Br. vet. }. (1977 ), 133-,432
SHORT COMMUNICATION
AN IMPROVED E ROSETIING TECHNIQUE FOR CATILE By R. WARDLEY
The Animal Virus Research Institute, Pirbright, Woking, Surrey
SUMMARY
Using media containing 6% w/v dextran, 70% of bovine peripheral blood leukocytes formed E rosettes with sheep red blood cells. The number of rosetting cells found in enriched populations of thymus-derived lymphocytes indicates that the rosetting cells are T lymphocytes. INTRODUCTION
Thymus-derived lymphocytes (T cells) can be distinguished from other lymphocytes and mononuclear cells by their ability to spontaneously bind heterologous erythrocytes (E rosettes). This immunologically non-specific characteristic differs from the binding of B lymphocytes to specifically sensitized erythrocytes. Recently an E rosette marker was established for bovine T cells, although it only labelled up to 38% of peripheral blood leukocytes (PBL) as T cells (Grewal, Rouse & Babiuk, 1976) compared to about 70% in other species. This report describes an improved technique which identifies up to 70% of bovine PBL as presumptive T cells. MATERIALS AND METHODS
PBL were prepared from heparinized venous blood, taken from adult Devon steers, by ficoll-hypaque flotation and carbonyl iron depletion as described previously (Rouse, Wardley & Babiuk, 1976). Foetal thymocytes were prepared using the method of Grewal et at. (1976). PBL for rosetting were resuspended in HEPES buffered (20 mM) RPMI 1640 containing 10% heat inactivated foetal calf serum (FCS) at 10 7 cells per m!. Sheep red blood cells (SRBC) collected in heparin (10 i.u./mi) were washed threetimes in phosphate buffered saline (PBS) and used within one week. For rosetting 200 ~l of packed SRBC were added to 5 ml of 6% w/v dextran in 0·9% NaCI (Dextraven 150, Fisons Ltd, Loughborough, England) and 200 ~l of this suspension was mixed with 100 ~l of lymphocyte suspension, incubated for various periods, centrifuged at 200 g for 5 min in conical centrifuge tubes and stored overnight at 4°C. Before these pellets were resuspended the supernatant was drawn off and 1 ml of PBS containing fluorescein diacetate (Koch-Light Lab., U.K.) was added. (A stock solution of fluorescein diacetate in acetone, 10 mg/ml, was stored at - 20°C and 100 ~l of this was added to 20 ml of PBS at 4°C just prior to use). The cell pellet was gently resuspended
AN IMPROVED E ROSElTING TECHNIQUE FOR CAlTLE
433
by a slow stream of air bubbles introduced on to its surface by a Pasteur pipette. Individual slides were prepared and read immediately using a combination of incident UV illumination and transmitted white light. Under such conditions lymphocytes fluoresce brightly, easily distinguishing them from the SRBC. Lymphocytes with three or more SRBC adhering to their surfaces were considered as a rosette. Each sample was set up in duplicate and two slides were prepared from each pellet. At least 300 cells per slide were counted.
RES ULTS
The results are summarized in Tables I and II. As can be seen from Tabl e I, incubation prior to-tentrifugation affected the percentage of E rosettes formed . There appeared to be no difference between incubating at 37°C or 22°C for up to 30 min, whereas at 60 min rosette numbers decreased. At 4°C a consistently lower number of rosettes were found for all the incubation periods used . It was therefore decided to perform all subsequent assays using a 10 min incubation at 37° C except where membrane fluorescence of rosetted cells was to be examined in the same preparation. Here a 10 min period at 22°C was used (Grewal et ai., 1976 ).
TABLE! EFFECT OF TIME
AND TEMPERATURE
OF INC UBATION
ON THE
PERCENTAGE OF E ROSETTES FORMED BY BOVINE PBL
Percentage· of E rosettes
Temperature of incubation
37°C 22°C 4°C
IOmint
30min
60 min
72 (65-75) 67 (63-69) 54 (49-56)
69 (6 4-72) 70 (65-73 ) 56 (50-61)
53 (40-58) 48 (37 -52) 54 (5 1-57 )
* mean of four experiments, brackets give range.
t
time of incubation prior to centrifugation and overnight incubation at 4°C.
From the data presented in Table II it appears that the rosetted lymphocyte is probably a T cell. Only 0 ·5% rosetting cells have surface Ig. Nylon wool efRuent cells show an increased number of E rosettes and up to 99% of thymocytes form rosettes. Neuraminidase treatment of SRBC gave fewer rosettes, probably because the pellets were then more difficult to resuspend and rosettes might have been disrupted in the process. Monitoring of PBL from six other steers gave E rosette counts ranging from 66·9 to 78 ·5%, mean 72 ·0%.
BRITISH VETERINARY JOURNAL, 133 , 4
434
TABLE II CHARACTERISTICS OF DIFFERENT LYMPHOCYTE POPULATIONS
%
Cell type
% MF
E rosettes 71 *
PBL Nylon wool effiu ent cellt Nylon woo l adherent cellt Thymocyte PBL (neuraminidase treated SRBC):j:
(68-74) 85 (82-87 ) 49 (46-53 ) 96 (94-99 ) 52 (48-57)
21 (J8- 23 ) 6
% + ve rosettes +veMF
0·5 NO
(4-10) 43 (40-45 ) 0 ·5
NO
NO
NO
NO
MF Membrane fluorescence. * mean of four experiments ; brackets give range. NO not done. t prepared according to julius, Simpson & Hertzenberg (1973 ). :j: neura minidase treatment after Grewal et at. (1976).
DISCUSSION
A recently described method used for determining the number of E rosettes in bovine PBL preparations employed neuraminidase treated SRBC and a rosetting medium of 100% FCS (Grewal et at., 1976). Under such conditions, less than 40% of PBL form ed rosettes and it also appeared that this number varied depending upon the batch of FCS used (Grewal et aI., 1976 ). In the test described in this paper untreated SRBC and a rosetting medium of 6% dextran were used . About 70% of bovine PBL then formed E rosettes. This figure is comparable with that found in other species. This improved technique (i) requires less time to perform, as the SRBC do not have to be pretreated with neuraminidase, (ii ) overcomes the unreliability of different FCS batches and (iii ) consistently identifies 30% more PBL as presumptive T cells. To provide further evidence that these cells are indeed T cells, functional mitogen assays and the demonstration of helper effects will be needed. ACKNOWLEDGEMENTS
I would like to thank Dr R. Binns for suggesting the use of Dextraven 150. REFERENCES GR EWAL, A. S . , RO USE, B. T. & BABIUK, L. A. (1976). Can.]. compo Med. 40, 298. J ULI US , M. H ., SIMPSON, E. & HERTZENBERG, L. A. (J973). Eur.]. Immunol. 3, 645. RO USE , B. T., WARDLEY, R. C. & BABJUK, L. A. (1976 ). Infec. Immunity, 13, 1433.
(Acceptedfor publication.3 March J9 77)
SHORT COMMUNICATION
AN IMPROVED E ROSETIING TECHNIQUE FOR CATILE By R. WARDLEY
The Animal Virus Research Institute, Pirbright, Woking, Surrey
SUMMARY
Using media containing 6% w/v dextran, 70% of bovine peripheral blood leukocytes formed E rosettes with sheep red blood cells. The number of rosetting cells found in enriched populations of thymus-derived lymphocytes indicates that the rosetting cells are T lymphocytes. INTRODUCTION
Thymus-derived lymphocytes (T cells) can be distinguished from other lymphocytes and mononuclear cells by their ability to spontaneously bind heterologous erythrocytes (E rosettes). This immunologically non-specific characteristic differs from the binding of B lymphocytes to specifically sensitized erythrocytes. Recently an E rosette marker was established for bovine T cells, although it only labelled up to 38% of peripheral blood leukocytes (PBL) as T cells (Grewal, Rouse & Babiuk, 1976) compared to about 70% in other species. This report describes an improved technique which identifies up to 70% of bovine PBL as presumptive T cells. MATERIALS AND METHODS
PBL were prepared from heparinized venous blood, taken from adult Devon steers, by ficoll-hypaque flotation and carbonyl iron depletion as described previously (Rouse, Wardley & Babiuk, 1976). Foetal thymocytes were prepared using the method of Grewal et at. (1976). PBL for rosetting were resuspended in HEPES buffered (20 mM) RPMI 1640 containing 10% heat inactivated foetal calf serum (FCS) at 10 7 cells per m!. Sheep red blood cells (SRBC) collected in heparin (10 i.u./mi) were washed threetimes in phosphate buffered saline (PBS) and used within one week. For rosetting 200 ~l of packed SRBC were added to 5 ml of 6% w/v dextran in 0·9% NaCI (Dextraven 150, Fisons Ltd, Loughborough, England) and 200 ~l of this suspension was mixed with 100 ~l of lymphocyte suspension, incubated for various periods, centrifuged at 200 g for 5 min in conical centrifuge tubes and stored overnight at 4°C. Before these pellets were resuspended the supernatant was drawn off and 1 ml of PBS containing fluorescein diacetate (Koch-Light Lab., U.K.) was added. (A stock solution of fluorescein diacetate in acetone, 10 mg/ml, was stored at - 20°C and 100 ~l of this was added to 20 ml of PBS at 4°C just prior to use). The cell pellet was gently resuspended
AN IMPROVED E ROSElTING TECHNIQUE FOR CAlTLE
433
by a slow stream of air bubbles introduced on to its surface by a Pasteur pipette. Individual slides were prepared and read immediately using a combination of incident UV illumination and transmitted white light. Under such conditions lymphocytes fluoresce brightly, easily distinguishing them from the SRBC. Lymphocytes with three or more SRBC adhering to their surfaces were considered as a rosette. Each sample was set up in duplicate and two slides were prepared from each pellet. At least 300 cells per slide were counted.
RES ULTS
The results are summarized in Tables I and II. As can be seen from Tabl e I, incubation prior to-tentrifugation affected the percentage of E rosettes formed . There appeared to be no difference between incubating at 37°C or 22°C for up to 30 min, whereas at 60 min rosette numbers decreased. At 4°C a consistently lower number of rosettes were found for all the incubation periods used . It was therefore decided to perform all subsequent assays using a 10 min incubation at 37° C except where membrane fluorescence of rosetted cells was to be examined in the same preparation. Here a 10 min period at 22°C was used (Grewal et ai., 1976 ).
TABLE! EFFECT OF TIME
AND TEMPERATURE
OF INC UBATION
ON THE
PERCENTAGE OF E ROSETTES FORMED BY BOVINE PBL
Percentage· of E rosettes
Temperature of incubation
37°C 22°C 4°C
IOmint
30min
60 min
72 (65-75) 67 (63-69) 54 (49-56)
69 (6 4-72) 70 (65-73 ) 56 (50-61)
53 (40-58) 48 (37 -52) 54 (5 1-57 )
* mean of four experiments, brackets give range.
t
time of incubation prior to centrifugation and overnight incubation at 4°C.
From the data presented in Table II it appears that the rosetted lymphocyte is probably a T cell. Only 0 ·5% rosetting cells have surface Ig. Nylon wool efRuent cells show an increased number of E rosettes and up to 99% of thymocytes form rosettes. Neuraminidase treatment of SRBC gave fewer rosettes, probably because the pellets were then more difficult to resuspend and rosettes might have been disrupted in the process. Monitoring of PBL from six other steers gave E rosette counts ranging from 66·9 to 78 ·5%, mean 72 ·0%.
BRITISH VETERINARY JOURNAL, 133 , 4
434
TABLE II CHARACTERISTICS OF DIFFERENT LYMPHOCYTE POPULATIONS
%
Cell type
% MF
E rosettes 71 *
PBL Nylon wool effiu ent cellt Nylon woo l adherent cellt Thymocyte PBL (neuraminidase treated SRBC):j:
(68-74) 85 (82-87 ) 49 (46-53 ) 96 (94-99 ) 52 (48-57)
21 (J8- 23 ) 6
% + ve rosettes +veMF
0·5 NO
(4-10) 43 (40-45 ) 0 ·5
NO
NO
NO
NO
MF Membrane fluorescence. * mean of four experiments ; brackets give range. NO not done. t prepared according to julius, Simpson & Hertzenberg (1973 ). :j: neura minidase treatment after Grewal et at. (1976).
DISCUSSION
A recently described method used for determining the number of E rosettes in bovine PBL preparations employed neuraminidase treated SRBC and a rosetting medium of 100% FCS (Grewal et at., 1976). Under such conditions, less than 40% of PBL form ed rosettes and it also appeared that this number varied depending upon the batch of FCS used (Grewal et aI., 1976 ). In the test described in this paper untreated SRBC and a rosetting medium of 6% dextran were used . About 70% of bovine PBL then formed E rosettes. This figure is comparable with that found in other species. This improved technique (i) requires less time to perform, as the SRBC do not have to be pretreated with neuraminidase, (ii ) overcomes the unreliability of different FCS batches and (iii ) consistently identifies 30% more PBL as presumptive T cells. To provide further evidence that these cells are indeed T cells, functional mitogen assays and the demonstration of helper effects will be needed. ACKNOWLEDGEMENTS
I would like to thank Dr R. Binns for suggesting the use of Dextraven 150. REFERENCES GR EWAL, A. S . , RO USE, B. T. & BABIUK, L. A. (1976). Can.]. compo Med. 40, 298. J ULI US , M. H ., SIMPSON, E. & HERTZENBERG, L. A. (J973). Eur.]. Immunol. 3, 645. RO USE , B. T., WARDLEY, R. C. & BABJUK, L. A. (1976 ). Infec. Immunity, 13, 1433.
(Acceptedfor publication.3 March J9 77)
