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Detection of B and T cells, with lectins or antibodies, in healthy and bovine leukemia virus-infected cattle
Veterinary Immunology and Immunopathology, 18 (1988) 269-278 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
269
D e t e c t i o n of B and T Cells, w i t h Lectins or Antibodies, in Healthy and B o v i n e L e u k e m i a Virus-Infected Cattle CAROLINE FOSSUM 1, A. BURNY 2, D. PORTETELLE 2, M. MAMMERICKX3 and B. MOREIN'
1Department of Veterinary Microbiology, Section of Virology, Swedish University of Agricultural Sciences, BMC, Box 585, S-75 123 Uppsala (Sweden) 2Department of Molecular Biology, University of Brussels, B 1640 Rhode-Souint-Gen~se and Faculty of Agronomy, B 5800 Gembloux (Belgium) 3National Institute for Veterinary Research, B 1180 Vecle (Belgium) {Accepted 13 July 1987)
ABSTRACT
Fossum, C., Burny, A., Portetelle, D., Mammerickx, M. and Morein, B., 1988. Detection of B and T cells, with lectins or antibodies, in healthy and bovine leukemia virus-infected cattle. Vet. Immunol. Immunopathol., 18: 269-278. 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 immunoglobulinmolecules 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.
INTRODUCTION
Infection with the retrovirus bovine leukemia virus (BLV) induces the chronic disease enzootic bovine leukosis (EBL). Although BLV is believed to 0165-2427/88/$03.50
© 1988 Elsevier Science Publishers B.V.
270
infect the B lymphocytes (Paul et al., 1977) or a subpopulation thereof (Kenyon and Piper, 1977), antibodies to viral proteins are readily produced. An increased proliferation of a cell population bearing several B cell characteristics (Muscoplat et al., 1974; Wieland and Straub, 1975; Kenyon and Piper, 1977; Kumar et al., 1978) can, in 30-70% of the infected cattle, be manifested as a persistent lymphocytosis (PL). At a later stage the BLV infection gives rise to lymphoid tumors of a clonal B cell origin in 0.1-10% of the infected cattle (for reviews, see Burny et al., 1980, 1985). The implications of a key position for the B lymphocytes at BLV infections justify more detailed studies of bovine lymphocyte subpopulations. A reliable quantitation of bovine T and B cells has emerged from the extension of membrane markers for bovine cells during recent years with monoclonal antibodies (Pinder, 1980a,b; Lalor et al., 1986; Rabinovsky and Yang, 1986). Nevertheless, previously used markers, such as lectins, still have an important role in these studies. The binding of lectins is based on their specificity for sugars and can therefore reveal whether the lymphocytes alter the expression of sugar residues on their surface during maturation, activation and infection of the cells. In the present study we compared MAbs to subpopulations of bovine lymphocytes with lectins and polyclonal antibodies, in their binding to lymphocytes obtained from healthy cattle and from cattle at various stages of BLV infection. M A T E R I A L AND M E T H O D S
Animals The initial comparative studies on the binding of various lymphocyte markers were carried out on peripheral blood lymphocytes (PBL) isolated from 12 healthy animals of the Swedish Red and White breed. In the following study seven animals were included. Four were seropositive to BLV as measured by ELISA and had persistent lymphocytosis (BLV+PL + ). Two of the animals were seropositive to BLV but had no lymphocytosis ( B L V + P L - ). One animal, seronegative to BLV and with normal lymphocyte count ( B L V - P L - ) , was included as a control.
Isolation of P B L The lymphocytes were isolated by the Ficoll-Paque method initially described by BSyum (1968). Details for isolation of bovine mononuclear cells (MNC) were given previously {Johnsson and Morein, 1977). Briefly, the MNC were collected from the interface between Ficoll-Paque and plasma, generated after centrifugation of the blood. After three washings of the cells in phos-
271
phate-buffered saline (PBS) pH 7.2, supplemented with 0.2% human serum albumin (HSA) and 0.02% NAN3, the cells were counted and used in the labelling experiments.
Labelling techniques Approximately 3 × 106 MNC were used in each labelling experiment. Appropriate dilutions of the reagents were first established with MNC obtained from healthy cattle and then used throughout the experiment.
B cells. Two B cell markers were used; a fluoresceinated F (ab')2 fragment from polyclonal rabbit anti-bovine IgM serum and a mouse MAb to bovine IgM, designated B5.4 (Pinder et al., 1980b). The labelling procedure with the polyclonal F (ab')2 fragment has been described before (Johansson and Morein, 1983). Briefly, the MNC were incubated for 1 h at 37°C in serum-free medium to detach cytophilic antibodies (Watson, 1976) and washed twice in PBS, supplemented as described above, before 50/~l of the fluoresceinated F (ab')e antibodies to IgM were added. The cell suspension was incubated for 30 min at + 4 ° C with the antiserum, washed twice in PBS (supplemented) and fixed in 1.8% paraformaldehyde solution in PBS for 5 min at room temperature. After an additional washing the cells were ready for analysis. Labelling of B cells with the MAb B5.4 (kindly donated by Dr. M. Pinder, Gabon) was done with an indirect immunofluorescence (IF) technique. The first steps were performed similarly as for the polyclonal reagent. After washing away non-bound MAbs, the preparations were incubated with rhodamineconjugated goat F (ab')2 fragments of antibodies to mouse IgG heavy and light chains (Jackson Immuno-Research Laboratories, Inc. Avondale, PA) for another 30 min at ÷ 4 ° C. Thereafter the cells were washed and fixed as described above.
T cells. These were labelled with four different marker systems. Two MAbs, designated P5 and BLT-1 (kindly supplied by Dr. P. Lalor, ILRAD, Kenya and Dr. T. Yang, Storrs, U.S.A. ) and two lectins, Helix pomatia A hemagglutinin (HP) and Peanut agglutinin (PNA), were used. Labelling with the MAbs P5 and BLT-1 was performed with indirect IF as described above. The detection of receptors for the lectin HP was carried out as described before (Johansson and Morein, 1983 ). To reveal the receptors for HP, the cells were treated with 2/tg neuraminidase (Clostridium perfringens type VI, 1-3 IU/ml, Sigma) for 45 min at ÷ 4 ° C . After washing, the cells were incubated with fluorochrome-conjugated HP (direct method) or with HP followed by fluorochrome-conjugated F (ab') 2 antibodies to H P (indirect method ).
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Fig. 1. Two-parameter histogram generated by flow cytometry showing the distribution of bovine lymphocytes according to cell size (forward light scatter) on the horizontal axis and according to fluorescence intensity of the cells on the vertical axis. The lymphocytes were isolated from blood of a non-infected control animal and labelled with fluoresceinated antibodies to bovine IgM as described in Material and Methods. Exclusion limits for fluorescence positive and negative cells were set between channels 9 and 10 on the vertical axis and for small and large lymphocytes between channels 38 and 39 on the horizontal axis, dividing the histogram into four areas designated A-D. The B lymphocytes fall into areas B and C. Large B lymphocytes are found in area C. The border lines for fluorescence positive and negative cells and for small and large cells were established with cells from the non-infected animal and thereafter used for the analysis of lyrephocytes from the BLV-infected animals. L a b e l l i n g of cell m e m b r a n e r e c e p t o r s for P N A w a s p e r f o r m e d u n d e r n o n c a p p i n g c o n d i t i o n s w i t h direct I F a c c o r d i n g to P e a r s s o n et al. (1979). A f t e r w a s h i n g a n d fixation, t h e s p e c i m e n s were a n a l y s e d in a f l u o r e s c e n c e m i c r o s c o p e ( N i k o n , L a b o p h o t ) or b y flow c y t o m e t r y .
Flow cytometer analysis L y m p h o c y t e s labelled w i t h f l u o r e s c e i n a t e d a n t i b o d i e s to b o v i n e I g M as described a b o v e were a n a l y s e d in a flow c y t o m e t e r ( F A C S III, B e c t o n D i c k i n s o n ) e q u i p p e d w i t h a 5 - W a r g o n ion laser ( S p e c t r a P h y s i c s ). F o r e x c i t a t i o n t h e 488n m laser line was u s e d at a p o w e r of 250 m W . D a t a were a c c u m u l a t e d in t h e f o r m of h i s t o g r a m s t h a t c o r r e l a t e d f l u o r e s c e n c e i n t e n s i t y a n d f o r w a r d light s c a t t e r of t h e cells w i t h t h e n u m b e r of cells a n a l y s e d . T h e t w o - p a r a m e t e r hist o g r a m was divided into f o u r a r e a s w h i c h d i s c r i m i n a t e d p o s i t i v e l y s t a i n e d l y m p h o c y t e s f r o m t h e n e g a t i v e cells a n d s m a l l l y m p h o c y t e s f r o m large cells (Fig. 1 ). T h e p r o p o r t i o n of cells in e a c h a r e a was t h e n calculated.
273 RESULTS
Comparison of B cell markers in healthy animals The MAb B5.4 was compared with the polyclonal antibody to bovine IgM. The average proportion of B cells detected by the MAb B5.4 (16.2%) was close to that value detected by the polyclonal antibody (18.0% Ig + cells). Discrepancies between the two markers were found in individual lymphocyte samples, with a greater variation in the values of PBL samples stained positive with the MAb B5.4 (SD--8.2, n = 12 ) than in the values obtained with the polyclonal antibody (SD=4.0, n = 1 2 ) .
Comparison of T cell markers in healthy animals The two MAbs to bovine T cells, P5 and BLT-1, were compared in labelling experiments of bovine PBL with the indirect IF labelling with HP (anti-HP). The monoclonal antibodies attached to 66.1+3.8%, n--12 (P5) and 59.8_+ 7.1%, n = 5 (BLT-1). The average proportion of T cells as detected by indirect HP labelling of PBL from the same animals was in the same range (67.8+ 6.2%, n= 10, anti-HP + cells).
Elevation of the number orB and T lymphocytes in BL V-infected cattle The proportions of B and T lymphocytes were measured in BLV-infected and normal animals. Details are given in Fig. 2. The proportion of B cells detected was increased more than three times in the BLV-infected animals with lymphocytosis compared to non-infected animals, regardless of the B cell marker used. The proportion of T cells detected by the monoclonal antibody P5 was less than one-third of that in normal animals (Fig. 2). A decreased proportion of T cells was also recorded in the BLV-infected animals using the direct HP technique for their detection (Fig. 2 ), but a great variation was found between samples from different animals. Similarly, a great variation in the proportion of cells with HP receptors detected by the indirect HP method was recorded, with values ranging from 20 to 80% anti-HP positive cells compared to 60 to 75% in the normal animals (Fig. 2). One of the two cattle with antibodies to BLV but without lymphocytosis had an increased proportion of B lymphocytes, 49% and 58%, when tested on two occasions at an interval of 4 weeks. The corresponding values for the other BLV + P L - animal were 30% and 16% of B lymphocytes.
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Flow cytometer analysis of B lymphocytes from BL V-infected animals The samples labelled with fluoresceinated antibodies to bovine IgM were further analysed by flow cytometry (FCM). The proportion of cells identified as B lymphocytes by fluorescence microscopy and FCM analysis was in good agreement (r = 0.84). An increased intensity of the fluorescence was observed both by fluorescence microscopy and by FCM in a substantial proportion of the B lymphocytes from BLV-infected cattle compared to B lymphocytes from healthy cattle. The distribution of cells according to fluorescence intensity is visualized in the FCM-generated histogram (Fig. 3). From this diagram it can be seen that the Ig-positive cells obtained from BLV-infected cattle are distributed throughout the histogram (channels 1-64) while the fluorescence intensity of Ig-bearing cells isolated from the control animal did not exceed channel number 15. In the same histogram, the light scatter distribution, indicating the size of the cells, showed that 3.4 to 9.7% of the Ig-bearing cells obtained from B L V + P L + animals were enlarged. The corresponding value for the control animal was 0.3%.
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LIGHT SCATTER Fig. 3. Distribution of bovine lymphocytes, labelled with fluoresceinated antibodies to bovine IgM, in a histogram generated by FCM. The lymphocytes were obtained from one control animal (A), one animal seropositive to BLV but with normal lymphocyte count (B) and from two animals with antibodies to BLV and persistent lymphocytosis (C and D ). For evaluation of data see Material and Methods• DISCUSSION T h e lectins H P a n d P N A are reliable m a r k e r s for b o v i n e l y m p h o c y t e s origi n a t i n g f r o m h e a l t h y a n i m a l s ( M o r e i n et al., 1979; P e a r s s o n et al., 1979 ). T h a t was also c o n f i r m e d in t h e p r e s e n t work w h e r e a good a g r e e m e n t was f o u n d b e t w e e n t h e p r o p o r t i o n of cells d e t e c t e d b y i n d i r e c t i m m u n o f l u o r e s c e n c e with the lectin H P a n d with two m o n o c l o n a l antibodies, P 5 a n d B L T - 1 , b o t h claimed to detect bovine T l y m p h o c y t e s (Lalor et al., 1986; R a b i n o v s k y a n d Yang, 1986 ).
276 Similarly, both polyclonal and monoclonal antibodies to bovine IgM detected the same average proportion of B lymphocytes in blood from healthy cattle. In blood from BLV-infected cattle, these two B cell markers both indicated a strong increase in the number of B cells. In contrast, results obtained with the T cell markers HP and P5 did not correspond to each other when the markers were applied to lymphocytes obtained from BLV-infected cattle. While the MAb P5 detected a clear-cut decrease in the proportion o f T cells (x= 17% ) in BLV-infected cattle with a small variation between samples, the lectin HP indicated the same tendency with a lower mean value than in healthy cattle, but inconsistently and with great individual variations. As regards the indirect HP method, no clear-cut decrease in the proportion of T cells was found (Fig. 2 ). Further, the proportion of HP-positive cells plus the proportion of Ig-bearing lymphocytes exceeded 100% in samples from some of the BLV-infected cattle. Obviously, some Ig-bearing cells from BLV-infected animals also attach to the HP lectin, indicating that the surface of some of the B cells (HP+Ig + cells) found in BLV-infected animals has an altered sugar composition. These cells might be arrested at an early stage of maturation in accord with results from patients with chronic lymphatic leukemia (HellstrSm et al., 1976). Both the direct and indirect labelling procedures with HP include enzymatic removal of sialic acid to reveal the receptor for HP prior to addition of the lectin (see Material and Methods). The optimal concentration of neuraminidase needed to reveal the receptors for HP varies between species, and plateau values of the concentration of neuraminidase have been established for lymphocytes isolated from man (HellstrSm et al., 1976), rat (Swanborg et al., 1977), mouse (Haller et al., 1978), cattle (Morein et al., 1979), and horse (BrostrSm et al., 1985). No increase in the proportion of HP-positive cells is attained when lymphocytes from healthy cattle are treated with concentrations of neuraminidase up to ten times higher than the concentration selected as optimal (Morein et al., 1979). However, when lymphocytes obtained from BLV-infected cattle with lymphocytosis were tested, the proportion of HPpositive cells increased from 29 to 70%, from 50 to 62% and from 17 to 52%, even by doubling the selected concentration of neuraminidase. There was no increase in the proportion of HP-positive cells from the control animals by the same treatment (unpublished results). The lectin PNA was also tested at the standard dilution used for detection of bovine T cells. This concentration, however, caused aggregation of a substantial proportion of the lymphocytes obtained from BLV-infected animals (data not shown ), again indicating that these lymphocytes express an altered sugar composition on their surfaces compared to cells obtained from non-infected animals. Flow cytometer analysis of the lymphocytes labelled with fluoresceinated antibodies to bovine IgM confirmed the proportional results obtained from counting the cells in a fluorescence microscope. Again a greater individual vari-
277
ation in the proportion of B cells was obtained by the MAb than by the polyclonal antibodies which could be explained by a heterogeneity in the binding of B5.4 to serum IgM shown by Pinder et al. (1980b). Further, the fluorescence intensity of the B lymphocytes from the BLV-infected animals with lymphocytosis was proven be strongly elevated, indicating an increased number of surface immunoglobulin molecules. Also the proportion of "large" Ig-bearing cells was up to 30 times higher among blood lymphocytes from the BLV-infected animals than from the control animal, indicating an increased number of blastoid B lymphocytes in the blood from BLV-infected animals. From these results we conclude and confirm that the proportion of B cells increases and the proportion of T cells decreases following BLV infection (for review see Burny et al., 1980). Further, it should be taken into account that lectins are reliable markers only for the identification of T lymphocytes isolated from healthy cattle. In contrast to the lectins, the MAb P5 seems to recognize T cells with good reproducibility both in blood from cattle infected with BLV and in blood from healthy cattle. Thus, the accessibility of MAbs as markers for subsets of bovine lymphocytes has provided us with better tools to study lymphopoietic disorders in cattle. ACKNOWLEDGEMENTS
The monoclonal antibodies were kindly donated by Dr. Paul Lalor, ILRAD, Nairobi, Kenya (P5); Dr. Margaret Pinder, Gabon (B5.4) and Dr. Thomas Yang, Storrs (BLT-1). The flow cytometer analyses were carried out by Dr. Hugo Fellner-Feldegg, Department of Pathology, Swedish University of Agricultural Sciences, Uppsala. This work was supported by grants from the Swedish Council for Forestry and Agricultural Research.
REFERENCES BSyum, A., 1968. Isolation of leukocytes from human blood. A two phase system for removal of red cells with methyl cellulose as erythrocyte-aggregating agent. Scand. J. Clin. Lab. Invest., 21, Suppl. 9: 4-29. BrostrSm, H., HellstrSm, U., HammarstrSm, S., Obel, N. and Perlmann, P., 1985. A new surface marker on equine peripheral blood lymphocytes. I. Subpopulations of lymphocytes with receptors for Helix pomatia A hemagglutinin (HP). Vet. Immunol. Immunopathol., 8: 35-46. Burny, A., Bruck, C., Chantrenne, Y., Cleuter, Y., Dekegel, D., Ghysdael, J., Kettmann, R., Leclercq, M., Leunen, J., Mammerickx, M. and Portetelle, D., 1980. Bovine leukemia virus: molecular biology and epidemiology. In: G. Klein (Editor), Viral Oncology. Raven Press, New York, NY, pp. 231-290. Burny, A., Bruck, C., Cleuter, Y., Couez, D., Deschamps, J., Gregoire, D., Ghysdael, J., Kettmann, R., Mammerickx, M., Marbaix, G. and Portetelle, D., 1985. Bovine leukemia virus and enzootic bovine leukosis. Onderstepoort J. Vet. Res., 52: 133-144. Haller, 0., Gidlund, M., HellstrSm, U., HammarstrSm, S. and Wigzell, H., 1978. A new surface
278 marker on mouse natural killer cells: receptors for Helix pomatia A hemagglutinin. Eur. J. Immunol., 8: 765-771. Hellstrbm, U., Mellstedt, H., Perlmann, P., Holm, G. and Pettersson, D., 1976. Receptors for Helix pomatia A hemagglutinin on leukaemic lymphocytes from patients with chronic lymphocytic leukaemia (CLL). Clin. Exp. Immunol., 26: 196-203. Johansson, C. and Morein, B., 1983. Evaluation of labelling methods for bovine T and B lymphocytes. Vet. Immunol. Immunopathol., 4: 345-359. Johnsson, K. and Morein, B., 1977. In vitro stimulation of bovine circulating lymphocytes by parainfluenza type 3 virus. Res. Vet. Sci., 22: 83-85. Kenyon, S.J. and Piper, C.E., 1977. Cellular basis of persistent lymphocytosis in cattle infected with bovine leukemia virus. Infect. Immun., 16: 891-897. Kumar, S.P., Paul, P.S., Pomeroy, K.A., Johnson, D.W., Muscoplat, C.C., Van der Maaten, M.J., Miller, J.M. and SSrensen, D.K., 1978. Frequency of lymphocytes bearing Fc receptors and surface membrane immunoglobulins in normal, persistent lymphocytotic and leukemic cows. Am. J. Vet. Res., 39: 45-49. Lalor, P.A., Morrison, W.I., Goddeeris, R.M., Jack, R.M. and Black, S.J., 1986. Monoclonal antibodies identify phenotypically and functionally distinct cell types in the bovine lymphoid system. Vet. Immunol. Immunopathol., 13: 121-140. Morein, B., HellstrSm, U., Axelsson, L.-G., Johansson, C. and HammarstrSm, S., 1979. Helix pomatia A hemagglutinin, a surface marker for bovine T lymphocytes. Vet. Immunol. Immunopathol., 1: 27-36. Muscoplat, C.C., Johnson, D.W., Pomeroy, K.A., Olson, J.M., Larson, V.L., Stevensen, J.B. and SSrensen, D.K., 1974. Lymphocyte surface immunoglobulin: frequency in normal and lymphocytotic cattle. Am. J. Vet. Res., 35: 593-595. Paul, P.S., Pomeroy, K.A., Johnson, D.W., Muscoplat, C.C., Handwerger, B.S., Soper, F.F. and SSrensen, D.K., 1977. Evidence for the replication of bovine leukemia virus in the B lymphocytes. Am. J. Vet. Res., 38: 873-876. Pearsson, T.W., Roelants, G.E., Lundin, L.B. and Mayor-Whitey, K.S., 1979. The bovine lymphoid system; binding and stimulation of peripheral blood lymphocytes by lectins. J. Immunol. Methods, 26: 271-282. Pinder, M., Pearsson, T.W. and Roelants, G.E., 1980a. The bovine lymphoid system. II. Derivation and partial characterization of monoclonal antibodies against bovine peripheral blood lymphocytes. Vet. Immunol. Immunopathol., 1: 303-316. Pinder, M., Muscoe, A.J., Morrison, W.I. and Roelants, G.E., 1980b. The bovine lymphoid system. III. A monoclonal antibody specific for bovine cell surface and serum IgM. Immunology, 40: 359-365. Rabinovsky, E.D. and Yang, T., 1986. Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen. J. Immunol., 136: 609-615. Swanborg, R.H., HellstrSm, U., Perlmann, H., HammarstrSm, S. and Perlmann, P., 1977. A new rat lymphocyte surface marker: characterization and separation of cells with receptors for Helix pomatia A hemagglutinin. Scand. J. Immunol., 6: 235-239. Watson, D.L., 1976. The effect of cytophilic IgG2 on phagocytosis by ovine polymorphonuclear leukocytes. Immunology, 31: 159-165. Wieland, F. and Straub, O.C., 1975. Frequency of surface immunoglobulin bearing blood lymphocytes in cattle affected with bovine leukosis. Res. Vet. Sci., 19: 100-102.
269
D e t e c t i o n of B and T Cells, w i t h Lectins or Antibodies, in Healthy and B o v i n e L e u k e m i a Virus-Infected Cattle CAROLINE FOSSUM 1, A. BURNY 2, D. PORTETELLE 2, M. MAMMERICKX3 and B. MOREIN'
1Department of Veterinary Microbiology, Section of Virology, Swedish University of Agricultural Sciences, BMC, Box 585, S-75 123 Uppsala (Sweden) 2Department of Molecular Biology, University of Brussels, B 1640 Rhode-Souint-Gen~se and Faculty of Agronomy, B 5800 Gembloux (Belgium) 3National Institute for Veterinary Research, B 1180 Vecle (Belgium) {Accepted 13 July 1987)
ABSTRACT
Fossum, C., Burny, A., Portetelle, D., Mammerickx, M. and Morein, B., 1988. Detection of B and T cells, with lectins or antibodies, in healthy and bovine leukemia virus-infected cattle. Vet. Immunol. Immunopathol., 18: 269-278. 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 immunoglobulinmolecules 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.
INTRODUCTION
Infection with the retrovirus bovine leukemia virus (BLV) induces the chronic disease enzootic bovine leukosis (EBL). Although BLV is believed to 0165-2427/88/$03.50
© 1988 Elsevier Science Publishers B.V.
270
infect the B lymphocytes (Paul et al., 1977) or a subpopulation thereof (Kenyon and Piper, 1977), antibodies to viral proteins are readily produced. An increased proliferation of a cell population bearing several B cell characteristics (Muscoplat et al., 1974; Wieland and Straub, 1975; Kenyon and Piper, 1977; Kumar et al., 1978) can, in 30-70% of the infected cattle, be manifested as a persistent lymphocytosis (PL). At a later stage the BLV infection gives rise to lymphoid tumors of a clonal B cell origin in 0.1-10% of the infected cattle (for reviews, see Burny et al., 1980, 1985). The implications of a key position for the B lymphocytes at BLV infections justify more detailed studies of bovine lymphocyte subpopulations. A reliable quantitation of bovine T and B cells has emerged from the extension of membrane markers for bovine cells during recent years with monoclonal antibodies (Pinder, 1980a,b; Lalor et al., 1986; Rabinovsky and Yang, 1986). Nevertheless, previously used markers, such as lectins, still have an important role in these studies. The binding of lectins is based on their specificity for sugars and can therefore reveal whether the lymphocytes alter the expression of sugar residues on their surface during maturation, activation and infection of the cells. In the present study we compared MAbs to subpopulations of bovine lymphocytes with lectins and polyclonal antibodies, in their binding to lymphocytes obtained from healthy cattle and from cattle at various stages of BLV infection. M A T E R I A L AND M E T H O D S
Animals The initial comparative studies on the binding of various lymphocyte markers were carried out on peripheral blood lymphocytes (PBL) isolated from 12 healthy animals of the Swedish Red and White breed. In the following study seven animals were included. Four were seropositive to BLV as measured by ELISA and had persistent lymphocytosis (BLV+PL + ). Two of the animals were seropositive to BLV but had no lymphocytosis ( B L V + P L - ). One animal, seronegative to BLV and with normal lymphocyte count ( B L V - P L - ) , was included as a control.
Isolation of P B L The lymphocytes were isolated by the Ficoll-Paque method initially described by BSyum (1968). Details for isolation of bovine mononuclear cells (MNC) were given previously {Johnsson and Morein, 1977). Briefly, the MNC were collected from the interface between Ficoll-Paque and plasma, generated after centrifugation of the blood. After three washings of the cells in phos-
271
phate-buffered saline (PBS) pH 7.2, supplemented with 0.2% human serum albumin (HSA) and 0.02% NAN3, the cells were counted and used in the labelling experiments.
Labelling techniques Approximately 3 × 106 MNC were used in each labelling experiment. Appropriate dilutions of the reagents were first established with MNC obtained from healthy cattle and then used throughout the experiment.
B cells. Two B cell markers were used; a fluoresceinated F (ab')2 fragment from polyclonal rabbit anti-bovine IgM serum and a mouse MAb to bovine IgM, designated B5.4 (Pinder et al., 1980b). The labelling procedure with the polyclonal F (ab')2 fragment has been described before (Johansson and Morein, 1983). Briefly, the MNC were incubated for 1 h at 37°C in serum-free medium to detach cytophilic antibodies (Watson, 1976) and washed twice in PBS, supplemented as described above, before 50/~l of the fluoresceinated F (ab')e antibodies to IgM were added. The cell suspension was incubated for 30 min at + 4 ° C with the antiserum, washed twice in PBS (supplemented) and fixed in 1.8% paraformaldehyde solution in PBS for 5 min at room temperature. After an additional washing the cells were ready for analysis. Labelling of B cells with the MAb B5.4 (kindly donated by Dr. M. Pinder, Gabon) was done with an indirect immunofluorescence (IF) technique. The first steps were performed similarly as for the polyclonal reagent. After washing away non-bound MAbs, the preparations were incubated with rhodamineconjugated goat F (ab')2 fragments of antibodies to mouse IgG heavy and light chains (Jackson Immuno-Research Laboratories, Inc. Avondale, PA) for another 30 min at ÷ 4 ° C. Thereafter the cells were washed and fixed as described above.
T cells. These were labelled with four different marker systems. Two MAbs, designated P5 and BLT-1 (kindly supplied by Dr. P. Lalor, ILRAD, Kenya and Dr. T. Yang, Storrs, U.S.A. ) and two lectins, Helix pomatia A hemagglutinin (HP) and Peanut agglutinin (PNA), were used. Labelling with the MAbs P5 and BLT-1 was performed with indirect IF as described above. The detection of receptors for the lectin HP was carried out as described before (Johansson and Morein, 1983 ). To reveal the receptors for HP, the cells were treated with 2/tg neuraminidase (Clostridium perfringens type VI, 1-3 IU/ml, Sigma) for 45 min at ÷ 4 ° C . After washing, the cells were incubated with fluorochrome-conjugated HP (direct method) or with HP followed by fluorochrome-conjugated F (ab') 2 antibodies to H P (indirect method ).
272
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Fig. 1. Two-parameter histogram generated by flow cytometry showing the distribution of bovine lymphocytes according to cell size (forward light scatter) on the horizontal axis and according to fluorescence intensity of the cells on the vertical axis. The lymphocytes were isolated from blood of a non-infected control animal and labelled with fluoresceinated antibodies to bovine IgM as described in Material and Methods. Exclusion limits for fluorescence positive and negative cells were set between channels 9 and 10 on the vertical axis and for small and large lymphocytes between channels 38 and 39 on the horizontal axis, dividing the histogram into four areas designated A-D. The B lymphocytes fall into areas B and C. Large B lymphocytes are found in area C. The border lines for fluorescence positive and negative cells and for small and large cells were established with cells from the non-infected animal and thereafter used for the analysis of lyrephocytes from the BLV-infected animals. L a b e l l i n g of cell m e m b r a n e r e c e p t o r s for P N A w a s p e r f o r m e d u n d e r n o n c a p p i n g c o n d i t i o n s w i t h direct I F a c c o r d i n g to P e a r s s o n et al. (1979). A f t e r w a s h i n g a n d fixation, t h e s p e c i m e n s were a n a l y s e d in a f l u o r e s c e n c e m i c r o s c o p e ( N i k o n , L a b o p h o t ) or b y flow c y t o m e t r y .
Flow cytometer analysis L y m p h o c y t e s labelled w i t h f l u o r e s c e i n a t e d a n t i b o d i e s to b o v i n e I g M as described a b o v e were a n a l y s e d in a flow c y t o m e t e r ( F A C S III, B e c t o n D i c k i n s o n ) e q u i p p e d w i t h a 5 - W a r g o n ion laser ( S p e c t r a P h y s i c s ). F o r e x c i t a t i o n t h e 488n m laser line was u s e d at a p o w e r of 250 m W . D a t a were a c c u m u l a t e d in t h e f o r m of h i s t o g r a m s t h a t c o r r e l a t e d f l u o r e s c e n c e i n t e n s i t y a n d f o r w a r d light s c a t t e r of t h e cells w i t h t h e n u m b e r of cells a n a l y s e d . T h e t w o - p a r a m e t e r hist o g r a m was divided into f o u r a r e a s w h i c h d i s c r i m i n a t e d p o s i t i v e l y s t a i n e d l y m p h o c y t e s f r o m t h e n e g a t i v e cells a n d s m a l l l y m p h o c y t e s f r o m large cells (Fig. 1 ). T h e p r o p o r t i o n of cells in e a c h a r e a was t h e n calculated.
273 RESULTS
Comparison of B cell markers in healthy animals The MAb B5.4 was compared with the polyclonal antibody to bovine IgM. The average proportion of B cells detected by the MAb B5.4 (16.2%) was close to that value detected by the polyclonal antibody (18.0% Ig + cells). Discrepancies between the two markers were found in individual lymphocyte samples, with a greater variation in the values of PBL samples stained positive with the MAb B5.4 (SD--8.2, n = 12 ) than in the values obtained with the polyclonal antibody (SD=4.0, n = 1 2 ) .
Comparison of T cell markers in healthy animals The two MAbs to bovine T cells, P5 and BLT-1, were compared in labelling experiments of bovine PBL with the indirect IF labelling with HP (anti-HP). The monoclonal antibodies attached to 66.1+3.8%, n--12 (P5) and 59.8_+ 7.1%, n = 5 (BLT-1). The average proportion of T cells as detected by indirect HP labelling of PBL from the same animals was in the same range (67.8+ 6.2%, n= 10, anti-HP + cells).
Elevation of the number orB and T lymphocytes in BL V-infected cattle The proportions of B and T lymphocytes were measured in BLV-infected and normal animals. Details are given in Fig. 2. The proportion of B cells detected was increased more than three times in the BLV-infected animals with lymphocytosis compared to non-infected animals, regardless of the B cell marker used. The proportion of T cells detected by the monoclonal antibody P5 was less than one-third of that in normal animals (Fig. 2). A decreased proportion of T cells was also recorded in the BLV-infected animals using the direct HP technique for their detection (Fig. 2 ), but a great variation was found between samples from different animals. Similarly, a great variation in the proportion of cells with HP receptors detected by the indirect HP method was recorded, with values ranging from 20 to 80% anti-HP positive cells compared to 60 to 75% in the normal animals (Fig. 2). One of the two cattle with antibodies to BLV but without lymphocytosis had an increased proportion of B lymphocytes, 49% and 58%, when tested on two occasions at an interval of 4 weeks. The corresponding values for the other BLV + P L - animal were 30% and 16% of B lymphocytes.
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Flow cytometer analysis of B lymphocytes from BL V-infected animals The samples labelled with fluoresceinated antibodies to bovine IgM were further analysed by flow cytometry (FCM). The proportion of cells identified as B lymphocytes by fluorescence microscopy and FCM analysis was in good agreement (r = 0.84). An increased intensity of the fluorescence was observed both by fluorescence microscopy and by FCM in a substantial proportion of the B lymphocytes from BLV-infected cattle compared to B lymphocytes from healthy cattle. The distribution of cells according to fluorescence intensity is visualized in the FCM-generated histogram (Fig. 3). From this diagram it can be seen that the Ig-positive cells obtained from BLV-infected cattle are distributed throughout the histogram (channels 1-64) while the fluorescence intensity of Ig-bearing cells isolated from the control animal did not exceed channel number 15. In the same histogram, the light scatter distribution, indicating the size of the cells, showed that 3.4 to 9.7% of the Ig-bearing cells obtained from B L V + P L + animals were enlarged. The corresponding value for the control animal was 0.3%.
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LIGHT SCATTER Fig. 3. Distribution of bovine lymphocytes, labelled with fluoresceinated antibodies to bovine IgM, in a histogram generated by FCM. The lymphocytes were obtained from one control animal (A), one animal seropositive to BLV but with normal lymphocyte count (B) and from two animals with antibodies to BLV and persistent lymphocytosis (C and D ). For evaluation of data see Material and Methods• DISCUSSION T h e lectins H P a n d P N A are reliable m a r k e r s for b o v i n e l y m p h o c y t e s origi n a t i n g f r o m h e a l t h y a n i m a l s ( M o r e i n et al., 1979; P e a r s s o n et al., 1979 ). T h a t was also c o n f i r m e d in t h e p r e s e n t work w h e r e a good a g r e e m e n t was f o u n d b e t w e e n t h e p r o p o r t i o n of cells d e t e c t e d b y i n d i r e c t i m m u n o f l u o r e s c e n c e with the lectin H P a n d with two m o n o c l o n a l antibodies, P 5 a n d B L T - 1 , b o t h claimed to detect bovine T l y m p h o c y t e s (Lalor et al., 1986; R a b i n o v s k y a n d Yang, 1986 ).
276 Similarly, both polyclonal and monoclonal antibodies to bovine IgM detected the same average proportion of B lymphocytes in blood from healthy cattle. In blood from BLV-infected cattle, these two B cell markers both indicated a strong increase in the number of B cells. In contrast, results obtained with the T cell markers HP and P5 did not correspond to each other when the markers were applied to lymphocytes obtained from BLV-infected cattle. While the MAb P5 detected a clear-cut decrease in the proportion o f T cells (x= 17% ) in BLV-infected cattle with a small variation between samples, the lectin HP indicated the same tendency with a lower mean value than in healthy cattle, but inconsistently and with great individual variations. As regards the indirect HP method, no clear-cut decrease in the proportion of T cells was found (Fig. 2 ). Further, the proportion of HP-positive cells plus the proportion of Ig-bearing lymphocytes exceeded 100% in samples from some of the BLV-infected cattle. Obviously, some Ig-bearing cells from BLV-infected animals also attach to the HP lectin, indicating that the surface of some of the B cells (HP+Ig + cells) found in BLV-infected animals has an altered sugar composition. These cells might be arrested at an early stage of maturation in accord with results from patients with chronic lymphatic leukemia (HellstrSm et al., 1976). Both the direct and indirect labelling procedures with HP include enzymatic removal of sialic acid to reveal the receptor for HP prior to addition of the lectin (see Material and Methods). The optimal concentration of neuraminidase needed to reveal the receptors for HP varies between species, and plateau values of the concentration of neuraminidase have been established for lymphocytes isolated from man (HellstrSm et al., 1976), rat (Swanborg et al., 1977), mouse (Haller et al., 1978), cattle (Morein et al., 1979), and horse (BrostrSm et al., 1985). No increase in the proportion of HP-positive cells is attained when lymphocytes from healthy cattle are treated with concentrations of neuraminidase up to ten times higher than the concentration selected as optimal (Morein et al., 1979). However, when lymphocytes obtained from BLV-infected cattle with lymphocytosis were tested, the proportion of HPpositive cells increased from 29 to 70%, from 50 to 62% and from 17 to 52%, even by doubling the selected concentration of neuraminidase. There was no increase in the proportion of HP-positive cells from the control animals by the same treatment (unpublished results). The lectin PNA was also tested at the standard dilution used for detection of bovine T cells. This concentration, however, caused aggregation of a substantial proportion of the lymphocytes obtained from BLV-infected animals (data not shown ), again indicating that these lymphocytes express an altered sugar composition on their surfaces compared to cells obtained from non-infected animals. Flow cytometer analysis of the lymphocytes labelled with fluoresceinated antibodies to bovine IgM confirmed the proportional results obtained from counting the cells in a fluorescence microscope. Again a greater individual vari-
277
ation in the proportion of B cells was obtained by the MAb than by the polyclonal antibodies which could be explained by a heterogeneity in the binding of B5.4 to serum IgM shown by Pinder et al. (1980b). Further, the fluorescence intensity of the B lymphocytes from the BLV-infected animals with lymphocytosis was proven be strongly elevated, indicating an increased number of surface immunoglobulin molecules. Also the proportion of "large" Ig-bearing cells was up to 30 times higher among blood lymphocytes from the BLV-infected animals than from the control animal, indicating an increased number of blastoid B lymphocytes in the blood from BLV-infected animals. From these results we conclude and confirm that the proportion of B cells increases and the proportion of T cells decreases following BLV infection (for review see Burny et al., 1980). Further, it should be taken into account that lectins are reliable markers only for the identification of T lymphocytes isolated from healthy cattle. In contrast to the lectins, the MAb P5 seems to recognize T cells with good reproducibility both in blood from cattle infected with BLV and in blood from healthy cattle. Thus, the accessibility of MAbs as markers for subsets of bovine lymphocytes has provided us with better tools to study lymphopoietic disorders in cattle. ACKNOWLEDGEMENTS
The monoclonal antibodies were kindly donated by Dr. Paul Lalor, ILRAD, Nairobi, Kenya (P5); Dr. Margaret Pinder, Gabon (B5.4) and Dr. Thomas Yang, Storrs (BLT-1). The flow cytometer analyses were carried out by Dr. Hugo Fellner-Feldegg, Department of Pathology, Swedish University of Agricultural Sciences, Uppsala. This work was supported by grants from the Swedish Council for Forestry and Agricultural Research.
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