Use of alpha-naphthyl acetate esterase staining to identify T lymphocytes in cattle

Peripheral blood lymphocytes (PBL), lymph nodes, and/or spleens from clinically normal cattle were examined for cytochemical staining of alpha-naphthyl acetate esterase (ANAE). Two types of positive-staining patterns in ANAE staining resulted. By a combination of ANAE staining and latex-ingesting test, diffuse ANAE-positive cells were considered as mononuclear phagocytic cells. Using erythrocyte rosettes, erythrocyte antibody complement rosettes, nylon-wool column technique, surface immunoglobulin (SIg) staining, and the ANAE staining technique, granular ANAE-positive lymphocytes were shown to be T lymphocytes. The frequency of T and B lymphocytes in PBL, spleens, and lymph nodes of clinically normal cattle was measured, using ANAE staining and SIg staining. In PBL, 47.7% were ANAE-positive and 26.9% were SIg-positive; in spleens, 22.4% were ANAE-positive and 53.7% were SIg-positive; and in lymph nodes, 38.5% were ANAE-positive and 28.3% were SIg-positive. The frequencies of T and B lymphocytes in PBL, spleens, and/or lymph nodes from cattle with enzootic bovine leukosis (EBL) and cattle with persistent lymphocytosis and in tumor cells from cattle with EBL were measured. When compared with those of clinically normal cattle, PBL, spleens, and lymph nodes of cattle with EBL and the PBL of cattle with persistent lymphocytosis contained numerous SIg-positive cells and few ANAE-positive cells. Tumor cells from cattle with EBL contained 7.3% ANAE-positive and 78.0% SIg-positive cells.     

Alteration in lymphocyte subpopulations in bovine leukosis virus-infected cattle.

Alterations in peripheral blood lymphocyte subpopulations were examined in bovine leukosis virus (BLV)-infected cattle using antibodies specific for differentiation antigens in conjunction with analytical flow cytometry. Animals considered to be aleukemic and lymphocytotic were included in the study. Significantly fewer numbers of circulating B-lymphocytes (surface Ig-positive) and T-helper lymphocytes (BoCD4-positive) were identified in BLV-infected aleukemic cattle compared to non-infected controls while no significant differences were established for T-cytotoxic/suppressor lymphocytes (BoCD8-positive). In contrast, BLV-infected animals with persistent lymphocytosis had elevated numbers of circulating B-lymphocytes with no significant perturbation in circulating T-lymphocyte subsets identified when compared as a group with the negative control cattle. Application of regression analysis to data from individual lymphocytotic cattle demonstrated a significant correlation between absolute numbers of B- and T-lymphocytes. Increased numbers of B-lymphocytes were correlated with increased numbers of T-helper and T-cytotoxic/suppressor lymphocytes.     

Ki-67 antigen expression in lymphocytes of cattle infected with bovine leukemia virus (BLV).

The Ki-67 monoclonal antibody, which recognizes an antigen present on the nuclear membrane surface of mammalian cells in the replication phase, has been used for the determination of the cellular cycle of peripheral blood lymphocytes on a group of cattle positive for bovine leukemia virus (BLV) and with blood values showing a persistent lymphocytosis. The results obtained have shown that: 1. Both of the techniques used (immunofluorescence and immunoperoxidase) are easily applicable and give uniform results; 2. Cattle with a persistent lymphocytosis show an absolute number of cells in cycle significantly more elevated compared with cattle positive for BLV with normal blood values.     

Factors affecting the infectivity of lymphocytes from cattle with bovine leukosis virus.

Peripheral blood mononuclear cells were obtained from 13 bovine leukosis virus infected cattle and inoculated subcutaneously into 29 recipient adult steers to determine (a) the number of mononuclear cells (equivalent amount of blood) necessary to cause infection and (b) factors influencing infectivity of mononuclear cells from bovine leukosis virus-infected animals. A total of 55 inoculations were made. Inoculation of 1 X 10(4), 2 X 10(4) and 5 X 10(4) mononuclear cells caused seroconversion in 12%, 57% and 62% of steers, respectively. No infections occurred with 1 X 10(3) or 2 X 10(3) mononuclear cells. Cattle infected for longer than 24 months and those animals greater than three years of age were more likely to cause infection with 1 to 5 X 10(4) mononuclear cells than were cattle infected for less than 24 months or animals less than three years of age. Lymphocytes from cattle with persistent lymphocytosis caused more infections when 1 X 10(4) or 2 X 10(4) mononuclear cells were inoculated, than did lymphocytes from nonpersistent lymphocytosis cattle; however, both groups were equally infectious when 5 X 10(4) mononuclear cells were inoculated. No differences were found in infectivity of experimentally vs naturally exposed animals.     

Frequency of lymphocytes bearing Fc receptors and surface membrane immunoglobulins in normal, persistent lymphocytotic and leukemia cows.

Fluoresceinated, heat-aggregated bovine immunoglobulins (B-IgG) and human immunoglobulins (H-IgG) were used to detect a receptor for the crystallizable fragment (Fc) of the immunoglobulin molecule on peripheral blood lymphocytes (PBL) of cattle. The aggregated and B-IgG and H-IgG bound to the bovine PBL, but aggregated H-IgG was found to be more sensitive for the detection of Fc receptors. The specificity of aggregated H-IgG binding to the Fc receptors was established by demonstrating that antigen-antibody complexes inhibited this binding, and unaggregated H-IgG did not bind significantly to PBL. Double-labeling experiments suggested that all Fc+ cells have surface immunoglobulins (SIg), a marker for B lymphocytes. The percentage of Fc+ and SIg+ cells in normal animals was 9.5% (range 4-15%) and 16.2% (range 4.5-30.2%), respectively. Persistent lymphocytotic cows had 2.71 times more Fc+ and 3.85 times more SIg+ lymphocytes than did normal cows. Cows with lymphosarcoma had a lower percentage of Fc+ and SIg+ cells than did cows with persistent lymphocytosis. Cases with thymic lymphosarcoma and those with the skin form of leukemia had normal percentages of Fc+ and SIg+ cells.     

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on B-1a cell from persistent lymphocytosis (PL) cows and lymphoma cell induced by bovine leukemia virus

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on B lymphocytes from persistent lymphocytosis (PL) cattle and lymphoma cells induced by bovine leukemia virus (BLV) was studied in vitro. Flow cytometric analysis showed that high levels of receptors to GM-CSF were expressed on these cell types. Proliferation of these B cells was induced in response to bovine GM-CSF. In tumor cell lines, the rate of cell proliferation was correlated with expression of GM-CSF receptors. A monoclonal antibody to GM-CSF inhibited lymphocyte proliferation and blocked the GM-CSF binding of lymphocytes. Cells expressing GM-CSF receptor were Ig positive and both CD5 and CD11 positive (B-1a cell). These results suggest that an abnormal expression of GM-CSF receptors on B lymphocytes from PL and lymphoma cells induced by BLV plays important roles in the PL and proliferation of lymphoma.     

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.     

Bovine leukosis. IV. Trypanosomiasis, lymphocytosis and DNA synthesis.

The possible influence of natural trypanosome infection on lymphocytosis and DNA synthesizing lymphocyte counts in peripheral blood was determined on 220 cows from two leukosis herds and 25 cows from leukosis free control herd. Trypanosome incidences were determined during summers of 1969 and 1970 by inoculating whole blood onto blood agar slants and incubating at room temperature. Incidence of trypanosomiasis in cattle was found to be variable, possibly due to factors affecting the primary isolation of Trypanosoma theileri. A small trypanosome resembling Trypanosoma uniforme was found occasionally as a concomitant infection with T. theileri. Trypanosomiasis occurred with equal frequency in the animals of the leukosis and the control herds. No correlations were noted between trypanosomiasis, lymphocytosis and DNA synthesizing lymphocytes in peripheral circulation.     

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.     

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.     

Erythrocyte rosettes--a marker for bovine T cells.

Many species of erythrocytes were investigated for their ability to form spontaneous rosette with bovine peripheral blood leukocytes and fetal thymocytes. Only sheep and chicken red blood cells gave rosettes. Using conditions shown optimum for the demonstration of human rosette forming cells, only low numbers of bovine rosettes were demonstrable. By changing culture conditions to include 100% fetal calf serum, neuraminidase treated erythrocytes and/or lymphocytes and optimizing the incubation times and temperature, up to 38% of peripheral blood leukocytes and 52% of thymocytes formed rosettes. A thymic origin of rosetting cells was ascribed to T cells for the following reasons: 1) thymocytes gave higher numbers than did peripheral blood leukocytes, 2) rosette forming cell numbers were increased in peripheral blood leukocyte subpopulations enriched in T cells by nylon column separation and 3) only very few rosette forming cells had surface immunoglobulin, a marker of B lymphocytes. The reasons why all T cells were not detected by the technique were discussed.     

Abnormal clonalities of B-lymphocytes in bovine leukemia virus-infected cattle with persistent lymphocytosis

Peripheral B-lymphocyte clonality of 274 bovine leukemia virus-infected cattle with lymphocytosis was analyzed using clonality PCR based on sequences of the variable region of the bovine immunoglobulin H chain. None of the cattle showed monoclonal proliferation, while 10, 31, and 233 showed minor-clonal, oligoclonal, and polyclonal proliferation, respectively. A total of 163 cattle were analyzable the following year, and lymphocytosis was maintained in 157, indicating persistent lymphocytosis (PL). B-lymphocyte clonality of the 157 PL cattle was minor-clonal in 6 (3.8%), oligoclonal in 8 (5.1%), and polyclonal in 143 (91.1%). A higher rate of enzootic bovine leukosis (EBL) onset within a year was observed in PL cattle with minor-clonal (50.0% (3/6)) and oligoclonal (25.0% (2/8)) proliferation compared to those with polyclonal (5.6% (8/143)) proliferation. Minor-clonal and oligoclonal proliferation in PL cattle may be a prognosis factor for developing EBL.     

Changes in B cell and T cell subsets in bovine leukaemia virus-infected cattle

Direct immunofluorescence and fluorescence-activated cell sorter techniques were used for the detection of surface immunoglobulin positive (SIg+) cells in peripheral blood lymphocytes (PBL's) of bovine leukaemia virus (BLV) infected cattle with or without persistent lymphocytosis (PL+, PL-) and in BLV-free cattle. The percentage of SIg+ cells was more than twice as high in BLV+PL+ cattle than in BLV-free and BLV+PL- cattle. Bovine T cells, and T cell subsets were identified indirectly by the same techniques using three monoclonal antibodies (MAb's) specific for all T cells (IL-A43), T helper (BoT4) cells (IL-A12) and T cytotoxic (BoT8) cells (IL-A17). The major histocompatibility complex (MHC) determinants of both class II (BoT4) and class I (BoT8) as well as all T cells were significantly reduced in BLV+PL+ compared to BLV-free cattle. The actual decrease in the BoT8 cell subset or the dilution effect that would change effector:target cell ratio suggests that a resultant decrease in cytotoxic activity in BLV+PL+ cattle may play an important role in the progress of BLV infection in cattle.     

Detection of B and T cells, with lectins or antibodies, in healthy and bovine leukemia virus-infected cattle

Lectins, polyclonal antibodies and monoclonal antibodies (MAbs) were evaluated as markers for bovine lymphocytes obtained from healthy animals and from cattle infected with bovine leukemia virus (BLV). In the blood from healthy cattle the proportion of cells identified as T lymphocytes with the lectin Helix pomatia (HP) (67.8 +/- 6.2%) using the indirect immunofluorescence technique was similar to the proportion of cells identified by the MAbs P5 (66.1 +/- 3.8%) and BLT-1 (59.8 +/- 7.1%). The proportion of B cells in blood from healthy animals identified with a polyclonal antibody to bovine IgM (18.0%) was similar to that identified with a MAb to bovine IgM (16.2%). However, greater variation between individual values was detected with the MAb (SD = 8.2) than with the polyclonal antibody (SD = 4.0). In the blood from BLV-infected cattle with persistent lymphocytosis, both the polyclonal and the MAb revealed a threefold increase of B cells. A proportion of the B cells had an increased amount of immunoglobulin molecules in their plasma membrane as indicated by flow cytometry. The proportion of T lymphocytes, identified by the MAb P5, was reduced to one-third of that in non-infected cattle. The indirect HP labelling gave inconsistent results and seems not to detect solely T lymphocytes among blood lymphocytes from BLV-infected cattle.     

Preparation and characterization of monoclonal antibodies against bovine B lymphocyte surface antigens

Two monoclonal antibodies (MoAbs; BLMo-4 and BLMo-10) were prepared by immunizing with a cell line established from peripheral blood mononuclear cells (PBMC) of enzootic bovine leukosis (EBL) cattle. The specificities of these MoAbs were assayed using bovine PBMC. BLMo-4 reacted with all surface immunoglobulin-positive cells (SIg+ cells; B lymphocytes) and also recognized monocytes, but did not react with T lymphocytes. BLMo-10 recognized a majority, although not all, B lymphocytes, but did not react with either T lymphocytes or monocytes. The antigens recognized by BLMo-4 and BLMo-10 were not Ig, Fc or C3 receptors on the surface of B lymphocytes. The reactivity of the MoAbs with mononuclear cells from the lymphoid organs of adult cattle was studied. BLMo-4 and BLMo-10 did not react with any bone marrow cells. BLMo-10 reacted with 7.4% of thymocytes, and stained the medulla of the thymus in the immunoperoxidase assay. In the case of PBMC, spleen and lymph node cells, the percentage of cells positive for BLMo-4 was slightly higher than that of SIg+ cells, but BLMo-10 showed a slightly lower value.     

Detection of monoclonal integration of bovine leukemia virus proviral DNA as a malignant marker in two enzootic bovine leukosis cases with difficult clinical diagnosis

Monoclonal integration of bovine leukemia virus (BLV) proviral DNA into bovine genomes was detected in peripheral blood from two clinical cases of enzootic bovine leukosis (EBL) without enlargement of superficial lymph nodes. A BLV-specific probe hybridized with 1 to 3 EcoRI and HindIII fragments in these 2 atypical EBL cattle by Southern blotting and hybridization, as well as in 3 typical EBL cattle. The probe also hybridized to a large number of EcoRI and HindIII fragments in 5 cattle with persistent leukosis. These results suggest that the detection of monoclonal integration of BLV provirus into the host genome may serve as a marker of monoclonal proliferation and malignancy in difficult to diagnose EBL cattle.     

Properties of nine continuous B-cell lines established from enzootic bovine leukosis tumors.

We established 9 cell lines from 63 tumor cases of enzootic bovine leukosis and studied their properties. Cells of all lines formed small clumps and floated in culture medium, indicating growth. Four of the 9 cell lines were surface immunoglobulin (SIg)-positive, but the remaining 5 line cells were negative for SIg or, if SIg was detected, the percentage of SIg-positive cells was very low. Tests for the properties of the cells with monoclonal antibodies to lymphocytes revealed that the established line cells are B-lymphocytes. Morphological observation also revealed that they had the morphology of B-lymphoblastic cell. The results of E and EAC rosette assay were negative, but 6 of 8 cell lines were positive for EA rosetting. All the 9 cell lines reacted with MoAb C-143, which recognizes the tumor-associated antigen (TAA) of the EBL tumor cell. All 9 cell lines produced bovine leukosis virus (BLV). These results suggest that the 9 cell lines are tumor cells derived from B-lymphocytes of EBL.     

Immature T cell neoplasms in three young cattle

Immature T cell neoplasms in three young Holstein cattle with neoplastic involvement of the thymus are described. Case 1, with a precursor T lymphoblastic leukemia (calf form of leukosis), was an 86-day-old female calf. The leukemia was characterized by replacement of the bone marrow and spleen by leukemia cells, but preservation of epithelial frameworks throughout the thymus. The other two neoplasms were thymic γδ T cell lymphomas, which were observed in a 246-day-old steer (case 2) and a 16-month-old heifer (case 3). Histological examination revealed obliteration of the normal thymic architecture and stromal fibrosis, with the spleen and liver far less severely affected than in case 1. There were cytological differences bewteen the tumors in case 1 and cases 2 and 3. Additionally, WC1 and CD8 were expressed only in the latter. Thus, the leukemia and these lymphomas should be regarded as independent disease entities on the basis of histological and immunohistochemical characteristics.     

Suppression of natural cytotoxic activity of lymphocytes from cattle and sheep during the progress of bovine leukosis

Peripheral blood lymphocytes (PBL) from cattle and sheep exhibited natural cytotoxicity on a fetal lamb kidney (FLK) cell line that was persistently infected with bovine leukemia virus (BLV) by 20-h 51Cr release assay. This cytotoxic activity was tested using PBL from normal cattle and sheep or BLV-infected animals. Although cytotoxic activity was also found in PBL from normal animals and from BLV-infected, but clinically healthy animals, the activity in PBL from animals with persistent lymphocytosis or leukemic animals was markedly decreased. The cytotoxic activity of PBL from 3 sheep sequentially tested before and during the disease was found to decrease gradually with the progress of the disease, and finally no cytotoxic activity was found at the time of death due to leukemia. These results suggest that the natural cytotoxic activity in PBL may have a role in immunosurveillance for progress of the tumor.