Journal of Immunological Methods, 26 (1979) 1--10
1
© Elsevier/North-Holland Biomedical Press
PURIFICATION OF RABBIT B SPLEEN CELLS BY REMOVAL OF A D H E R E N T A N D OF T CELLS
JEAN-MARC CAVAILLON, B E R N H A R D CINADER, CHI-TAO CHOU and STANISLAW DUBISKI
Institute o f Immunology, Medical Sciences Building, University of Toronto, Toronto, Ont. M5S 1A8, Canada (Received 5 June 1978, accepted 28 August 1978)
B lymphocytes from the rabbit spleen were freed of T cells by removal of cells which formed rosettes with papain-treated rabbit erythrocytes. Additional purification could be achieved if fractionation by rosette removal was preceded by removal with a magnet of cells which adhered to or ingested poly L-lysine coated iron core particles. Cell yield and purification were assessed by complement mediated cytotoxic kill of B and T cells with antibody directed against R A B E L A and RTLA, respectively. Other criteria depehded on determination of the number of Fc receptor bearing cells and of thymidine uptake by cells which were stimulated with concanavalin A, PHA or with antibody directed against the allotypic specificity of receptor Ig light chains. Purified preparations of B cells were obtained in a yield of about 20% of the B cells in the original spleen and contained less than 10% of cells which were not B cells. This method allows purification which does not interfere with the membrane of the isolated cells.
INTRODUCTION
The analysis of the rabbit's lymphoid system is dependent on the developm e n t of reagents which are highly specific for individual cell populations (Fradelizi et al., 1973; Redelman et al., 1976; Wilson et al., 1977; Chou et al., 1977) and on techniques which permit isolation of cells by processes which minimize stimulation or inhibition of the isolated cells. Several methods have been used for purification of or enrichment with T cells, e.g. B cells have been removed on nylon wool (Redelman et al., 1976; Krawinkel et al., 1977), by rosetting with anti-Ig antibody-coated sheep red blood cells, or by mixed agglutination technique (Teodorescu et al., 1976; Cavaillon et al., 1977; Jensenius et al., 1977). B cells have also been removed by passage Abbreviations: BUSC, B cells isolated from unfractionated spleen cells;
B N A , B cells isolated from spleen cells from which adherent cells were removed; RTLA, rabbit thymus l y m p h o c y t e antigen; RABELA, rabbit bursal equivalent lymphocyte antigen; Con A, concanavalin A; PHA, phytohaemagglutinin; EA rosettes, rosettes between rabbit spleen cells and sheep erythrocytes coated with rabbit IgG antibody directed against the sheep erythrocytes.
over beads coated with antibody directed against Ig or against allotypic specificity (Bell and Wigzell, 1977; Rivas-Alcala et al., 1979). B cells have been purified by isolation of Ig+ cell rosettes (Teodorescu et al., 1976), and removal of T cells, lysed with complement and antibody, specific for T cells, such as RTLA antisera (Shek et al., 1976; l~edelman et al., 1976; Teodorescu et al., 1976). We shall present, in this paper, a m e t h o d by which rabbit B cells can be produced in reasonable yield and fairly free of T cells and macrophages. This technique is based on the discovery by Wilson et al. (1975) that papain-treated rabbit erythrocytes rosetted with T cells. We could adapt this analytical approach to a procedure for purification by removing T cell rosettes on Ficoll-Hypaque gradients and thus recovering B cells which do not rosette. Highly purified B cell preparations were obtained, if spleen cell suspensions were freed of adherent cells, prior to rosetting, and were then freed of T cells. MATERIALS AND METHODS
Rabbits New Zealand white rabbits (Riemens Fur Rancher Ltd., St. Agathe, Ontario) of either sex, 3--6 months old, allotype b4/b4, were employed. Rabbit red blood cells (RRBC) Blood was collected on 1000 U.S.P. units/ml of heparin (Organon, Canada) from the artery of the rabbit's ear and washed 3X in PBS pH 7.2; care was taken to remove leucocytes. Preparation of spleen lymphocyte suspensions Rabbits were killed by i.v. injection of Nembutal (Abbott Lab., Montreal). Spleens were excised and rinsed in modified McCoy's 5a medium (MCM; Grand Island Biological Company, Grand Island, NY). Spleens were cut into small pieces, gently pressed in MCM and the suspension was passed through a sterile 80 mesh stainless steel screen, were centrifuged for 8 min at 1100 rev/ min and room temperature and the cells were washed in MCM. Non-adherent cells (NA ) NA cells were prepared, as described by Shek et al. (1974). In brief, spleen cells were freed, with a magnet, of cells which adhered to or ingested iron core particles, coated with poly L-lysine (Technicon Corp., Tarrytown, NY 10591); the remaining cells are designated as NA cells (Rivas Alcala et al., 1979). Papain treatment of rabbit red blood cells (RRBC) RRBC were treated with papain as described by Wilson et al. (1975). Briefly, after washing, the erythrocytes were resuspended to 20% v/v in PBS and an equal volume of a mixture was added which consisted of I part
0.25% papain (N.B.C. Lab., Cleveland, OH) in PBS, 1 part Na2HPO4 (3.6 g in 100 ml distilled water) and 2 parts 0.2% L-cysteine HC1. RRBC were incubated at 37°C for 18 min, were then centrifuged, washed once with PBS, twice with RPMI-1640 medium (Gibco Lab., Grand Island, NY) and were finally resuspended to 0.7% v/v in PBS. Removal o f lymphoid cells which form rosettes with papain-treated rabbit ery th rocy tes We employed a m e t h o d based on an analytical technique devised by Wilson et al. (1975). Spleen cell suspension (3 ml, 6 × 106 cells/ml in RPMI1640) were mixed with papain-treated rabbit erythrocytes (3 ml, 0.7%) and with 3 ml of RPMI-1640 supplemented with 15% fetal calf serum (FCS). Thereafter, the cell mixture was centrifuged for 4 min at 1700 rev/min and 4°C; the centrifuged mixture was kept for 15 min at 4 ° C. Cells pelleted from the centrifuged mixture were gently resuspended in the original fluid, pooled and kept at 4°C; 1.8 ml per tube of FCS were added. In r o u n d - b o t t o m e d test tubes (17 mm × 100 mm, Falcon, Oxnard, CA, U.S.A.), the cell suspension (7 ml) was carefully layered on a Ficoll-Hypaque mixture (3 ml of 25 parts of 12% Ficoll, Pharmacia, Uppsala, Sweden, and 10 parts of 33% Hypaque, Winthrop, Aurora, Ontario, Canada; final density at room temperature = 1.08 g/ml + 5 × 10-4). The tubes were centrifuged for 25 min at 4°C and 1500 rev/min. Lymphocytes, depleted of rosettes, were recovered at the interphase and collected with a Pasteur pipette. The recovered cells were centrifuged for 5 min at 4°C and 1800 rev/min, and were washed twice in RPMI medium. Determination o f T cell content with R T L A antiserum R T L A antiserum was raised by immunization of goats with rabbit thymus cells and was made T-cell specific by suitable absorption (Fradelizi et al., 1973). By means of immunofiuorescence, it could be shown that RTLAbearing cells were localized in thymus-dependent areas of the spleen (Fujiwara et al., 1974). Complement-mediated cell kill with I~TLA antiserum did n o t reduce B cell responses, i.e., did not interfere with thymidine incorporation after stimulation with antibody against light-chain allotypic specificity (Shek et al., 1974) or with polyclonal activation by a B mitogen extracted from Nocardia (Bona et al., 1975). On the other hand, cell kill of RTLAbearing cells abolished response to phytohaemagglutinin and concanavalin A. R T L A antiserum was employed for complement-mediated c y t o t o x i c kill of T cells; dead cells were identified and counted by Trypan blue exclusion test, as described by Fradelizi et al. (1973). Determination o f B cell content with R A B E L A antiserum R A B E L A antiserum was raised by immunization of goats with rabbit appendix cells and was absorbed so as to make it specific for B cells (Chou et al., 1977). The specificity of R A B E L A antiserum does not overlap with that
of RTLA antiserum since the arithmetic sum of per cent cell kill by RABELA antiserum alone, and by RTLA antiserum, alone, is equal to the per cent of cell kill by a mixture of the two antisera (Chou et al., 1977). Finally, the percentage of RABELA positive and of Ig-bearing spleen cells is in good agreement. RABELA antiserum was employed for complement mediated c y t o t o x i c i t y kill of B cells, as described in an earlier publication (Chou et al., 1977).
Determination of macrophage number Macrophages were enumerated by counting cells stained by the m e t h o d of Tucker et al. (1977). Stimulation of lymphocytes with lectins and with antibody against Ig receptors o f B cells Cells were stimulated with Con A and PHA in Multi-Dish Disposo Trays (Linbro Scientific, Inc., Hamden, CT): each well contained 2 × 105 cells in RPMI medium supplemented with 20% normal rabbit serum b4/b5. Stimulation with antibody directed against allotypic specificity b4 (Chou et al., 1974) was carried out in the presence of normal rabbit serum b9/b9; the total serum content was 0.2 ml/ml of mixture. Optimal dilutions of lectins were 30 pg/well of Con A (Miles Yeda Ltd., Kankakee, IL), 3 pg/well of PHA (Difco Lab., Detroit, MI); anti-4 (prepared as described by Dubiski (1969)) was used in a final dilution of 1 : 10. After 24 h of incubation at 37°C in an atmosphere of 5% CO2 and 95% air, [3H]thymidine (0.25 pCi/ well; specific activity 20 Ci/mmol) (Amersham-Searle Corp.) was added and the incubation was continued for a further 24 h. Thereafter, cells were harvested on glass fibre filters with a Skatron machine (Flow Lab., Rockville, MD, U.S.A.). Thymidine incorporation was determined by liquid scintillation counting and was expressed as net cpm, i.e., as counts in the presence of mitogen less counts in the absence of mitogen. EA rosettes The percentage of Fc receptor-bearing cells was determined by enumeration of EA rosettes. Rabbit IgG antibody directed against sheep red blood cells (SRBC) was prepared from antisera by precipitation with 18% sodium sulphate and subsequent chromatography on DEAE-cellulose (Weir, 1973). SRBC were exposed to a subagglutinating dose of this rabbit anti-SRBC IgG for 30 min at 37°C. After incubation, the sensitized SRBC were washed in PBS and resuspended to 1% v/v in RPMI-1640 medium. L y m p h o c y t e s (106 cells in 0.2 ml) were mixed with 0.2 ml of the sensitized SRBC and the cell mixture was centrifuged at 4°C for 5 min at 800 rev/min. The centrifuged cells were kept for 1 h at 4°C before the pellet was resuspended. Rosetteforming cells (RFC) were defined as being attached to 3 or more SRBC; such rosettes were not observed when lymphocytes were mixed with untreated SRBC. Rosettes were counted after addition of trypan blue.
RESULTS In adapting the analytical technique for T cell enumeration with papaintreated isologous red cells (Wilson et al., 1975), two technical problems had to be solved: we had to scale up the cell numbers and find a density at which we would cleanly separate rosetted cells from those which did not rosette. With equal volumes of l y m p h o c y t e suspensions of 6 × 106 cells/ml and with 0.7% papain-treated rabbit erythrocyte suspensions, the yield of B cells remained constant as between 6 and 60 × 106 cells per mixture of a final volume of 3--30 ml, respectively. Separation of rosettes from non-rosetted cells was carried out by centrifugation on a Ficoll-Hypaque or L y m p h o p r e p mixture. It was our aim to select a density at which rosetted cells would be at the b o t t o m of the tube and the cells which had not rosetted would collect, in good yield, in the interphase. At densities of 1.08 g/ml (Ficoll-Hypaque), the yield of non-rosetted cells, obtained from unfractionated spleen cells was 17 + 5%, whereas at a density of 1.077 g/ml (Lymphoprep, Nyegard and Co, Norway) it was 7.5%; the higher density and Ficoll-Hypaque were chosen for routine purification of B cells. The experiment arrangement finally selected is described in Materials and Methods. In the following, we shall characterize the cells which are obtained by removing rosetted cells either from unfractionated spleen cells (USC) or from suspensions which had been freed of cells that adhered to or ingested poly L-lysine-coated magnetic particles (NA).
Enumeration o f B and T cells by c o m p l e m e n t mediated cytotoxic kill B cells were isolated either from untreated suspensions of spleen cells (USC) or from suspensions from which adherent cells were removed (NA); the B cell preparations were designated Bvsc o r BNA , respectively. The T cell content, as established by complement mediated cytotoxic kill with RTLA antiserum, was 5 -+ 3% in Bvsc and 4 -+ 4% in BNA. The B cell c o n t e n t of these two preparations, as determined with RABELA antiserum, was 89 + 6% for Busc and 91 -+ 7% for BNA (Fig. 1). This is an approximately 10-fold reduction in T cells by comparison with USC which contained 39-+ 11% RTLA positive cells and 56 -+ 8% RABELA positive cells. While there is no significant difference in the T and B cell c o n t e n t of Bvsc and BNA, there was a difference in 60% of the cases when the comparison was made between preparations from the same spleen cells; the content of RABELA positive cells was somewhat higher in the BNA than in the Bus c (Fig. 1). Bus c and BNA were obtained in a yield of 26 -+ 7% and 21 -+ 7%, respectively, of the B cells, contained in unfractionated starting material. The relative low yield of BNA was due to a loss of 47 -+ 7% in the step in which adherent cells were removed. Mitogen-induced thymidine uptake as a measure o f cell purification Since concanavalin A (Con A) and phytohaemagglutinin (PHA) stimulate
V
USC
2 tO
W 10
u) ..Q
0
Busc
8 6 4
..Q
E -'I z
2
10! 8
V
6
BNA
4
2 i
40
,
,
60
e
80
100
% RABELA positive cells
0
20
40
60
% RTLA positive cells
Fig. 1. Rabbit B and T cells in unfractionated and in separated subpopulations of spleen cells. Spleen cells were freed of cells which formed rosettes with papain-treated rabbit erythrocytes. The cells were treated with rabbit complement, antiserum against RABELA or RTLA and cytotoxic kill was measured and plotted as a percentage against the number of animals in which this percentage of RABELA or RTLA bearing cells was found. The point of the arrow indicates mean values; the width of the arrow is equal to 2 S.D.
r a b b i t T derived l y m p h o c y t e s ( O z e r and W a k s m a n , 1 9 7 4 ; S h e k et al., 1 9 7 4 , 1 9 7 5 , 1 9 7 6 a ) a n d since a n t i b o d y d i r e c t e d against light-chain a l l o t y p i c m a r k e r b 4 s t i m u l a t e s B derived l y m p h o c y t e s (Fradelizi et al., 1 9 7 3 ; C h o u et al., 1 9 7 4 ; S h e k et al., 1 9 7 6 b ) , it is possible to c o m p a r e t h e B a n d T cell cont e n t o f t w o p o p u l a t i o n s in t e r m s o f lectin o r a n t i b o d y - i n d u c e d t h y m i d i n e i n c o r p o r a t i o n . We h a v e d o n e so, b y e x p o s i n g c o n s t a n t n u m b e r s o f Busc, BNA and USC t o e a c h o f t h e s e agents; t h e u n t r e a t e d spleen cells w e r e first passed t h r o u g h a Ficoll gradient, similar to t h a t used f o r r o s e t t e d e p l e t i o n , so as t o e l i m i n a t e this p r e p a r a t i v e step f r o m a f f e c t i n g t h e c o m p a r i s o n b e t w e e n s t a r t i n g m a t e r i a l a n d t h e cells isolated f r o m it. T h y m i d i n e u p t a k e , a f t e r s t i m u l a t i o n w i t h a n t i b o d y d i r e c t e d against b4, increased in p r o p o r t i o n to t h e increase in B cells (Table 1). T h e r e s p o n s e to t h e T m i t o g e n s was greatly d i m i n i s h e d , b u t t h e degree o f r e d u c t i o n was m u c h g r e a t e r in t h e r e s p o n s e t o
7 TABLE 1 R E L A T I V E T H Y M I D I N E U P T A K E ( u n f r a c t i o n a t e d spleen cells = 100%) O F B SPLENOCYTES, S T I M U L A T E D WITH T A N D B M I T O G E N S M i t o g e n r e s p o n s e o f f r a c t i o n s as p e r c e n t of u n f r a c t i o n a t e d spleen cells
Cells s t i m u l a t e d w i t h
ConA PHA Anti-b4
Busc
BNA
5 + 3(11) a 13 -+ 9 (10) 162 -+ 37 (6)
5 -+ 4 ( 7 ) 14 -+ 10 (7) 160 -+ 48 (3)
a N u m b e r of d e t e r m i n a t i o n s .
Con A than in the response to PHA: the Con A/PHA t h y m i d i n e ratio of unfractionated spleen cells was 3.9 -+ 1.8; it was 1.6 + 0.9 for Bvsc and 1.0 -+ 0.7 f o r BNA. There were no significant differences between the response of BNA and Bvsc to either of these two mitogens.
Enumeration o f cells with Fc receptors The n u m b e r o f Fc-bearing cells was det erm i ned by the EA rosette assay. In every case we f ound a greater n u m b e r of EA rosette-forming cells in preparation o f Bvsc than in preparations of BNA. On the average, the n u m b e r o f Fc-bearing cells was 70 + 4% in Bvsc and 58 -+ 9% in BNA.In order to determine w h e t h e r or n o t this difference was attributable to macrophages, the n u m b e r o f macrophages was det er m i ned by staining for non-specific esterase. As d eter min ed by this technique, the vast majority of macrophages from the peritoneal cavity can be shown to form EA rosettes and 7 + 3% o f stained cells are f o u n d in USC. T he difference in macrophage c o n t e n t between Bvsc and BNA is much smaller (5 + 3% and 2 + 1% respectively) than the difference between the n u m b e r of cells with Fc receptors (71 -+ 4% and 58 + 9%
TABLE2 PERCENTAGE OF Fc RECEPTOR-BEARING CELLS(determinedbyEArosette method) AND OF MACROPHAGES(determined b y non-specific e s t e r a s e s t a i n i n g ) IN ISOLATED B SPLENOCYTES Experiment number
1 2 3 4
% EA rosettes
% macrophages
Busc
BNA
BUSC
BNA
68 67 72 76
46 57 69 61
9.9 5 2.4 4.5
3.5 1.4 1.7 0.4
respectively) so that removal of macrophages cannot be the only factor responsible for the differences between EA rosette-forming cells in Bvsc and BNA (Table 2). DISCUSSION It is clear from the foregoing results that considerable enrichment in B cells can be obtained by removal of T cells which form rosettes with papain-treated rabbit erythrocytes. In the resulting preparations the contamination with T cells is less than 5% and the content in B cells is 90%, as judged by cytotoxic kill with antibodies directed against RTLA and RABELA, respectively. The remaining contaminating cells are, presumably, macrophages and this impurity can be reduced further by preliminary removal, with a magnet, of cells which adhere to or ingest iron particles, coated with poly L-lysine. By comparison with RTLA and RABELA antibody-induced complement-mediated cell death, the assessment of purification by stimulation with mitogens and with antisera against Ig receptors represents a somewhat complex relation between purification factor and measurement. Thymidine incorporation is affected by shifts in subpopulations of T and B cells and by regulatory cell interactions and thus, the resulting data are more difficult to interpret quantitatively than is the enumeration of RTLA and RABELA positive cells. We know from earlier work that there are suppressor cells for the response of B cells (Shek et al., 1975), that there are helper cells which participate in the response of T cells to stimulation with Con A and PHA (Kim and Herscowitz, 1978; Persson et al., 1978; Rosenstreich and Mizel, 1978; Rivas-Alcala et al., 1979) and that there are T suppressor cells which regulate the response to Con A target cells (RivasAlcala et al., 1979}. The relative thymidine incorporation after stimulation with antibody against Ig receptors of USC, on the one hand, and of BNA and Bvsc, on the other hand, would indicate that the balance between suppressor and responder cells is not altered by purification. In short, thymidine incorporation of B cells, stimulated with antibody against ~ light-chain allotypic specificities, simply reflects the purification, achieved in the preparation of BNA and Bus c. Interesting conclusions as to subpopulation changes in B cells can be deduced from the assay for Fc receptor-bearing cells. In this respect a difference between Bvsc and BNA is observed: there is a lower content in BNA of cells with Fc receptors. The difference between Bvsc and BNA is clearly attributable to removal of adherent cells, but cannot be attributable to depletion in macrophages, alone, since the estimate of the number of macrophages is much lower than the difference of cells with Fc receptors in Bvsc and BNA (Table 2). It is apparent that a subpopulation of adherent cells has been removed in the preliminary step for the preparation of BNA. It is, however, not known whether this adherent Fc receptor-bearing cell is a B or T cell.
The relation b e t w e e n cell n u m b e r and m e a s u r e d p a r a m e t e r is clearly m o r e c o m p l e x in t h e m i t o g e n i c s t i m u l a t i o n o f T t h a n o f B cells. C o m p a r e d t o USC, t h y m i d i n e u p t a k e b y the T cell c o m p o n e n t o f purified B cells is less t h a n w o u l d be e x p e c t e d f r o m the n u m b e r o f R T L A bearing cells (Table 1). F u r t h e r m o r e t h e decrease o f the response t o P H A is m u c h less than is t h a t o f the response t o Con A. This m a y be due to a c h a n g e in the relative n u m b e r o f B cell or o f T cell s u b p o p u l a t i o n s , i.e. t o a c h a n g e in help or suppression. The p u r i f i c a t i o n m e t h o d , p r e s e n t e d in these pages, has the advantage o f simplicity, o f a r e a s o n a b l e yield. Purified cell p o p u l a t i o n s o f this t y p e are n e e d e d if f u r t h e r i n f o r m a t i o n as to f u n c t i o n a l p r o p e r t i e s o f B cell s u b p o p u lations are to be o b t a i n e d in n o n - i n b r e d animals w h e r e the strategies o f r e c o n s t i t u t i o n with cells o f syngeneic d o n o r s are n o t applicable. As a consequence, criteria for cell i d e n t i f i c a t i o n and f o r n o n - i n t e r f e r i n g p u r i f i c a t i o n m u s t be rigidly defined and m u s t be u n a m b i g u o u s since the weight a t t a c h e d to d a t a f r o m this t y p e o f a p p r o a c h is necessarily m u c h greater than it is in species w h e r e m e t h o d s d e p e n d e n t o n availability o f inbred animals can be used to c o n t r o l c o n c l u s i o n s reached b y o t h e r a p p r o a c h e s . ACKNOWLEDGEMENT T h a n k s are due to MRC (MT-3390, M T - 8 3 2 ) and to NCI for financial support. REFERENCES Bell, C. and H. Wigzell, 1977, Eur. J. Immunol. 7,726. Bona, C., L. Chedid, C. Damais, R. Ciorbaru, P.N. Shek, S. Dubiski and B. Cinader, 1975, J. Immunol. 114, 348. Cavaillon, J.M., C. Bona, P.A. Cazenave and B. Cinader, 1977, J. Immunol. Methods 14, 355. Chou, C.-T., B. Cinader and S. Dubiski, 1974, Cell. Immunol. 11, 304. Chou, C.-T., B. Cinader and S. Dubiski, 1977, Cell. Immunol. 28,334. Dubiski, S., 1969, J. Immunol. 103, 120. Fradelizi, D.P., C.-T. Chou, B. Cinader and S. Dubiski, 1973, Cell. Immunol. 7,484. Fujiwara, S., R.M. Armstrong and B. Cinader, 1974, Jpn. J. Exp. Med. 44,359. Jensenius, J.C., A.F. Williams and L.E. Mole, 1977, Eur. J. Immuno]. 7, 104. Kim, K.J. and H.B. Herscowitz, 1978, J. Reticuloendothel. Soc. 23, 75. Krawinkel, U., M. Cramer, R.G. Mage, A.S. Kelus and K, Rajewsky, 1977, J. Exp. Med. 146, 792. Ozer, Jr., H. and B.H. Waksman, 1974, J.'Immunol. 113, 1780. Persson, U., L. HammarstrSm, E. MSller, G. MSller and C.I.E. Smith, 1978, Immunol. Rev. 40, 78. Redelman, D., C.B. Scott, H.W. Sheppard Jr. and S. Sell, ]976, Cell. Immunol. 24, 11. Rivas Alcala, A.R., C.-T. Chou, B. Cinader and S. Dubiski, 1979, in press. Rosenstreich, D.L. and S.B. Mizel, 1978, Immuno]. Rev. 40, 102. Shek, P.N., C.-T. Chou, S. Dubiski and B. Cinader, 1974, Int. Arch. Allergy Appl. Immunol. 46, 753. Shek, P.N., C.-T. Chou, S. Dubiski and B. Cinader, 1975, Cell. Immunol. 18, 331. Shek, P.N., C.-T. Chou, S. Dubiski and B. Cinader, 1976a, Immunology 31, 129.
10 Shek, P.N., C.-T. Chou, S. Dubiski and B. Cinader, 1976b, Immunology 30,549. Teodorescu, M., E.P. Mayer, H. Reiter and S. Dray, 1976, Cell. Immunol. 22, 66. Tucker, S.B., R.V. Pierre and R.E. Jordon, 1977, J. Immunol. Methods 14,267. Weir, D.M. (Ed.), 1973, Handbook of Experimental Immunology (Blackwell Scientific Publications, Oxford) pp. 7.7--7.11. Wilson, A.B., B.W. Gurner and R.R.A. Coombs, 1975, Int. Arch. Allergy Appl. Immunol. 48, 383. Wilson, B.S., A.J. Strelkauskas and S. Dray, 1977, Cell. Immunol. 30, 162.
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© Elsevier/North-Holland Biomedical Press
PURIFICATION OF RABBIT B SPLEEN CELLS BY REMOVAL OF A D H E R E N T A N D OF T CELLS
JEAN-MARC CAVAILLON, B E R N H A R D CINADER, CHI-TAO CHOU and STANISLAW DUBISKI
Institute o f Immunology, Medical Sciences Building, University of Toronto, Toronto, Ont. M5S 1A8, Canada (Received 5 June 1978, accepted 28 August 1978)
B lymphocytes from the rabbit spleen were freed of T cells by removal of cells which formed rosettes with papain-treated rabbit erythrocytes. Additional purification could be achieved if fractionation by rosette removal was preceded by removal with a magnet of cells which adhered to or ingested poly L-lysine coated iron core particles. Cell yield and purification were assessed by complement mediated cytotoxic kill of B and T cells with antibody directed against R A B E L A and RTLA, respectively. Other criteria depehded on determination of the number of Fc receptor bearing cells and of thymidine uptake by cells which were stimulated with concanavalin A, PHA or with antibody directed against the allotypic specificity of receptor Ig light chains. Purified preparations of B cells were obtained in a yield of about 20% of the B cells in the original spleen and contained less than 10% of cells which were not B cells. This method allows purification which does not interfere with the membrane of the isolated cells.
INTRODUCTION
The analysis of the rabbit's lymphoid system is dependent on the developm e n t of reagents which are highly specific for individual cell populations (Fradelizi et al., 1973; Redelman et al., 1976; Wilson et al., 1977; Chou et al., 1977) and on techniques which permit isolation of cells by processes which minimize stimulation or inhibition of the isolated cells. Several methods have been used for purification of or enrichment with T cells, e.g. B cells have been removed on nylon wool (Redelman et al., 1976; Krawinkel et al., 1977), by rosetting with anti-Ig antibody-coated sheep red blood cells, or by mixed agglutination technique (Teodorescu et al., 1976; Cavaillon et al., 1977; Jensenius et al., 1977). B cells have also been removed by passage Abbreviations: BUSC, B cells isolated from unfractionated spleen cells;
B N A , B cells isolated from spleen cells from which adherent cells were removed; RTLA, rabbit thymus l y m p h o c y t e antigen; RABELA, rabbit bursal equivalent lymphocyte antigen; Con A, concanavalin A; PHA, phytohaemagglutinin; EA rosettes, rosettes between rabbit spleen cells and sheep erythrocytes coated with rabbit IgG antibody directed against the sheep erythrocytes.
over beads coated with antibody directed against Ig or against allotypic specificity (Bell and Wigzell, 1977; Rivas-Alcala et al., 1979). B cells have been purified by isolation of Ig+ cell rosettes (Teodorescu et al., 1976), and removal of T cells, lysed with complement and antibody, specific for T cells, such as RTLA antisera (Shek et al., 1976; l~edelman et al., 1976; Teodorescu et al., 1976). We shall present, in this paper, a m e t h o d by which rabbit B cells can be produced in reasonable yield and fairly free of T cells and macrophages. This technique is based on the discovery by Wilson et al. (1975) that papain-treated rabbit erythrocytes rosetted with T cells. We could adapt this analytical approach to a procedure for purification by removing T cell rosettes on Ficoll-Hypaque gradients and thus recovering B cells which do not rosette. Highly purified B cell preparations were obtained, if spleen cell suspensions were freed of adherent cells, prior to rosetting, and were then freed of T cells. MATERIALS AND METHODS
Rabbits New Zealand white rabbits (Riemens Fur Rancher Ltd., St. Agathe, Ontario) of either sex, 3--6 months old, allotype b4/b4, were employed. Rabbit red blood cells (RRBC) Blood was collected on 1000 U.S.P. units/ml of heparin (Organon, Canada) from the artery of the rabbit's ear and washed 3X in PBS pH 7.2; care was taken to remove leucocytes. Preparation of spleen lymphocyte suspensions Rabbits were killed by i.v. injection of Nembutal (Abbott Lab., Montreal). Spleens were excised and rinsed in modified McCoy's 5a medium (MCM; Grand Island Biological Company, Grand Island, NY). Spleens were cut into small pieces, gently pressed in MCM and the suspension was passed through a sterile 80 mesh stainless steel screen, were centrifuged for 8 min at 1100 rev/ min and room temperature and the cells were washed in MCM. Non-adherent cells (NA ) NA cells were prepared, as described by Shek et al. (1974). In brief, spleen cells were freed, with a magnet, of cells which adhered to or ingested iron core particles, coated with poly L-lysine (Technicon Corp., Tarrytown, NY 10591); the remaining cells are designated as NA cells (Rivas Alcala et al., 1979). Papain treatment of rabbit red blood cells (RRBC) RRBC were treated with papain as described by Wilson et al. (1975). Briefly, after washing, the erythrocytes were resuspended to 20% v/v in PBS and an equal volume of a mixture was added which consisted of I part
0.25% papain (N.B.C. Lab., Cleveland, OH) in PBS, 1 part Na2HPO4 (3.6 g in 100 ml distilled water) and 2 parts 0.2% L-cysteine HC1. RRBC were incubated at 37°C for 18 min, were then centrifuged, washed once with PBS, twice with RPMI-1640 medium (Gibco Lab., Grand Island, NY) and were finally resuspended to 0.7% v/v in PBS. Removal o f lymphoid cells which form rosettes with papain-treated rabbit ery th rocy tes We employed a m e t h o d based on an analytical technique devised by Wilson et al. (1975). Spleen cell suspension (3 ml, 6 × 106 cells/ml in RPMI1640) were mixed with papain-treated rabbit erythrocytes (3 ml, 0.7%) and with 3 ml of RPMI-1640 supplemented with 15% fetal calf serum (FCS). Thereafter, the cell mixture was centrifuged for 4 min at 1700 rev/min and 4°C; the centrifuged mixture was kept for 15 min at 4 ° C. Cells pelleted from the centrifuged mixture were gently resuspended in the original fluid, pooled and kept at 4°C; 1.8 ml per tube of FCS were added. In r o u n d - b o t t o m e d test tubes (17 mm × 100 mm, Falcon, Oxnard, CA, U.S.A.), the cell suspension (7 ml) was carefully layered on a Ficoll-Hypaque mixture (3 ml of 25 parts of 12% Ficoll, Pharmacia, Uppsala, Sweden, and 10 parts of 33% Hypaque, Winthrop, Aurora, Ontario, Canada; final density at room temperature = 1.08 g/ml + 5 × 10-4). The tubes were centrifuged for 25 min at 4°C and 1500 rev/min. Lymphocytes, depleted of rosettes, were recovered at the interphase and collected with a Pasteur pipette. The recovered cells were centrifuged for 5 min at 4°C and 1800 rev/min, and were washed twice in RPMI medium. Determination o f T cell content with R T L A antiserum R T L A antiserum was raised by immunization of goats with rabbit thymus cells and was made T-cell specific by suitable absorption (Fradelizi et al., 1973). By means of immunofiuorescence, it could be shown that RTLAbearing cells were localized in thymus-dependent areas of the spleen (Fujiwara et al., 1974). Complement-mediated cell kill with I~TLA antiserum did n o t reduce B cell responses, i.e., did not interfere with thymidine incorporation after stimulation with antibody against light-chain allotypic specificity (Shek et al., 1974) or with polyclonal activation by a B mitogen extracted from Nocardia (Bona et al., 1975). On the other hand, cell kill of RTLAbearing cells abolished response to phytohaemagglutinin and concanavalin A. R T L A antiserum was employed for complement-mediated c y t o t o x i c kill of T cells; dead cells were identified and counted by Trypan blue exclusion test, as described by Fradelizi et al. (1973). Determination o f B cell content with R A B E L A antiserum R A B E L A antiserum was raised by immunization of goats with rabbit appendix cells and was absorbed so as to make it specific for B cells (Chou et al., 1977). The specificity of R A B E L A antiserum does not overlap with that
of RTLA antiserum since the arithmetic sum of per cent cell kill by RABELA antiserum alone, and by RTLA antiserum, alone, is equal to the per cent of cell kill by a mixture of the two antisera (Chou et al., 1977). Finally, the percentage of RABELA positive and of Ig-bearing spleen cells is in good agreement. RABELA antiserum was employed for complement mediated c y t o t o x i c i t y kill of B cells, as described in an earlier publication (Chou et al., 1977).
Determination of macrophage number Macrophages were enumerated by counting cells stained by the m e t h o d of Tucker et al. (1977). Stimulation of lymphocytes with lectins and with antibody against Ig receptors o f B cells Cells were stimulated with Con A and PHA in Multi-Dish Disposo Trays (Linbro Scientific, Inc., Hamden, CT): each well contained 2 × 105 cells in RPMI medium supplemented with 20% normal rabbit serum b4/b5. Stimulation with antibody directed against allotypic specificity b4 (Chou et al., 1974) was carried out in the presence of normal rabbit serum b9/b9; the total serum content was 0.2 ml/ml of mixture. Optimal dilutions of lectins were 30 pg/well of Con A (Miles Yeda Ltd., Kankakee, IL), 3 pg/well of PHA (Difco Lab., Detroit, MI); anti-4 (prepared as described by Dubiski (1969)) was used in a final dilution of 1 : 10. After 24 h of incubation at 37°C in an atmosphere of 5% CO2 and 95% air, [3H]thymidine (0.25 pCi/ well; specific activity 20 Ci/mmol) (Amersham-Searle Corp.) was added and the incubation was continued for a further 24 h. Thereafter, cells were harvested on glass fibre filters with a Skatron machine (Flow Lab., Rockville, MD, U.S.A.). Thymidine incorporation was determined by liquid scintillation counting and was expressed as net cpm, i.e., as counts in the presence of mitogen less counts in the absence of mitogen. EA rosettes The percentage of Fc receptor-bearing cells was determined by enumeration of EA rosettes. Rabbit IgG antibody directed against sheep red blood cells (SRBC) was prepared from antisera by precipitation with 18% sodium sulphate and subsequent chromatography on DEAE-cellulose (Weir, 1973). SRBC were exposed to a subagglutinating dose of this rabbit anti-SRBC IgG for 30 min at 37°C. After incubation, the sensitized SRBC were washed in PBS and resuspended to 1% v/v in RPMI-1640 medium. L y m p h o c y t e s (106 cells in 0.2 ml) were mixed with 0.2 ml of the sensitized SRBC and the cell mixture was centrifuged at 4°C for 5 min at 800 rev/min. The centrifuged cells were kept for 1 h at 4°C before the pellet was resuspended. Rosetteforming cells (RFC) were defined as being attached to 3 or more SRBC; such rosettes were not observed when lymphocytes were mixed with untreated SRBC. Rosettes were counted after addition of trypan blue.
RESULTS In adapting the analytical technique for T cell enumeration with papaintreated isologous red cells (Wilson et al., 1975), two technical problems had to be solved: we had to scale up the cell numbers and find a density at which we would cleanly separate rosetted cells from those which did not rosette. With equal volumes of l y m p h o c y t e suspensions of 6 × 106 cells/ml and with 0.7% papain-treated rabbit erythrocyte suspensions, the yield of B cells remained constant as between 6 and 60 × 106 cells per mixture of a final volume of 3--30 ml, respectively. Separation of rosettes from non-rosetted cells was carried out by centrifugation on a Ficoll-Hypaque or L y m p h o p r e p mixture. It was our aim to select a density at which rosetted cells would be at the b o t t o m of the tube and the cells which had not rosetted would collect, in good yield, in the interphase. At densities of 1.08 g/ml (Ficoll-Hypaque), the yield of non-rosetted cells, obtained from unfractionated spleen cells was 17 + 5%, whereas at a density of 1.077 g/ml (Lymphoprep, Nyegard and Co, Norway) it was 7.5%; the higher density and Ficoll-Hypaque were chosen for routine purification of B cells. The experiment arrangement finally selected is described in Materials and Methods. In the following, we shall characterize the cells which are obtained by removing rosetted cells either from unfractionated spleen cells (USC) or from suspensions which had been freed of cells that adhered to or ingested poly L-lysine-coated magnetic particles (NA).
Enumeration o f B and T cells by c o m p l e m e n t mediated cytotoxic kill B cells were isolated either from untreated suspensions of spleen cells (USC) or from suspensions from which adherent cells were removed (NA); the B cell preparations were designated Bvsc o r BNA , respectively. The T cell content, as established by complement mediated cytotoxic kill with RTLA antiserum, was 5 -+ 3% in Bvsc and 4 -+ 4% in BNA. The B cell c o n t e n t of these two preparations, as determined with RABELA antiserum, was 89 + 6% for Busc and 91 -+ 7% for BNA (Fig. 1). This is an approximately 10-fold reduction in T cells by comparison with USC which contained 39-+ 11% RTLA positive cells and 56 -+ 8% RABELA positive cells. While there is no significant difference in the T and B cell c o n t e n t of Bvsc and BNA, there was a difference in 60% of the cases when the comparison was made between preparations from the same spleen cells; the content of RABELA positive cells was somewhat higher in the BNA than in the Bus c (Fig. 1). Bus c and BNA were obtained in a yield of 26 -+ 7% and 21 -+ 7%, respectively, of the B cells, contained in unfractionated starting material. The relative low yield of BNA was due to a loss of 47 -+ 7% in the step in which adherent cells were removed. Mitogen-induced thymidine uptake as a measure o f cell purification Since concanavalin A (Con A) and phytohaemagglutinin (PHA) stimulate
V
USC
2 tO
W 10
u) ..Q
0
Busc
8 6 4
..Q
E -'I z
2
10! 8
V
6
BNA
4
2 i
40
,
,
60
e
80
100
% RABELA positive cells
0
20
40
60
% RTLA positive cells
Fig. 1. Rabbit B and T cells in unfractionated and in separated subpopulations of spleen cells. Spleen cells were freed of cells which formed rosettes with papain-treated rabbit erythrocytes. The cells were treated with rabbit complement, antiserum against RABELA or RTLA and cytotoxic kill was measured and plotted as a percentage against the number of animals in which this percentage of RABELA or RTLA bearing cells was found. The point of the arrow indicates mean values; the width of the arrow is equal to 2 S.D.
r a b b i t T derived l y m p h o c y t e s ( O z e r and W a k s m a n , 1 9 7 4 ; S h e k et al., 1 9 7 4 , 1 9 7 5 , 1 9 7 6 a ) a n d since a n t i b o d y d i r e c t e d against light-chain a l l o t y p i c m a r k e r b 4 s t i m u l a t e s B derived l y m p h o c y t e s (Fradelizi et al., 1 9 7 3 ; C h o u et al., 1 9 7 4 ; S h e k et al., 1 9 7 6 b ) , it is possible to c o m p a r e t h e B a n d T cell cont e n t o f t w o p o p u l a t i o n s in t e r m s o f lectin o r a n t i b o d y - i n d u c e d t h y m i d i n e i n c o r p o r a t i o n . We h a v e d o n e so, b y e x p o s i n g c o n s t a n t n u m b e r s o f Busc, BNA and USC t o e a c h o f t h e s e agents; t h e u n t r e a t e d spleen cells w e r e first passed t h r o u g h a Ficoll gradient, similar to t h a t used f o r r o s e t t e d e p l e t i o n , so as t o e l i m i n a t e this p r e p a r a t i v e step f r o m a f f e c t i n g t h e c o m p a r i s o n b e t w e e n s t a r t i n g m a t e r i a l a n d t h e cells isolated f r o m it. T h y m i d i n e u p t a k e , a f t e r s t i m u l a t i o n w i t h a n t i b o d y d i r e c t e d against b4, increased in p r o p o r t i o n to t h e increase in B cells (Table 1). T h e r e s p o n s e to t h e T m i t o g e n s was greatly d i m i n i s h e d , b u t t h e degree o f r e d u c t i o n was m u c h g r e a t e r in t h e r e s p o n s e t o
7 TABLE 1 R E L A T I V E T H Y M I D I N E U P T A K E ( u n f r a c t i o n a t e d spleen cells = 100%) O F B SPLENOCYTES, S T I M U L A T E D WITH T A N D B M I T O G E N S M i t o g e n r e s p o n s e o f f r a c t i o n s as p e r c e n t of u n f r a c t i o n a t e d spleen cells
Cells s t i m u l a t e d w i t h
ConA PHA Anti-b4
Busc
BNA
5 + 3(11) a 13 -+ 9 (10) 162 -+ 37 (6)
5 -+ 4 ( 7 ) 14 -+ 10 (7) 160 -+ 48 (3)
a N u m b e r of d e t e r m i n a t i o n s .
Con A than in the response to PHA: the Con A/PHA t h y m i d i n e ratio of unfractionated spleen cells was 3.9 -+ 1.8; it was 1.6 + 0.9 for Bvsc and 1.0 -+ 0.7 f o r BNA. There were no significant differences between the response of BNA and Bvsc to either of these two mitogens.
Enumeration o f cells with Fc receptors The n u m b e r o f Fc-bearing cells was det erm i ned by the EA rosette assay. In every case we f ound a greater n u m b e r of EA rosette-forming cells in preparation o f Bvsc than in preparations of BNA. On the average, the n u m b e r o f Fc-bearing cells was 70 + 4% in Bvsc and 58 -+ 9% in BNA.In order to determine w h e t h e r or n o t this difference was attributable to macrophages, the n u m b e r o f macrophages was det er m i ned by staining for non-specific esterase. As d eter min ed by this technique, the vast majority of macrophages from the peritoneal cavity can be shown to form EA rosettes and 7 + 3% o f stained cells are f o u n d in USC. T he difference in macrophage c o n t e n t between Bvsc and BNA is much smaller (5 + 3% and 2 + 1% respectively) than the difference between the n u m b e r of cells with Fc receptors (71 -+ 4% and 58 + 9%
TABLE2 PERCENTAGE OF Fc RECEPTOR-BEARING CELLS(determinedbyEArosette method) AND OF MACROPHAGES(determined b y non-specific e s t e r a s e s t a i n i n g ) IN ISOLATED B SPLENOCYTES Experiment number
1 2 3 4
% EA rosettes
% macrophages
Busc
BNA
BUSC
BNA
68 67 72 76
46 57 69 61
9.9 5 2.4 4.5
3.5 1.4 1.7 0.4
respectively) so that removal of macrophages cannot be the only factor responsible for the differences between EA rosette-forming cells in Bvsc and BNA (Table 2). DISCUSSION It is clear from the foregoing results that considerable enrichment in B cells can be obtained by removal of T cells which form rosettes with papain-treated rabbit erythrocytes. In the resulting preparations the contamination with T cells is less than 5% and the content in B cells is 90%, as judged by cytotoxic kill with antibodies directed against RTLA and RABELA, respectively. The remaining contaminating cells are, presumably, macrophages and this impurity can be reduced further by preliminary removal, with a magnet, of cells which adhere to or ingest iron particles, coated with poly L-lysine. By comparison with RTLA and RABELA antibody-induced complement-mediated cell death, the assessment of purification by stimulation with mitogens and with antisera against Ig receptors represents a somewhat complex relation between purification factor and measurement. Thymidine incorporation is affected by shifts in subpopulations of T and B cells and by regulatory cell interactions and thus, the resulting data are more difficult to interpret quantitatively than is the enumeration of RTLA and RABELA positive cells. We know from earlier work that there are suppressor cells for the response of B cells (Shek et al., 1975), that there are helper cells which participate in the response of T cells to stimulation with Con A and PHA (Kim and Herscowitz, 1978; Persson et al., 1978; Rosenstreich and Mizel, 1978; Rivas-Alcala et al., 1979) and that there are T suppressor cells which regulate the response to Con A target cells (RivasAlcala et al., 1979}. The relative thymidine incorporation after stimulation with antibody against Ig receptors of USC, on the one hand, and of BNA and Bvsc, on the other hand, would indicate that the balance between suppressor and responder cells is not altered by purification. In short, thymidine incorporation of B cells, stimulated with antibody against ~ light-chain allotypic specificities, simply reflects the purification, achieved in the preparation of BNA and Bus c. Interesting conclusions as to subpopulation changes in B cells can be deduced from the assay for Fc receptor-bearing cells. In this respect a difference between Bvsc and BNA is observed: there is a lower content in BNA of cells with Fc receptors. The difference between Bvsc and BNA is clearly attributable to removal of adherent cells, but cannot be attributable to depletion in macrophages, alone, since the estimate of the number of macrophages is much lower than the difference of cells with Fc receptors in Bvsc and BNA (Table 2). It is apparent that a subpopulation of adherent cells has been removed in the preliminary step for the preparation of BNA. It is, however, not known whether this adherent Fc receptor-bearing cell is a B or T cell.
The relation b e t w e e n cell n u m b e r and m e a s u r e d p a r a m e t e r is clearly m o r e c o m p l e x in t h e m i t o g e n i c s t i m u l a t i o n o f T t h a n o f B cells. C o m p a r e d t o USC, t h y m i d i n e u p t a k e b y the T cell c o m p o n e n t o f purified B cells is less t h a n w o u l d be e x p e c t e d f r o m the n u m b e r o f R T L A bearing cells (Table 1). F u r t h e r m o r e t h e decrease o f the response t o P H A is m u c h less than is t h a t o f the response t o Con A. This m a y be due to a c h a n g e in the relative n u m b e r o f B cell or o f T cell s u b p o p u l a t i o n s , i.e. t o a c h a n g e in help or suppression. The p u r i f i c a t i o n m e t h o d , p r e s e n t e d in these pages, has the advantage o f simplicity, o f a r e a s o n a b l e yield. Purified cell p o p u l a t i o n s o f this t y p e are n e e d e d if f u r t h e r i n f o r m a t i o n as to f u n c t i o n a l p r o p e r t i e s o f B cell s u b p o p u lations are to be o b t a i n e d in n o n - i n b r e d animals w h e r e the strategies o f r e c o n s t i t u t i o n with cells o f syngeneic d o n o r s are n o t applicable. As a consequence, criteria for cell i d e n t i f i c a t i o n and f o r n o n - i n t e r f e r i n g p u r i f i c a t i o n m u s t be rigidly defined and m u s t be u n a m b i g u o u s since the weight a t t a c h e d to d a t a f r o m this t y p e o f a p p r o a c h is necessarily m u c h greater than it is in species w h e r e m e t h o d s d e p e n d e n t o n availability o f inbred animals can be used to c o n t r o l c o n c l u s i o n s reached b y o t h e r a p p r o a c h e s . ACKNOWLEDGEMENT T h a n k s are due to MRC (MT-3390, M T - 8 3 2 ) and to NCI for financial support. REFERENCES Bell, C. and H. Wigzell, 1977, Eur. J. Immunol. 7,726. Bona, C., L. Chedid, C. Damais, R. Ciorbaru, P.N. Shek, S. Dubiski and B. Cinader, 1975, J. Immunol. 114, 348. Cavaillon, J.M., C. Bona, P.A. Cazenave and B. Cinader, 1977, J. Immunol. Methods 14, 355. Chou, C.-T., B. Cinader and S. Dubiski, 1974, Cell. Immunol. 11, 304. Chou, C.-T., B. Cinader and S. Dubiski, 1977, Cell. Immunol. 28,334. Dubiski, S., 1969, J. Immunol. 103, 120. Fradelizi, D.P., C.-T. Chou, B. Cinader and S. Dubiski, 1973, Cell. Immunol. 7,484. Fujiwara, S., R.M. Armstrong and B. Cinader, 1974, Jpn. J. Exp. Med. 44,359. Jensenius, J.C., A.F. Williams and L.E. Mole, 1977, Eur. J. Immuno]. 7, 104. Kim, K.J. and H.B. Herscowitz, 1978, J. Reticuloendothel. Soc. 23, 75. Krawinkel, U., M. Cramer, R.G. Mage, A.S. Kelus and K, Rajewsky, 1977, J. Exp. Med. 146, 792. Ozer, Jr., H. and B.H. Waksman, 1974, J.'Immunol. 113, 1780. Persson, U., L. HammarstrSm, E. MSller, G. MSller and C.I.E. Smith, 1978, Immunol. Rev. 40, 78. Redelman, D., C.B. Scott, H.W. Sheppard Jr. and S. Sell, ]976, Cell. Immunol. 24, 11. Rivas Alcala, A.R., C.-T. Chou, B. Cinader and S. Dubiski, 1979, in press. Rosenstreich, D.L. and S.B. Mizel, 1978, Immuno]. Rev. 40, 102. Shek, P.N., C.-T. Chou, S. Dubiski and B. Cinader, 1974, Int. Arch. Allergy Appl. Immunol. 46, 753. Shek, P.N., C.-T. Chou, S. Dubiski and B. Cinader, 1975, Cell. Immunol. 18, 331. Shek, P.N., C.-T. Chou, S. Dubiski and B. Cinader, 1976a, Immunology 31, 129.
10 Shek, P.N., C.-T. Chou, S. Dubiski and B. Cinader, 1976b, Immunology 30,549. Teodorescu, M., E.P. Mayer, H. Reiter and S. Dray, 1976, Cell. Immunol. 22, 66. Tucker, S.B., R.V. Pierre and R.E. Jordon, 1977, J. Immunol. Methods 14,267. Weir, D.M. (Ed.), 1973, Handbook of Experimental Immunology (Blackwell Scientific Publications, Oxford) pp. 7.7--7.11. Wilson, A.B., B.W. Gurner and R.R.A. Coombs, 1975, Int. Arch. Allergy Appl. Immunol. 48, 383. Wilson, B.S., A.J. Strelkauskas and S. Dray, 1977, Cell. Immunol. 30, 162.
