Characterization of surface markers of bovine gut mucosal leukocytes using monoclonal antibodies

The surface phenotypes of bovine intestinal leukocytes isolated from the intraepithelium (IEL), lamina propria (LPL) and Peyer's patches (PPL) of the small intestinal mucosa of normal adult cows were determined using monoclonal antibodies (mAb) specific to adult bovine peripheral blood leukocytes (PBL). Laser flow cytometric (LFC) analysis demonstrated that IEL contained significantly (P less than 0.1 to 0.02) fewer cells (26%) expressing the pan T cell phenotype in comparison to LPL (38%) and PPL (44%). Similarly, significantly (P less than 0.01 to 0.001) lower numbers of B cells were observed among IEL (10%) compared to LPL (28%) and PPL (33%). While approximately equal numbers of B7A1+ "null" cells (10%) and DH59B+ "Ia+ monocytes/granulocytes" (16.5%) were observed among the three intestinal cell populations, IEL contained significantly (P less than 0.1 to 0.05) lower numbers (19%) of T helper (Th) cells in comparison to LPL (44%) and PPL (38%). In contrast, lymphocytes with the T cytotoxic/suppressor (Tc/s) phenotype were significantly lower (P less than 0.01 to 0.001) among LPL (14.5%) compared to IEL (25%) and PPL (23%). While the numbers of cells expressing class I major histocompatibility complex (MHC) surface antigens (H58A+) were approximately equal among LPL (79%) and PPL (87%), a significant difference (P less than 0.02) was observed between IEL (71%) and PPL. Similarly, while approximately equal numbers of cells expressing the MHC class II surface phenotype were observed among LPL (42%) and PPL (46%), IEL contained significantly (P less than 0.01) fewer (31%) MHC class II cells in comparison to PPL. Enrichment for T cells by plastic adherence and Sephadex G-10/nylon wool fractionation revealed a significant (P less than 0.01) and proportional increase in T lymphocyte subsets expressing pan T, Th and Tc/s phenotypes among the three cell populations. Similarly, enrichment for B cells by the same techniques showed a significant (P less than 0.01) and proportional increase in cells expressing the panB cell phenotype among LPL and PPL. Marked differences in cell size distribution and cell surface density were observed when the three intestinal leukocyte populations were compared by LFC using monoclonal antibodies directed at various cell surface markers. Furthermore, considerable quantitative variations of each cell surface marker were observed among the individual animals tested. The results of this study indicate that bovine IEL, which contain a high percentage of cells (greater than 30%) with no known phenotype are significantly different from LPL and PPL which are phenotypically similar cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

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+).     

T and B peripheral blood lymphocytes in normal and lymphocytotic sheep

Surface immunoglobulins (SIg), Peanut Agglutinin (PNA), spontaneous erythrocyte rosette (E-rosette) and Helix pomatia (HP) marker were investigated in normal and Bovine leukemia virus (BLV)-infected sheep. In normal sheep, 19.3% +/- 4.9 of peripheral blood lymphocytes (PBL) were SIg+, whereas 58% +/- 5.69 were PNA+, and 19.6 +/- 5.2 were E-rosette forming cells (E-RFC). In BLV-induced lymphocytotic sheep, SIg+ cells in PBL reached 59.4% +/- 15.06. In the same animals, PNA bound to 20.6% +/- 9.69 and E-RFC were 8.7% +/- 4.5. A panning technique was applied with an anti sheep-immunoglobulins coated plates to separate SIg+ (adherent cells = A) and SIg- cells (non-adherent cells = NA). The (A) population was 94-95% SIg+ cells and 2-3% PNA+, while the (NA) population was 0-4% SIg+ and 79-85% PNA+ cells. Thus PNA is a T cell marker in sheep species. HP, a marker for bovine T lymphocytes was also studied. Sheep PBL do not bind to HP. However, after panning separation about 50% of NA cells became HP+.     

Distribution of T and B lymphocytes in mammary dry secretions, colostrum and blood of adult dairy cattle

The distribution of lymphocyte subpopulations from dry secretions, colostrum and blood from 10 healthy adult Hostein-Fresian cows was studied using the TH21A and B26A mouse monoclonal antibodies (MAb) to adult bovine B and T lymphocytes, respectively. The mammary gland lymphocytes (MGL) were isolated from composite sample of all four quarters by density centrifugation over discontinuous gradient of ficoll-diatrizoate. The peripheral blood lymphocytes (PBL) were purified using the ficoll-thrombin method. Isolated PBL and MGL were analyzed using the two fluorochromes method (TFM) and laser flow cytometry (LFC). The mean viability of isolated PBL and MGL from dry secretions and colostrum after the TFM and LFC were 92.4% +/- 3.2%, 91.4% +/- 6.0% and 87.1% +/- 6.1%, respectively. There was a good correlation between the two MAbs and the percentage of surface immunoglobulin (SIg) positive cells in the peripheral blood using the TFM. The PBL yielded a mean percentage of 21.2% B cells, 66.4% T cells and 9.4% "Null cells" (TH21A+; SIg-). The TFM on MGL from dry secretions and colostrum indicated two distinct patterns (group I and II) of SIg and reactivity to MAb markers (p less than 0.001). The MGL data included in group I and group II were gathered from both colostral and dry secretions. In comparison to the distribution of lymphocyte subsets within peripheral blood the mean percentages of B cells, T cells and "Null cells" in the mammary gland were respectively, 2.8%, 88.1% and 5.4% for group I and 3.5%, 89.0% and 15.1% for group II. In the mammary secretions, the use of SIg alone was not considered to be a good marker for B cells; in four animals a mean percentage of 15.6% (13.9/89.0 X 100) of the mammary gland T lymphocytes were also SIg+. Of the TH21A+ MGL, only 18.8% were SIg+ in group II compared with 34.1% for MGL from group I and 69.3% for the PBL. Marked differences in cell size distribution and cell surface antigen density were found when PBL and MGL from dry secretions were compared by LFC using the B26A MAb. The results of this study demonstrate a difference in the percentages of peripheral blood and mammary gland B and T lymphocytes and confirm previous findings in which the T lymphocytes were found to represent the major subpopulation of lymphocytes in bovine mammary secretions. This may represent an essential event in the adoptive transfer of cellular immunity through the colostrum in cattle.     

Non-immune erythrocyte rosette formation of bovine peripheral blood and thymus lymphocytes

An E-rosetting reaction is described which gave 92.1%±2.4 (mean±S.D.) E-rosettes with bovine fetal thymocytes and 48.2%±8.4 with with bovine peripheral blood leukocyte (PBL) preparations. Both culture conditions and culture medium were critical factors in obtaining maximal and reproducible E-rosette numbers. Optimum rosette formation occurred when bovine PBL and neuraminidase treated sheep erythrocytes (nSRBC) were reacted in L-15 culture medium supplemented with 10% fetal calf serum (FCS). Other media including 100% FCS, MEM with 10% FCS, and RPMI-1640 with 10% FCS were less satisfactory. Cultural conditions found to be optimal for enumeration of bovine E-rosettes are similar to those reported as optimal for detection of human T cells. The specificity of rosette formation by bovine thymus derived (T) lymphocytes was shown by demonstration of (1) rosettes and surface membrane immunoglobulins (mIg) on different cells in PBL, (2) rosette formation by the majority of fetal thymocytes, and (3) no inhibition of rosette formation by anti-immunoglobulin serum. Using the E-rosette and mIg assays for presumptive bovine T and B lymphocytes, respectively, it was possible to differentiate from 57.5 to 90% (75.2%±9.3) of cells in bovine PBL preparations, and from 90.2 to 97.5% (94.2%±2.1) of cells in bovine fetal thymocyte preparations into T and B cells.     

Long term substitution and specific immune responses after transfer of bovine peripheral blood lymphocytes into severe combined immunodeficient mice.

The long term immune responsiveness of bovine peripheral blood lymphocytes engrafted into severe combined immunodeficient mice (bovine PBL SCID mice) was analyzed. After intraperitoneal transfer (i.p.) of 2x10(7) bovine PBL into SCID mice, FACS analysis revealed successful engraftment of bovine CD4 and CD8+ T cells in the peritoneal cavity, the peripheral blood, spleen, lymph nodes, bone marrow, and thymus of reconstituted mice for up to 13 weeks. As shown by immunocytochemistry in sections of spleens from SCID mice 16 weeks after substitution, bovine T and B cells were localized perivasculary forming pseudofollicular structures. Nevertheless, histopathology of spleen and liver from bovine PBL SCID mice revealed pathological alterations indicating rejection of xenogenic cells or graft versus host disease (GVHD). On the functional level, i.p. transfer of bovine PBL into SCID mice induced increasing levels of bovine IgM and IgG in the sera of recipients. Bovine Ig could be detected up to 20 weeks. Immunization of SCID mice reconstituted with PBL of normal donors with dinitrophenol (DNP)-edestin induced a weak specific bovine antibody response in recipient mice. In contrast, a secondary specific bovine IgG response was observed after antigen restimulation of SCID mice reconstituted with PBL from calves preimmunized either with DNP-edestin or keyhole limpet hemocyanin (KLH) showing functional T cell-independent and -dependent antibody responses of bovine PBL SCID mice. Our data demonstrate that transfer of bovine PBL into SCID mice leads to a long term engraftment of bovine cells in lymphatic and non-lymphatic organs inducing a functional substitution of T and B cell immune response of SCID mice. Therefore, bovine PBL SCID chimera can serve as a small animal model for the analysis of bovine lymphopoiesis and infectious diseases of cattle.     

Bovine T cells do not require auxiliary cells for response to selected mitogens

Bovine peripheral blood mononuclear cells (PBM's) were depleted of monocytes by three techniques: plastic adherence, passage through Sephadex G-10, and carbonyl iron treatment followed by buoyant density separation over Ficoll-Hypaque (FH). Although the resulting cell populations differed in their T and B cell ratios and percentages of residual monocytes, these preparations were generally more responsive to phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) than control cells containing monocytes. Passage of PBM's over a column of Sephadex G-10 and subsequent negative selection on plastic dishes previously coated with F(ab′)₂ anti-immunoglobulin or peanut agglutinin (PNA) resulted in highly enriched populations of T cells bearing receptors for PNA (99% PNAR⁺) and B cells (84% surface-immunoglobulin⁺, 10% PNAR⁺, 6% null), respectively. The percentage of monocytes remaining in either cell preparation was less than 0.1%. Reactions of these isolated lymphocyte subpopulations demonstrated that bovine T cells can be strongly activated by PHA, Con A and PWM without apparent need for auxiliary B cells or monocytes. Stimulation of T and B cell populations with PWM produced a pattern of reactivity which was interpreted to indicate that PWM may also activate B cells slightly, perhaps requiring T cell help. The use of this simple panning technique for lymphocyte separation, with its large capacity and specificity, has general application to the further study of cellular interactions.     

Bovine T lymphocyte response to bovine herpesvirus-1: cell phenotypes, viral recognition and acid-labile interferon production

The phenotype of bovine cells that proliferate to bovine herpesvirus (BHV-1) were identified by peanut agglutinin (PNA) and monoclonal antibodies with specificity for Pan T cells (B29), a T cell subset (B24), and an MHC class II (Ia-like) antigen (R-1). PNA+ cells but not PNA- cells separated by fluorescent activated cell sorting responded to BHV-1. Virally-activated T cells expressed MHC class II antigens, and possibly antigen specific receptors for virus. Binding of radiolabeled virus increased six-fold beyond the expected value for activated cells suggesting specificity of the lymphocytes for the virus. Finally, cells that responded to BHV-1 produced high levels of acid sensitive interferon. Taken together these results characterize the phenotypes of bovine lymphocytes that interact with BHV-1.     

Cattle infected with bovine leukaemia virus may not only develop persistent B-cell lymphocytosis but also persistent B-cell lymphopenia

We investigated the distribution of B and T cells in the peripheral blood of haematologically inconspicuous (non-persistent lymphocytotic, PL-) cattle infected with the bovine leukaemia virus (BLV). Flow cytometric data were obtained from six PL- cattle and compared with six age-matched animals with persistent lymphocytosis (PL+) and five non-infected healthy controls (BLV-). In the PL- group, the percentage and number of surface immunoglobulin-positive (sIg+) B cells were significantly reduced. Whereas in BLV-cattle, about 40% of the peripheral blood lymphocytes (PBL) were sIg + and 24% were sIgM + B cells. In the PL- group, less than 20% of the PBL were sIg+ and sIgM+ B cells. Only 5% of the PBL co-expressed sIgM+ and CD5+ versus 16% in BLV-. This decrease was persistent over 3 years and predominantly affected: (i) B cells that did not express sIgM; (ii) sIgM + B cells co-expressing CD5 and CD11b; and (iii) equally both lambda- and K-type light chain B-cell subpopulations. In contrast, the number of all circulating lymphocytes, CD5- and CD11b- sIgM+ B cells and CD2+ T cells did not differ. In PL+ animals, about 75% of the PBL were sIgM+ CD5+ B cells. These cells were of polyclonal origin, as light chains of the lambda- and K-type were expressed in a ratio of 4:1 (57.7% of PBL lambda+, 14% kappa+) as in BLV- animals (33.6% of PBL lambda+, 8.7% kappa+). In PL+ cattle the absolute number of B-cells and, therefore, their relative percentage is significantly increased. For this reason, even in case of absolutely increased T-cell numbers, the relative percentage of T-cells could be lower than in normal controls. The cause for the observed B cell decrease in PL- cattle is unknown, but it can be assumed that cytotoxic T cells are involved in this B-cell lymphopenia.     

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.     

Cattle Infected with Bovine Leukaemia Virus may not only Develop Persistent B‐cell Lymphocytosis but also Persistent B‐cell Lymphopenia

We investigated the distribution of B and T cells in the peripheral blood of haematologically inconspicuous (non‐persistent lymphocytotic, PL^?) cattle infected with the bovine leukaemia virus (BLV). Flow cytometric data were obtained from six PL^? cattle and compared with six age‐matched animals with persistent lymphocytosis (PL⁺) and five non‐infected healthy controls (BLV^?). In the PL^? group, the percentage and number of surface immunoglobulin‐positive (sIg⁺) B cells were significantly reduced. Whereas in BLV^? cattle, about 40% of the peripheral blood lymphocytes (PBL) were sIg⁺ and 24% were sIgM⁺ B cells. In the PL^? group, less than 20% of the PBL were sIg⁺ and sIgM⁺ B cells. Only 5% of the PBL co‐expressed sIgM⁺ and CD5⁺ versus 16% in BLV^?. This decrease was persistent over 3 years and predominantly affected: (i) B cells that did not express sIgM; (ii) sIgM⁺ B cells co‐expressing CD5 and CD11b; and (iii) equally both λ‐ and κ‐type light chain B‐cell subpopulations. In contrast, the number of all circulating lymphocytes, CD5^? and CD11b^? sIgM⁺ B cells and CD2⁺ T cells did not differ. In PL⁺ animals, about 75% of the PBL were sIgM⁺CD5⁺ B cells. These cells were of polyclonal origin, as light chains of the λ‐ and κ‐type were expressed in a ratio of 4:1 (57.7% of PBL λ⁺, 14% κ⁺) as in BLV^? animals (33.6% of PBL λ⁺, 8.7% κ⁺). In PL⁺ cattle the absolute number of B‐cells and, therefore, their relative percentage is significantly increased. For this reason, even in case of absolutely increased T‐cell numbers, the relative percentage of T‐cells could be lower than in normal controls. The cause for the observed B cell decrease in PL^? cattle is unknown, but it can be assumed that cytotoxic T cells are involved in this B‐cell lymphopenia.     

Increase of intestinal intraepithelial lymphocytes expressing CD8 antigen following challenge infection with Eimeria acervulina.

Eimerian infection-induced changes in the intestinal intraepithelial lymphocyte (IEL) subpopulations expressing CD8 antigen (cytotoxic/suppressor T cells) or antigen-specific T cell receptor (TCR) heterodimer alpha beta (TCR2) or gamma delta (TCR1) were investigated in F2 crosses of 15I5 B-congenic chickens differing for the major histocompatibility complex (MHC). Duodenum TCR2+ IEL were increased in B2 B2 and B5 B5 chickens 7 days following secondary infection. Two-color immunofluorescence revealed that the majority of CD8+ cells in the duodenum intraepithelium of immune chickens expressed TCR2. A significant increase in the duodenum TCR2+CD8+ and TCR1+CD8+ IEL occurred in B2 B2 chickens, which developed significantly less oocyst production than the B5 B5 chickens following challenge infection. The results suggest that a significant increase in the duodenum CD8+ IEL may reflect an enhanced acquired immunity of B2 B2 chickens.     

Predominant subpopulations of T lymphocytes in the mammary gland secretions during lactation and intraepithelial T lymphocytes in the intestine of dairy cows

Lymphocytes obtained from mammary gland secretions (MGS) during lactation or the dry period of dairy cows were simultaneously analyzed and compared to ileal intraepithelial lymphocytes (IEL) and peripheral blood lymphocytes (PBL) using monoclonal antibodies (mAb) specific for bovine leukocyte differentiation antigens. The T-lymphocytes of MGS during lactation and those in IEL were predominantly CD8(+), while T-cells in MGS during the dry period were predominantly CD4(+). In addition, the proportion of gamma delta T-cells in MGS during lactation and IEL was fairly high. A large percentage of CD8(+) cells and T-cells coexpressed the activation molecule, ACT2, yielding a high proportion of ACT2(+) CD8 T-cells and ACT2(+) gamma delta T-cells, in MGS during lactation and IEL. However, both types of cells were found at an extremely low level in MGS during the dry period and in PBL. Thus, the predominant T-cell populations in MGS during lactation are phenotypically similar to those in IEL in the intestine.     

Peanut agglutinin as a surface marker for canine T lymphocytes

Peanut agglutinin (PNA) and surface immunoglobulin (SIg) were investigated as markers for T and B lymphocytes in blood and lymphoid tissues of dogs of various ages. In the blood study, 4 age groups (n = 8 dogs/group) were used. The mean (+/- SD) percentages of PNA-positive (PNA+) cells were 68.4 +/- 8.6% (group 1, less than 1 year old), 70.3 +/- 9.2% (group 2, 1 to 2 years old), 72.0 +/- 3.7% (group 3, 5 to 6 years old), and 63.8 +/- 10.1% (group 4, 10 to 11 years old). The mean percentages of SIg-positive (SIg+) cells in blood were 32.1 +/- 10.6% (group 1), 43.2 +/- 7.0% (group 2), 34.3 +/- 4.8% (group 3), and 35.0 +/- 6.8% (group 4). The mean total percentages of PNA+ and SIg+ cells were 100 +/- 6.0% (group 1), 113.5 +/- 4.9% (group 2), 106.3 +/- 5.3% (group 3), and 98.9 +/- 9.2% (group 4). The proportions of PNA+ and SIg+ cells in dogs of group 2 were significantly (P less than 0.05) different from those in dogs of the other groups. Serial changes in PNA+ and SIg+ cells were investigated in blood of 6- to 29-week-old pups (n = 8). A significant (P less than 0.05) transient decrease in PNA+ cells and a corresponding increase in SIg+ cells was observed in pups between 14 and 17 weeks old. Lymphoid tissue specimens and blood samples were obtained from 2- to 6-month-old dogs (n = 11) and from 6- to 12-month-old dogs (n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of labelling methods for bovine T and B lymphocytes

Two lectins, one from Helix pomatia (HP) and one from Peanut (PN), were evaluated as bovine T cell markers. HP attaches to about 40% of the bovine peripheral blood lymphocytes (PBL). With an indirect technique about 60% of PBL are HP positive, while PN attaches to a slightly higher proportion ot the PBL.In double labelling experiments, HP was shown to bind only to Ig negative cells while 2% of the PBL were double labelled with PN and anti-Ig.The influence of different pretreatments of the PBL or the antibodies for labelling of the membrane bound Ig was studied. Preincubation for detaching cytophilic antibodies was important to avoid false Ig positive cells.     

Lymphocyte subpopulations in peripheral blood of lambs experimentally infected with bovine respiratory syncytial virus

The lymphocyte subpopulations of peripheral blood of normal lambs and lambs experimentally infected with bovine respiratory syncytial virus (RSV) were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with bovine RSV was characterized by a significant rise in SBU-T8+ (CD8+ or cytotoxic) T cells and a significant reduction in SBU-T4+ (CD4+ or helper) T cells and B (LCA p220+) lymphocytes (P less than 0.05). The helper/suppressor (CD4/CD8) ratio was reduced from 3.91 on the day of experimental infection to 1.13 on 10 days after experimental infection (P less than 0.001). The total number of SBU-T4+ (CD4+) and B cells returned to pre-inoculation values 14 days after experimental infection but the helper/suppressor ratio remained depressed up to 21 days post-inoculation.     

Spontaneously active suppressive cells in canine peripheral blood

The phenomenon of the unusually high spontaneous suppressive activity of cells in peripheral blood of dogs was analysed. The m/c (mitomycin C)-treated population of peripheral blood leucocytes (PBL) contained cells able to reduce the responsiveness of autologous cells by 48 +/- 15% (P less than 0.01) and their activity was not indomethacin dependent. Thoracic duct lymphocytes (TDL) did not reduce the response of PBL to PHA, neither did cell crowding. The supernatants from 24-h cultures of m/c-treated PBL did not affect the response to PHA, and parallelly precultured cells inhibited the proliferation of PBL to a lesser degree (24 +/- 9%) than the fresh cells (50 +/- 16%, P less than 0.05). Addition of m/c-treated polymorphonuclear cells at PMN to PBL ratios of 1:4 and 1:1 progressively inhibited PBL reactivity to PHA, from 29.5 +/- 3.5% to 68.5 +/- 9%, respectively, and the supernatants from 24-h cultures of PMN reduced the proliferation by 48 +/- 2.8%. The neutrophil-derived inhibitory factor(s) was non-cytotoxic and reduced the formation of blasts to 61.5 +/- 3.5% of the control values. These results indicate that dog PBL from Lymphoprep gradient contain a population of non-recirculating, short-lived, spontaneously suppressive cells, mainly PMN, which modulate T cell reactivity in vitro, suggesting that neutrophils may be able to exert a regulatory effect in vivo.     

Monoclonal antibodies that distinguish bovine T and B lymphocytes

The specificities of three monoclonal antibodies (MAbs) were investigated using microcytotoxicity, fluorescence microscopy and laser flow cytometry (LFC) techniques. By microcytotoxicity, bovine thymocytes (n = 4) were estimated to be 85% B26A+, 4% TH21A+, and 1% H4+. Nylon wool enriched peripheral blood T lymphocytes (n = 3) were 90% B26A+, 10% TH21A+ and 10% H4+. Adherent B cell enriched fractions (n = 3) were 10% B26A+, 90% TH21A+ and 90% H4+. The two fluorochrome method was used to simultaneously identify lymphocytes that were sIg+ and MAb+. In these experiments, 92% of all sIg+ cells were H4+. An identical result was obtained for TH21A. 85% of all sIg- cells were B26A+. Using LFC, the mean percentages of sIg+, H4+ and TH21A+ PBL (n = 5) were not significantly different. B26A recognized a significantly greater population of cells, equivalent to the expected percentage of T lymphocytes. LFC also revealed two relatively discrete sizes of B26A+ PBL. The larger population overlapped the size range in which sIg+, H4+, TH21A+ PBL were found. The more numerous smaller B26A+ PBL were in a size range in which few sIg+, H4+ and TH21A+ PBL were found. In a study of MAb reactions with PBL of 185 cows, it was shown that in 92% of the animals H4 and TH21A were positively correlated (r = +.93), when H4 and TH21A were negatively correlated with B26A (r = -.94 and r = -.92, respectively). These correlation coefficients indicate a converse relationship between B26A and both H4 and TH21A. The remaining 8% of the animals were heterogeneous in their expression of the H4 and TH21A markers but not the B26A marker. These results provide strong evidence that: 1) B26A is a pan-T lymphocyte MAb in cattle, 2) a small but significant degree of heterogeneity exists in the expression of the epitopes recognized by H4 and TH21A. However, both MAbs recognize all B lymphocytes of most individuals, and 3) using a variety of immunological methods these three MAbs can now reliably be used to assay bovine T and B lymphocytes.     

T and B lymphocytes in horses persistently infected with equine infectious anaemia virus

The percentage of T and B lymphocytes in the peripheral blood of horses chronically infected with equine infectious anaemia (EIA) virus was determined and the results were compared with the percentage of these cells in healthy uninfected horses. Cells with membrane receptors for sheep erythrocytes (T and active T lymphocytes) were determined by E and A rosette techniques, while cells with receptors for the C3b component of complement and those with receptors for mouse erythrocytes (B lymphocytes), were determined by the EAC rosette method. The percentage of Fc positive cells was assayed by the EA rosette test.The majority of peripheral blood lymphocytes (PBL) from both uninfected and EIA-infected horses formed rosettes of each kind with only three erythrocytes indicating a low density of the corresponding receptors on the cell membrane under the condition of the assays used. The percentage of T lymphocytes in the peripheral blood of diseased horses (52.4±1.6%), as detected by E rosettes, was significantly (p<0.01) higher than in control animals (42.4±3.5%). In clinically healthy horses 8.9±1.1% of PBL were identified by A rosettes as active T cells, whereas animals with a chronic form of EIA had a much lower (p<0.001) percentage of these cells (4.7±0.7%). In the B lymphocyte subpopulations the percentages of cells bearing Fc and C3b receptors were markedly elevated (p<0.001) in EIA-infected horses (24.7±0.8% and 42.8±2.2% respectively) as compared to uninfected animals (15.1±1.4% and 29.6±1.2% respectively). Receptors for mouse erythrocytes, as yet undescribed on equine PBL, were demonstrated in approximately equal proportions on lymphocytes from EIA-infected (24.8±1.5%) and uninfected horses (24.3±2.1%).     

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.