Journal of Immunological Methods, 21 (1978) 197--210
197
© Elsevier/North-Holland Biomedical Press
S H E E P E R Y T H R O C Y T E R O S E T T E S IN PIGS, S H E E P , C A T T L E A N D G O A T S D E M O N S T R A T E D IN T H E P R E S E N C E O F D E X T R A N
R.M. BINNS Department of Immunology, A. R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, U.K.
(Received 19 August 1977, accepted 14 December 1977)
Formation of rosettes with sheep RBC by blood lymphocytes from young pigs was increased from 24.4% +- 2.2 (mean ± S.E.) in PBS to 54.1% +_1.9 in the presence of dextran. This increase was achieved without inducing appreciable rosette formation with other RBC which do not form rosettes in PBS. Lymphocytes which rosette only in dextran are predominant in pigs between 20 and 160 days old, when the peripheral lymphocyte pool is increasing very rapidly. The use of dextran revealed major populations of blood lymphocytes rosetting with SRBC in adult sheep (30.7% -+2.0), adult cattle (37.3% +-4.2) and adult goats (13.3% +-1.2). Proportions of rosette-forming lymphoeytes tended to increase with age. In calf lymphoid tissues the distribution of rosette-forming lymphocytes suggested that these were T cells. In the improvement of rosette formation with SRBC, dextran was more effective than foetal calf serum, papgin treatment of the SRBC or combinations of these treatments.
INTRODUCTION A high p r o p o r t i o n o f l y m p h o c y t e s o f several species f o r m r o s e t t e s with red b l o o d cells, usually o f a d i f f e r e n t species. F o r e x a m p l e l y m p h o c y t e s f r o m m a n and m o n k e y r o s e t t e with sheep, goat, pig and m o n k e y RBC ( C o o m b s et al., 1 9 7 0 ; K r e e f t e n b e r g and Leerling, 1 9 7 2 ; Bach, 1973; Terrell et al., 1 9 7 7 ) , dog with h u m a n and guinea pig RBC (Bowles et al., 1 9 7 5 ) , guinea pig with r a b b i t ( S t a d e c k e r e t al., 1 9 7 3 ; Wilson and C o o m b s , 1973), a n d cat with guinea pig, rat and m o u s e RBC ( T a y l o r e t al., 1975). H o w e v e r , r a b b i t l y m p h o c y t e s r o s e t t e with r a b b i t red b l o o d cells (Wilson et al., 1975). Where studied, r o s e t t e f o r m a t i o n has p r o v e d t o be a p r o p e r t y o f T l y m p h o cytes. R o s e t t e f o r m a t i o n was n o t e d b e t w e e n live pig l y m p h o c y t e s and sheep RBC (SRBC) in the c o u r s e o f studies o f surface Ig bearing l y m p h o c y t e s using t h e m i x e d antiglobulin test (Binns et al., 1 9 7 2 ) and o n analysis p r o v e d t o be a p r o p e r t y o f T l y m p h o c y t e s (Escajadillo and Binns, 1975a). B l o o d l y m p h o c y t e s f r o m r u m i n a n t s have n o t f o r m e d significant n u m b e r s o f r o s e t t e s with red cells f r o m a n y species. T h e i n t e r a c t i o n b e t w e e n h u m a n l y m p h o c y t e s and SRBC, the first t o be observed ( C o o m b s et al., 1 9 7 0 ) , is strong and d e t e c t s the m a j o r i t y o f T cells.
198 In some other species the percentage of lymphocytes forming rosettes m simple media with untreated erythrocytes is low and the interaction must be strengthened. Several enzymic, physical and chemical treatments of bott~ red cell and l y m p h o c y t e have been used to influence rosette formation i~ the improvement and analysis of the interactions, mainly for the human SRBC rosette (Bach, 1973; Dwyer, 1976). Care must be taken because somv treatments increase rosette numbers so that both B and T cells react. [~se of several such methods on pig SRBC rosettes will be considered elsewhere (m preparation). Mild papain treatment of the red cells increases rosette formation markedly with pig peripheral blood lymphocytes and still gives reactions only with sIg-negative ( " T " ) lymphocytes (Escajadillo and Binns, 1975b}. The numbers of rosette-forming cells (RFC) in each of the lymphoid tissues were increased but the relative numbers (thymus high ~ bone ram-row low~ were unchanged (unpublished). However, the presence of papain on ceil sur faces was undesirable when lymphocytes were prepared for culture and for surface marker experiments. It has been reported that dextran increases the interaction between human l y m p h o c y t e s and sheep RBC, while not causing rosette formation with B lymphoeytes (Brown et al., 1975). In this paper we confirm the value of dextran for increasing the interaction between lymphocytes and SRBC w i t h o u t altering the specificity of reaction. We demonstrate its value in detecting the majority of T lymphocytes in the pig and show that it can be used to reveal lymphoeytes with rosetting affinity for SRBC in ruminants. Subsequent papers will describe the confirmation of the t h y m u s dependence of rosetting lymphocytes. MATERIALS AND METHODS Young and adult pigs of the respiratory disease-free Babraham Large White herd were used. The sheep were non-pregnant adults of the Clun Forest, Finnish Landrace and Merino breeds. All but two were female, aged from 1.5 to 9.75 years. Sheep red cells were from the standard donor Clun Forest W60 used for all SRBC rosette studies. Babraham Jersey cattle and Saanen or Saanen cross-goats aged 0--10 years were also studied. Blood samples taken in pigs from the anterior vena cava or at slaughter and in sheep, cattle and goats from the jugular vein, were defibrinated by shaking with glass beads. Phagocytic cells were removed by incubation with carbonyl iron for 30--45 min (~15 mg/ml of blood turning on a Matburn rotator at 37°C). L y m p h o c y t e s were then isolated by our standard m e t h o d for pigs (Binns and Symons, 1974) and, for most sheep samples, by our usual m e t h o d (Symons and Binns, 1975). Briefly, these techniques were: the pig blood erythrocytes were sedimented with 0.25 vol 'Dextraven' 150 (Fisons) for 20--30 min at 37°C, the leucocyte-rich supernatant was removed and centrifuged (15 min at 5°C and ~ 4 0 0 × g) and the resulting pellet suspended in PBS and layered on Ficoll/
199 Triosil (F/T). After centrifuging at 600 X g for 30 min the l y m p h o c y t e layer on the F/T was removed, washed 3 times and suspended in medium (MEM/ HEPES Biocult) at 107/ml. For sheep and goats, 1 vol of blood was added to 5 vol of ammonium chloride/Tris (ACT) at 37°C, mixed and left for 3 min, and then 5 vol of phosphate-buffered saline pH 7.4 (PBS) at 4°C added. After centrifuging (at ~ 4 0 0 X g and 5°C for 15 min) the pellet, suspended in PBS was layered over F/T and a gradient made by drawing into a pipette ~ 0 . 5 ml each of F/T and supernatant and gently relayering. After centrifuging, the white layer of l y m p h o c y t e s (distinct from the ghost layer above) was harvested as for the pig. For some sheep samples the lyric step differed, a new technique developed for isolation of white cells in several species being employed (manuscript in preparation). Sheep blood l y m p h o c y t e s prepared by the glycerol and ACT methods gave similar results and accordingly the m e t h o d used is not differentiated for individual samples in the data. Viable white cells were prepared by incubating blood with glycerol PBS for 30 min at room temperature, centrifuging down the cellular elements and rapidly resuspending in PBS when the red cells lysed. The whole blood from these sheep was mixed with 5 vol of 4% glycerol in PBS (v/v) or the packed cells with 10 vol of 3.5% glycerol PBS. Lysis was with 10 vol of PBS. The l y m p h o c y t e s were then harvested from the lysate in the same-way as after ACT lysis. Bovine l y m p h o c y t e s were prepared from whole blood by the glycerol technique using 5 vol of 4.5% glycerol. Viability of cells by all techniques was high (typically >95%). L y m p h o c y t e s were prepared from lymphoid tissues by teasing into medium and centrifuging on F/T. Red cells were removed from sternal marrow and spleen suspensions by the glycerol lysis m e t h o d using 6% glycerol PBS. In our standard rosette test (modification of Escajadillo and Binns, 1975a) 1 drop of l y m p h o c y t e suspension at 1 X 107/ml was added to 2 drops of 2% SRBC in PBS or in the enhancing medium (6% dextran or FCS) in a narrow b o t t o m e d Dreyer agglutination tube, mixed vigorously, centrifuged at 200 X g for 5 min and incubated overnight ( ~ 1 8 h) at +4°C. When tests were read on the san~e day, the pellets had been left for more than 2 h at RT °. Rosette counts were made by removing the supernatant, gently resuspending the pellet in 4 drops fluorescein diacetate in PBS (Binns and Tonkin, 1974) with a pasteur pipette, placing 1.5 drops under a 20 mm 2 coverslip on a slide and counting the number of rosettes in 200--400 l y m p h o c y t e s by dark ground fluorescence microscopy. Rosette-forming l y m p h o c y t e s (RFC) were defined as bearing 5 or more adherent RBC. Usually duplicates were read and the results expressed as the mean. The agents used to enhance rosette formation were commercially available dextrans of molecular weights 40, 70, 110 and 150 X 103 at 6% in saline (Fison's " L o m o d e x 40", " L o m o d e x 70", "Dextraven 110" and "Dextraven
200
150"), foetal calf serum, both present during incubation of the lymphocytes and red cells, and Low's papain used to pretreat the SRBC (Escajadillo and Binns, 1975b). Values are shown as mean _+standard error (M ± S.E.). RESULTS
Enhancement by dextran of pig lymphocyte rosette for,nation with sheep RBC Preliminary tests using dextran and Ficoll indicated that dextran increases the proportion of pig blood l y m p h o c y t e s forming rosettes with SRBC. Dextran molecular weight and enhancement of rosette formation. The dextrans of different molecular weight between 70 and 150 × 103 gave similar good enhancement of rosette formation and 40 × 103 was only slightly less effective (Table 1). As in tests with SRBC in PBS, these tests with SRBC in dextran revealed a greater percentage of rosettes when read overnight ~nd the rosettes tended to be stronger than when read on the same day, an observation confirmed in subsequent use of the method. Dextravan 150 was used routinely in all other tests with dextran. Specificity of the increase in rosettes in dextran. The detection of large numbers of l y m p h o c y t e s rosetting with RBC in dextran was only found with RBC which form a significant n u m b e r of rosettes in saline. Thus of many species tested, only sheep and goat RBC, previously shown to rosette (Escajadillo and Binns, 1975a) detected a significant proportion of blood lymphocytes (Table 2). Table 3 shows that rosettes formed with another ruminant red cell, bovine RBC in dextran were 2 h RT °
Overnight
27 60 70 72 62
40 72 80 82 82
4°C
a F i n a l d e x t r a n concentration in all t e s t s = 4%.
201 TABLE 2 R O S E T T E F O R M A T I O N B E T W E E N PIG A N D G O A T L Y M P H O C Y T E S A N D V A R I O U S E R Y T H R O C Y T E S IN PBS A N D D E X T R A N Lymphocytes
Pig a Goat
Species RBC in Sheep PBS
Sheep DEX
Goat PBS
Goat DEX
Bovine DEX
Mouse DEX
50 0.5
76 a 28
25 < 0.3
75 8
2 5
1 1
a T h y m e c t o m i z e d pig: s u c h pigs lack null cells a n d h a v e a h i g h e r p r o p o r t i o n of d e x t r a n S R B C r o s e t t e - f o r m i n g l y m p h o c y t e s ( B i n n s et al., 1 9 7 7 ) .
strength of a pre-existing interaction between the l y m p h o c y t e and red cell (present with both SRBC and GRBC). Rosette formation by lymphocytes of 24 pigs of different ages with SRBC in PBS and in dextran PBS. Over a period of 18 months 28 blood samples were taken from 24 pigs varying from 7 to 300 days old. SRBC rosettes formed in PBS varied from 5 to 45% (M + S.E. 24.4 ± 2.2) and in dextran PBS from 28 to 67% (M +_S.E. 54.1 ± 1.9) of peripheral blood lymphocytes. Fig. 1 shows the percentage of blood l y m p h o c y t e s rosetting with SRBC in PBS and in dextran and the proportion of the latter rosetting in PBS (=S/DS) in relation to age at bleeding. This formula relating the two figures for SRBC rosette-forming cells is an index of the proportion of 'weak' and 'strong' rosette cells (S/DS = strong/weak + strong). The figure varied from 0.16 to 0.70 (0.445 + 0.034). Comparison of RFC in PBS (S) and in dextran (DS) in pigs of different age reveal various points on the subpopulations shown by this method. TABLE 3 R O S E T T E F O R M A T I O N BY P I G L Y M P H O C Y T E S WITH RBC IN D E X T R A N R E T A I N S SPECIFICITY FOR SRBC Lymphocytes from pig No.
Rosetted with S R B C in
6536 6538 B32 B34 a a Thymectomized. b B o v i n e RBC.
B R B C b in
PBS
Dextran
PBS
Dextran
5 12 36 58
31 50 66 80
197
© Elsevier/North-Holland Biomedical Press
S H E E P E R Y T H R O C Y T E R O S E T T E S IN PIGS, S H E E P , C A T T L E A N D G O A T S D E M O N S T R A T E D IN T H E P R E S E N C E O F D E X T R A N
R.M. BINNS Department of Immunology, A. R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, U.K.
(Received 19 August 1977, accepted 14 December 1977)
Formation of rosettes with sheep RBC by blood lymphocytes from young pigs was increased from 24.4% +- 2.2 (mean ± S.E.) in PBS to 54.1% +_1.9 in the presence of dextran. This increase was achieved without inducing appreciable rosette formation with other RBC which do not form rosettes in PBS. Lymphocytes which rosette only in dextran are predominant in pigs between 20 and 160 days old, when the peripheral lymphocyte pool is increasing very rapidly. The use of dextran revealed major populations of blood lymphocytes rosetting with SRBC in adult sheep (30.7% -+2.0), adult cattle (37.3% +-4.2) and adult goats (13.3% +-1.2). Proportions of rosette-forming lymphoeytes tended to increase with age. In calf lymphoid tissues the distribution of rosette-forming lymphocytes suggested that these were T cells. In the improvement of rosette formation with SRBC, dextran was more effective than foetal calf serum, papgin treatment of the SRBC or combinations of these treatments.
INTRODUCTION A high p r o p o r t i o n o f l y m p h o c y t e s o f several species f o r m r o s e t t e s with red b l o o d cells, usually o f a d i f f e r e n t species. F o r e x a m p l e l y m p h o c y t e s f r o m m a n and m o n k e y r o s e t t e with sheep, goat, pig and m o n k e y RBC ( C o o m b s et al., 1 9 7 0 ; K r e e f t e n b e r g and Leerling, 1 9 7 2 ; Bach, 1973; Terrell et al., 1 9 7 7 ) , dog with h u m a n and guinea pig RBC (Bowles et al., 1 9 7 5 ) , guinea pig with r a b b i t ( S t a d e c k e r e t al., 1 9 7 3 ; Wilson and C o o m b s , 1973), a n d cat with guinea pig, rat and m o u s e RBC ( T a y l o r e t al., 1975). H o w e v e r , r a b b i t l y m p h o c y t e s r o s e t t e with r a b b i t red b l o o d cells (Wilson et al., 1975). Where studied, r o s e t t e f o r m a t i o n has p r o v e d t o be a p r o p e r t y o f T l y m p h o cytes. R o s e t t e f o r m a t i o n was n o t e d b e t w e e n live pig l y m p h o c y t e s and sheep RBC (SRBC) in the c o u r s e o f studies o f surface Ig bearing l y m p h o c y t e s using t h e m i x e d antiglobulin test (Binns et al., 1 9 7 2 ) and o n analysis p r o v e d t o be a p r o p e r t y o f T l y m p h o c y t e s (Escajadillo and Binns, 1975a). B l o o d l y m p h o c y t e s f r o m r u m i n a n t s have n o t f o r m e d significant n u m b e r s o f r o s e t t e s with red cells f r o m a n y species. T h e i n t e r a c t i o n b e t w e e n h u m a n l y m p h o c y t e s and SRBC, the first t o be observed ( C o o m b s et al., 1 9 7 0 ) , is strong and d e t e c t s the m a j o r i t y o f T cells.
198 In some other species the percentage of lymphocytes forming rosettes m simple media with untreated erythrocytes is low and the interaction must be strengthened. Several enzymic, physical and chemical treatments of bott~ red cell and l y m p h o c y t e have been used to influence rosette formation i~ the improvement and analysis of the interactions, mainly for the human SRBC rosette (Bach, 1973; Dwyer, 1976). Care must be taken because somv treatments increase rosette numbers so that both B and T cells react. [~se of several such methods on pig SRBC rosettes will be considered elsewhere (m preparation). Mild papain treatment of the red cells increases rosette formation markedly with pig peripheral blood lymphocytes and still gives reactions only with sIg-negative ( " T " ) lymphocytes (Escajadillo and Binns, 1975b}. The numbers of rosette-forming cells (RFC) in each of the lymphoid tissues were increased but the relative numbers (thymus high ~ bone ram-row low~ were unchanged (unpublished). However, the presence of papain on ceil sur faces was undesirable when lymphocytes were prepared for culture and for surface marker experiments. It has been reported that dextran increases the interaction between human l y m p h o c y t e s and sheep RBC, while not causing rosette formation with B lymphoeytes (Brown et al., 1975). In this paper we confirm the value of dextran for increasing the interaction between lymphocytes and SRBC w i t h o u t altering the specificity of reaction. We demonstrate its value in detecting the majority of T lymphocytes in the pig and show that it can be used to reveal lymphoeytes with rosetting affinity for SRBC in ruminants. Subsequent papers will describe the confirmation of the t h y m u s dependence of rosetting lymphocytes. MATERIALS AND METHODS Young and adult pigs of the respiratory disease-free Babraham Large White herd were used. The sheep were non-pregnant adults of the Clun Forest, Finnish Landrace and Merino breeds. All but two were female, aged from 1.5 to 9.75 years. Sheep red cells were from the standard donor Clun Forest W60 used for all SRBC rosette studies. Babraham Jersey cattle and Saanen or Saanen cross-goats aged 0--10 years were also studied. Blood samples taken in pigs from the anterior vena cava or at slaughter and in sheep, cattle and goats from the jugular vein, were defibrinated by shaking with glass beads. Phagocytic cells were removed by incubation with carbonyl iron for 30--45 min (~15 mg/ml of blood turning on a Matburn rotator at 37°C). L y m p h o c y t e s were then isolated by our standard m e t h o d for pigs (Binns and Symons, 1974) and, for most sheep samples, by our usual m e t h o d (Symons and Binns, 1975). Briefly, these techniques were: the pig blood erythrocytes were sedimented with 0.25 vol 'Dextraven' 150 (Fisons) for 20--30 min at 37°C, the leucocyte-rich supernatant was removed and centrifuged (15 min at 5°C and ~ 4 0 0 × g) and the resulting pellet suspended in PBS and layered on Ficoll/
199 Triosil (F/T). After centrifuging at 600 X g for 30 min the l y m p h o c y t e layer on the F/T was removed, washed 3 times and suspended in medium (MEM/ HEPES Biocult) at 107/ml. For sheep and goats, 1 vol of blood was added to 5 vol of ammonium chloride/Tris (ACT) at 37°C, mixed and left for 3 min, and then 5 vol of phosphate-buffered saline pH 7.4 (PBS) at 4°C added. After centrifuging (at ~ 4 0 0 X g and 5°C for 15 min) the pellet, suspended in PBS was layered over F/T and a gradient made by drawing into a pipette ~ 0 . 5 ml each of F/T and supernatant and gently relayering. After centrifuging, the white layer of l y m p h o c y t e s (distinct from the ghost layer above) was harvested as for the pig. For some sheep samples the lyric step differed, a new technique developed for isolation of white cells in several species being employed (manuscript in preparation). Sheep blood l y m p h o c y t e s prepared by the glycerol and ACT methods gave similar results and accordingly the m e t h o d used is not differentiated for individual samples in the data. Viable white cells were prepared by incubating blood with glycerol PBS for 30 min at room temperature, centrifuging down the cellular elements and rapidly resuspending in PBS when the red cells lysed. The whole blood from these sheep was mixed with 5 vol of 4% glycerol in PBS (v/v) or the packed cells with 10 vol of 3.5% glycerol PBS. Lysis was with 10 vol of PBS. The l y m p h o c y t e s were then harvested from the lysate in the same-way as after ACT lysis. Bovine l y m p h o c y t e s were prepared from whole blood by the glycerol technique using 5 vol of 4.5% glycerol. Viability of cells by all techniques was high (typically >95%). L y m p h o c y t e s were prepared from lymphoid tissues by teasing into medium and centrifuging on F/T. Red cells were removed from sternal marrow and spleen suspensions by the glycerol lysis m e t h o d using 6% glycerol PBS. In our standard rosette test (modification of Escajadillo and Binns, 1975a) 1 drop of l y m p h o c y t e suspension at 1 X 107/ml was added to 2 drops of 2% SRBC in PBS or in the enhancing medium (6% dextran or FCS) in a narrow b o t t o m e d Dreyer agglutination tube, mixed vigorously, centrifuged at 200 X g for 5 min and incubated overnight ( ~ 1 8 h) at +4°C. When tests were read on the san~e day, the pellets had been left for more than 2 h at RT °. Rosette counts were made by removing the supernatant, gently resuspending the pellet in 4 drops fluorescein diacetate in PBS (Binns and Tonkin, 1974) with a pasteur pipette, placing 1.5 drops under a 20 mm 2 coverslip on a slide and counting the number of rosettes in 200--400 l y m p h o c y t e s by dark ground fluorescence microscopy. Rosette-forming l y m p h o c y t e s (RFC) were defined as bearing 5 or more adherent RBC. Usually duplicates were read and the results expressed as the mean. The agents used to enhance rosette formation were commercially available dextrans of molecular weights 40, 70, 110 and 150 X 103 at 6% in saline (Fison's " L o m o d e x 40", " L o m o d e x 70", "Dextraven 110" and "Dextraven
200
150"), foetal calf serum, both present during incubation of the lymphocytes and red cells, and Low's papain used to pretreat the SRBC (Escajadillo and Binns, 1975b). Values are shown as mean _+standard error (M ± S.E.). RESULTS
Enhancement by dextran of pig lymphocyte rosette for,nation with sheep RBC Preliminary tests using dextran and Ficoll indicated that dextran increases the proportion of pig blood l y m p h o c y t e s forming rosettes with SRBC. Dextran molecular weight and enhancement of rosette formation. The dextrans of different molecular weight between 70 and 150 × 103 gave similar good enhancement of rosette formation and 40 × 103 was only slightly less effective (Table 1). As in tests with SRBC in PBS, these tests with SRBC in dextran revealed a greater percentage of rosettes when read overnight ~nd the rosettes tended to be stronger than when read on the same day, an observation confirmed in subsequent use of the method. Dextravan 150 was used routinely in all other tests with dextran. Specificity of the increase in rosettes in dextran. The detection of large numbers of l y m p h o c y t e s rosetting with RBC in dextran was only found with RBC which form a significant n u m b e r of rosettes in saline. Thus of many species tested, only sheep and goat RBC, previously shown to rosette (Escajadillo and Binns, 1975a) detected a significant proportion of blood lymphocytes (Table 2). Table 3 shows that rosettes formed with another ruminant red cell, bovine RBC in dextran were 2 h RT °
Overnight
27 60 70 72 62
40 72 80 82 82
4°C
a F i n a l d e x t r a n concentration in all t e s t s = 4%.
201 TABLE 2 R O S E T T E F O R M A T I O N B E T W E E N PIG A N D G O A T L Y M P H O C Y T E S A N D V A R I O U S E R Y T H R O C Y T E S IN PBS A N D D E X T R A N Lymphocytes
Pig a Goat
Species RBC in Sheep PBS
Sheep DEX
Goat PBS
Goat DEX
Bovine DEX
Mouse DEX
50 0.5
76 a 28
25 < 0.3
75 8
2 5
1 1
a T h y m e c t o m i z e d pig: s u c h pigs lack null cells a n d h a v e a h i g h e r p r o p o r t i o n of d e x t r a n S R B C r o s e t t e - f o r m i n g l y m p h o c y t e s ( B i n n s et al., 1 9 7 7 ) .
strength of a pre-existing interaction between the l y m p h o c y t e and red cell (present with both SRBC and GRBC). Rosette formation by lymphocytes of 24 pigs of different ages with SRBC in PBS and in dextran PBS. Over a period of 18 months 28 blood samples were taken from 24 pigs varying from 7 to 300 days old. SRBC rosettes formed in PBS varied from 5 to 45% (M + S.E. 24.4 ± 2.2) and in dextran PBS from 28 to 67% (M +_S.E. 54.1 ± 1.9) of peripheral blood lymphocytes. Fig. 1 shows the percentage of blood l y m p h o c y t e s rosetting with SRBC in PBS and in dextran and the proportion of the latter rosetting in PBS (=S/DS) in relation to age at bleeding. This formula relating the two figures for SRBC rosette-forming cells is an index of the proportion of 'weak' and 'strong' rosette cells (S/DS = strong/weak + strong). The figure varied from 0.16 to 0.70 (0.445 + 0.034). Comparison of RFC in PBS (S) and in dextran (DS) in pigs of different age reveal various points on the subpopulations shown by this method. TABLE 3 R O S E T T E F O R M A T I O N BY P I G L Y M P H O C Y T E S WITH RBC IN D E X T R A N R E T A I N S SPECIFICITY FOR SRBC Lymphocytes from pig No.
Rosetted with S R B C in
6536 6538 B32 B34 a a Thymectomized. b B o v i n e RBC.
B R B C b in
PBS
Dextran
PBS
Dextran
5 12 36 58
31 50 66 80
