T lymphocytes recovered from 40 ml of blood was in the range of 20-40 X 10(6).     

Effect of infections with swine fever virus on immune functions I. Response of lymphocytes from blood and lymphoid organs from infected and normal pigs to anti-immunoglobulin serum and protein A

Pigs exposed to a low-virulent strain of swine fever virus (SFV) developed an inapparent infection. At times when a transient leucopenia occurred, the peripheral blood lymphocytes (PBL) were unresponsive to the mitogenic stimulus of anti-immunoglobulin serum (anti-Ig) and protein A.Pigs lethally infected with a virulent SFV showed leucopenia and unresponsiveness of PBL to anti-Ig and protein A from 2 days post infection until death.This suggests a defect in B lymphocyte function in pigs infected with SFV. The unresponsiveness to anti-Ig appeared not to be caused by a reduced ability of lymphocytes to redistribute their receptors into caps, the presence of suppressor cells or absence of surface immunoglobulin bearing lymphocytes in the peripheral blood. A direct action of the virus itself also seemed unlikely.Lymphocytes from spleen reacted as PBL. However, lymph node cells did not lose their capability to respond to anti-Ig.These data suggest that a change in the migration pattern of anti-Ig responsive lymphocytes could account for the observed unresponsiveness of PBL and spleen lymphocytes to anti-Ig.     

Cell surface markers on canine lymphocytes.

Reference values for T and B lymphocytes were determined on lymphocytes from canine thymus, spleen, lymph node, bone marrow, and peripheral blood by use of erythrocyte (E) and erythrocyte-antibody-complement (EAC) rosette assays, plus a direct fluorescent technique for assay of surface immunoglobulins. Numbers of T lymphocytes, indicated by E rosette formation with human erythrocytes, ranged from a low of 1% in the thymus to 13% in the peripheral blood, whereas B-lymphocyte numbers ranged from 3% (thymus) to 41% (bone marrow) and from 6% (thymus) to 36% (bone marrow), as indicated by EAC rosette formation or presence of surface immunoglobulins respectively. Stimulation of peripheral blood lymphocytes with either phytohemagglutinin or concanavalin A increased the total number of E-rosetting cells two to threefold, whereas the number of EAC-rosetting cells decreased by half. Further, the percentage of cells bearing Fc receptors increased after phytohemagglutinin stimulation. These results indicate the E rosette technique can be used to identify and to monitor a population of canine T lymphocytes.     

Surface markers on bovine fetal lymphocytes and immunoglobulin synthesis in a congenital infection related to epizootic bovine abortion

Immunological parameters were studied among 23 late-term bovine fetuses. Epizootic bovine abortion (EBA) disease was induced in fetuses by feeding Ornithodoros coriaceus ticks on pregnant heifers. A spirochaete-like microorganism was detected in the blood of diseased fetuses and in inapparent natural infections in some abattoir-collected fetuses. Fetuses were classified according to stages of disease: EBA diseased (n = 10), EBA infected (n = 7) and normal (n = 6). Using flow cytometry, the presence of surface immunoglobulins (sIg) and peanut agglutinin (PNA) receptors were used to detect B and T lymphocytes, respectively. In peripheral blood of normal fetuses, most lymphocytes were identified as T or B cells, whereas about 20 per cent of lymphocytes in EBA diseased fetuses did not reveal the sIg or PNA receptor markers (null cells). Size and shape analyses by flow cytometry detected a population of enlarged lymphocytes in the EBA diseased fetuses. The numbers of cells bearing determinants reactive with monoclonal antibodies specific for bovine T cells (B26A and B29A) and B cells (TH21A) were considerably less than those expressing the PNA receptor and sIg. These results suggested that the monoclonal antibodies were binding to differentiation antigens which were not consistently expressed on the fetal cells. Radio-immunodiffusion was used to measure bovine IgM, IgG1 and IgG2 in fetal serum. The quantities of immunoglobulins were markedly increased in animals infected with the spirochaete-like organism (groups 1 and 2) and were assumed to result from fetal antibody synthesis.     

Identification of canine T-lymphocyte subsets with monoclonal antibodies.

A panel of five murine monoclonal antibodies to canine T-lymphocytes were produced. Antibodies 4.78, 12.125 and 8.358 reacted with approximately 18%, 39% and 60% peripheral blood lymphocytes, respectively. Two color flow cytometric analysis showed that lymphocytes expressing 1.140, 4.78, 8.53 and 12.125 were subsets of lymphocytes expressing 8.358. The lymphocytes expressing 8.358 were negative for surface immunoglobulin. The subsets defined by 1.140, 4.78 or 8.53, 12.125 were mutually exclusive and together account for most cells expressing 8.358 in the peripheral blood, spleen, and lymph node. In the thymus, approximately 47% cells were positive for both 1.140/4.78 and 8.53/12.125. SDS-PAGE analysis of radiolabelled thymus cell lysates demonstrated that antibodies 1.140 and 4.78 immunoprecipitated a 32,35 kd heterodimer under reducing conditions and 12.125 immunoprecipitated a single 56 kd chain under reducing and non-reducing conditions. Antibodies 8.53/12.125 and 1.140/4.78 react with canine lymphocyte populations that occur in proportions similar to lymphocytes expressing CD4 and CD8 like molecules in several primate and non-primate species. The molecules recognized by 12.125 and 1.140/4.78 were similar in size and subunit composition to human CD4 and CD8.     

Enumeration of lymphocyte subpopulations in bronchoalveolar lavage of monkeys using an immunoenzymatic staining technique

Percentages of T-helper (OKT4), T-suppressor (OKTB), and B (B1) lymphocytes were determined in peripheral blood and bronchoalveolar lavage (BAL) from six cynomolgus monkeys using an alkaline phosphatase based immunoenzymatic staining technique. The percent of each lymphocyte subset in lung lavage fluids were 40 +/- 9%, 26 +/- 7% and 11 +/- 4% for OKT4, OKT8 and B1, respectively. This cell distribution is similar to that obtained from normal human BAL samples using fluorescence techniques to evaluate binding. Values for peripheral blood lymphocyte subsets were not statistically different from BAL. This immunoenzymatic technique avoids the necessity for cell separation procedures which are used to alleviate problems with alveolar macrophage autofluorescence that can be encountered in fluorescence based assays of BAL samples. This technique also can be used by laboratories interested in lymphocyte characterization, but not equipped for fluorescence microscopy or flow cytometry.     

Effect of infections with swine fever virus on immune functions II. Lymphocyte response to mitogens and enumeration of lymphocyte subpopulations

Peripheral blood and spleen lymphocytes from pigs infected with a low-virulent strain of swine fever virus (SFV) were transiently hyporesponsive to the mitogenic action of PHA, PWM and Con A. The mitogenic reactivity of lymphocytes from lymph nodes from such pigs appeared to be enhanced rather than depressed at that time. In addition, hyperresponsiveness of peripheral blood lymphocytes (PBL) to these mitogens occurred in some pigs.PBL from pigs lethally infected with virulent SFV showed a persistent depression of the response to these mitogens, whereas lymphocytes from lymph nodes had a high responding capacity.A lymphocyte response to SFV antigens could not be demonstrated in infected pigs.These SFV infections did not markedly affect the percentage of lymphocytes in the blood and most lymphoid organs rosetting with sheep red blood cells. On the other hand, surface immunoglobulin-bearing lymphocytes were markedly increased in lymph nodes from pigs exposed to virulent SFV. The sum of both lymphocyte subpopulations in the lymph nodes from these pigs often considerably exceeded the 100% value, which strongly suggests the presence of cells bearing both surface immunoglobulin and receptors for dextran-treated sheep red blood cells.Possible correlations between these findings are discussed. The results suggest that infections with SFV induce systemic alterations in the process of lymphocyte recirculation in the pig.     

Lymphocyte responsiveness to mitogens and quantitation of T and B lymphocytes in canine malignant lymphoma.

Two canine malignant lymphoma cases were studied, one from the time of detection of enlarged palpable lymph nodes through the terminal stage and another at the terminal stage. Hematologic and histopathologic studies were confirmative of leukemia. The lymphocyte subpopulations, T and B cells, were quantitated as identified by the presence or absence of surface immunoglobulin and erythrocyte-antibody-complement-rosette formation. The average number of B cells in the peripheral blood lymphocytes throughout the study were approximately 80%. The B cells in the lymph node lymphocytes were 82%. There was considerable fluctuation in the number of blood lymphocytes, but the percentage of T and B lymphocytes remained nearly constant. There was marked impairment in the lymphocytic response to mitogens. The results of this study indicate that the canine malignant lymphoma is predominantly a B-lymphocyte type.     

A comparison of the mitogenic effects of a phorbol ester on lymphocytes from bovine spleen,lymph node and peripheral bloodComparaison des effets mitogenes d'un ester de phorbol sur des lymphocytes bovins de rate,de ganglions lymphatiques et de sang peripherique

A comparison of the mitogenic effects of a phorbol ester on lymphocytes from bovine spleen, lymph node and peripheral blood.Bovine lymphocytes from three tissues, lymph node, spleen and peripheral blood were compared for their mitogenic responses to 12-O-tetraecanoylphorbol-13-acetate (TPA), a phorbol ester tumor promoter. TPA alone was found to be either not mitogenic or caused only a weak response when compared with the mitogenic lectin phytohemagglutinin (PHA). Of the three lymphocyte preparations, blood cells showed the greatest proliferative response to TPA. However, all three, lymph node, blood and spleen cells, showed a co-mitogenic response to TPA. That is, TPA synergistically enhanced DNA synthesis in cells stimulated with a suboptimal concentration of PHA.     

Changes in subpopulations of lymphocytes in peripheral blood, and supramammary and prescapular lymph nodes of cows with mastitis and normal cows

Direct immunofluorescence (IF) and indirect IF techniques were employed to analyze the distribution of B and T lymphocyte populations in peripheral blood, and in supramammary (draining), and prescapular (non-draining) lymph nodes of cows with mastitis and normal cows. In the peripheral blood there was a significant decrease in the percent and absolute number of B lymphocytes in mastitic cows (n = 29; 17.1 +/- 10.2%; 3.4 +/- 2.7 X 10(5) cells/ml) as compared to normal cows (n = 38; 25.2 +/- 7.8%; 9.3 +/- 5.4 X 10(5) cells/ml). The percent T lymphocyte count in mastitic cows (71.2 +/- 7.1%) was slightly increased over that of normals (65.8 +/- 7.2%), although the absolute number of T lymphocytes was decreased in mastitic cows (1.49 +/- 0.91 X 10(6) cells/ml vs. 2.47 +/- 1.28 X 10(6) cells/ml). In the prescapular lymph node the percent of B lymphocytes, but not T or "null lymphocytes", decreased significantly in mastitic cows as compared to that of normals. The decrease, i.e. 32%, paralleled the 32.1% decrease found in peripheral blood B lymphocytes. In contrast, in the supramammary lymph node of mastitic cows, the percent B lymphocytes increased over that of normals (35.1 +/- 2.0% vs. 20.4 +/- 9.4%), whereas the percent T lymphocytes decreased to 54.5 +/- 2.8% compared to 70.7 +/- 3.5% in normal cows. There was no significant change in percent "null lymphocytes". The weight of prescapular lymph nodes did not change in mastitic cows when compared to that of normals. As a result, the estimated number of B lymphocytes, but not of T and "null lymphocytes", decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exclusion of cytoplasmic fragments in flow cytometric analysis of lymph node samples from dogs with lymphoma using membrane-permeable violet laser-excitable DNA-binding fluorescent dye (DyeCycle Violet)

Background: Cytoplasmic fragments derived from fragile neoplastic lymphocytes are common in samples of lymph nodes collected from dogs with lymphoma. These cytoplasmic fragments interfere with accurate gating of target cells and quantification protocols used for flow cytometry because of their variable size and expression of lymphoid cell surface antigens on their membranes. Objective: The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments from flow cytometric analysis of canine lymph nodes in which lymphoma was present. Methods: Single‐cell suspensions of neoplastic cells were prepared from biopsy samples and fine‐needle aspirates of lymph nodes from 23 dogs with lymphoma. Suspensions were stained using a violet laser‐excitable (405 nm) membrane‐permeable DNA‐binding fluorescent dye (DyeCycle Violet [DCV]), incubated with antibodies against CD3, CD5, CD21, CD22, and CD45, and then stained with 7‐amino‐actinomycin D (7‐AAD), an argon‐excitable (488 nm) membrane‐impermeable DNA‐binding fluorescent dye. Multiparameter flow cytometry was used for analysis based on selective uptake and laser‐activated fluorescence of these dyes. Results: Cytoplasmic fragments, which were DCV‐negative and CD45‐positive, and dead cells, which were positive for 7‐AAD, were efficiently separated from neoplastic cells. Conclusion: Staining with DCV is a useful method to improve flow cytometric gating methods and quantitative analyses of lymph node samples from dogs with lymphoma.     

Monoclonal antibodies specific for pig serum and cell surface IgM

Two monoclonal antibodies were prepared which react specifically with pig serum immunoglobulin and with the population of B lymphocyte-bearing surface immunoglobulin. Comparison of our monoclonal antibodies with reagents specific for gamma, mu and alpha immunoglobulin chains in double immunodiffusion and immunoelectrophoresis revealed that the monoclonal antibodies recognise IgM in pig serum and mu chain or mu chain-like molecules on B lymphocytes. The monoclonal antibodies, designated LIG 2 and LIG 4, reacted positively with adult pig sera but not with fetal or precolostral sera or with sera from other animal species. LIG 2 and LIG 4 reacted with 15 per cent of cells from the peripheral blood lymphocyte population, 20.2 per cent of spleen cells and 20 per cent of lymph node cells, but did not react with pig erythrocytes, granulocytes or cells isolated from thymus, or with the lymphocytes of other species. Positive reactions were also found on lymphatic and intestinal tissue sections. No genetic polymorphism was found in the pig population revealed by the monoclonal antibodies. The monoclonal antibodies LIG 2 and LIG 4 may be useful for studying the pig immune system, especially as a standard reagent for measuring pig serum IgM and for the identification of positive B lymphocytes.     

Comparative studies on the distribution and population of immunocompetent cells in bovine hemal node, lymph node and spleen

The distribution and population of immunocompetent cells in bovine hemal node, mesenteric lymph node and spleen were analyzed comparatively by immunohistochemistry and flow cytometry. Many CD8(+) cells, CD172a(+) cells and γδ T cells were found in the lymphatic cord along the sinus of the hemal node and the splenic red pulp. A few CD8(+) cells and γδ T cells were distributed diffusely in the paracortex and medullary cord of the mesenteric lymph node. Many germinal centers were recognized in the lymphatic regions such as the cortex and white pulp of these lymphoid organs. The populations of CD8(+) cells and γδ T cells in the hemal node and the spleen were higher than those of the mesenteric lymph node. In addition, the populations of CD21(+) cells and MHC class II(+) cells in the hemal node and the mesenteric lymph node were higher than those of the spleen. The results suggest that the hemal node has an important role in both cellular and humoral immunity as well as the lymph node and the spleen in cattle.     

Evidence for the replication of bovine leukemia virus in the B lymphocytes.

Bovine peripheral blood lymphocytes from a cow with persistent lymphocytosis were separated on nylon wool columns into nylon-adherent and nonadherent populations. Nylon-adherent cells were highly enriched for surface immunoglobulin (SIg) bearing B lymphocytes (95.5%) and nonadherent cells for SIg negative non-B cells, presumably T lymphocytes (96.3%). The B lymphocytes were found to be the major producers for bovine leukemia virus. A total of 39% of the B-enriched cells, surviving after 72 hours in culture, produced bovine leukemia virus as compared with 0.5% of the non-B cells.     

A monoclonal antibody that identifies mature T lymphocytes of cattle

Hybridomas to bovine leukocytes were produced by immunization of BALB/C mice with bovine lymphoblasts and fusion of the mouse spleen cells with mouse myeloma cells. Monoclonal antibodies (MABs) were tested against various cell populations by indirect fluorescent microscopy using fluorochrome conjugated antibodies to mouse immunoglobulins. MAB-15, one of the resulting MABs obtained after cloning antibody-producing hybridomas, reacted with 56.8 +/- 8.4% of peripheral blood mononuclear cells (PBMC). MAB-15 did not react with monocytes or B cells, but did react with T cells (fluorescein isothiocyanate-conjugated peanut agglutinin positive cells). MAB-15 reacted with 3.2% of thymocytes from adult cattle. In addition to reacting with T cells, MAB-15 reacted with neutrophils and eosinophils. MAB-15 was characterized as an IgM antibody that was unable to lyse PBMC in the presence of complement. Thus, MAB-15 is a useful marker of mature T cells in the mononuclear cell population.     

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.     

Decreased expression of L-selectin (CD62L) on lymphocytes in enzootic bovine leukaemia

Expression of L-selectin was determined by single- and two-colour immunofluorescence on granulocytes, peripheral blood mononuclear cells (PBMC) and blasts of bovine origin by means of a monoclonal antibody IVA94 which recognizes bovine L-selectin (CD62L). Cells were separated from peripheral blood of healthy cattle and colleagues infected with bovine leukaemia virus (BLV). BLV-infected animals comprised lymphocytotic and non-lymphocytotic cows. L-selectin was expressed on 90-98% of granulocytes in all tested animals. The percentage of PBMC expressing L-selectin was lower in cattle with persistent lymphocytosis than in non-lymphocytotic or BLV-free cattle, and inversely correlated with lymphocyte counts. The ratio of B lymphocytes stained for L-selectin was significantly decreased from 60.2 +/- 1.9% in BLV-free cattle to 43.8 +/- 3.6 and 22.5 +/- 5.7% in non-lymphocytotic and lymphocytotic cattle, respectively. B-lymphocytes stained for L-selectin exhibited about 50% reduction in L-selectin expression in BLV-infected cattle compared with BLV-free cattle, as judged by the mean fluorescence intensity (MFI). The percentage of L-selectin-positive PBMC not bearing surface immunoglobulin M (predominantly T lymphocytes) was comparable in BLV-free and BLV-infected cattle. However, L-selectin expression on T lymphocytes was reduced (about 50%) in BLV-infected cattle, as judged by the MFI. We suppose that BLV infection results in a decreased L-selectin expression on lymphocytes, and accordingly, it may contribute to deregulation of the host immune system.     

Preparation of a bovine monoclonal antibody to testosterone by interspecies fusion

This communication reports the first successful attempt to produce a hybridoma cell line secreting bovine immunoglobulin to a small hapten, starting with peripheral blood lymphocytes, rather than spleen or lymph node cells. A heteromyeloma line, sensitive to selective media, was made by fusing NS1/1-Ag4-1 mouse myeloma cells with bovine peripheral blood lymphocytes. This cell line was then fused with blood lymphocytes from a steer immunised with a testosterone immunogen. Cell cultures were screened using an ELISA specific for bovine antibodies to testosterone. Following repeated cloning, a cell line was established which secretes moderate levels of a specific, high affinity antibody to testosterone. This particular cell line has significant potential for veterinary application and the successful fusion demonstrates the possibilities of heteromyelomas for the development of non-murine monoclonal antibodies.     

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.