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Enrichment of PHA transformed lymphocytes in samples containing mixed populations
Journal oflmmunological Methods, 13 (1976) 161--166 © Elsevier/North-Holland Biomedical Press
161
E NR I C HMENT OF PHA T R A N S F O R M E D LYMPHOCYTES IN SAMPLES CONTAINING MIXED POPULATIONS
BRUCE SHENKER and IRVING GRAY Department of Biology, Georgetown University, Washington, D.C. 20057. U.S.A.
(Received 5 February 1976, accepted 27 April ]976) A dextran density gradient centrifugation method has been used to separate PHA transformed lymphocytes from nontransformed lymphocytes obtained from rat blood. Fifty-four percent of the transformed lymphocytes were recovered in the fraction at a density of 1.0620 g/ml. Eighty-five percent of the cells in this fraction were transformed cells while the remainder were non-transformed lymphocytes. INTRODUCTION Stimulation of l y m p h o c y t e s by plant lectins such as phytohemagglutinin results in a n u m b e r of biochemical and morphological changes. These alterations include: increased turnover of m e m b r a n e phospholipids, increased protein, RNA and DNA synthesis as well as an increase in cell size (Sasaki et al., 1966; Parker et al., 1974). The action o f mitogens on l y m p h o c y t e s has been used for man y purposes, in particular, the study of the immune response as a correlate to antigen stimulation (Faguet et al., 1974). Since mitogen stimulation of l y m p h o c y t e s usually results in activation of a portion of the total cell population, depending on the experimental conditions (Faguet et al., 1974; Ling and Kay, 1975; Sell et al., 1975), this presents a problem for b o t h biochemical and physiological studies. In studies dealing with a heterogeneous population of cells, only a fraction of which has been transf o rmed , the resulting data are at best qualitative. Therefore, it was our purpose to develop a rapid technique for the c o n c e n t r a t i o n of transformed lymphocytes. Many m e t h o d s have been used for separating l y m p h o c y t e s into subpopulations, for example, electrophoresis (Serler et al., 1974), freezing and thawing (Knight et al., 1972), density gradient centrifugation (Newlin, 1973; Loos and Roos, 1974) and more recently, isoelectric focusing (Leise and LeSane, 1974). Density gradient centrifugation m e t h o d s seem to be the most rapid and require the least a m o u n t of elaborate e q u i p m e n t (Leise et al., 1970). Many types of gradients have been used to separate cells on the basis of small differences in specific gravity, for example, gelatin (Coulson, 1964), albumin (Turner, 1967), Ficoll (Uvnas and T hon, 1959) and dextran (Hilal et al., 1964). Dextran gradient centrifugation was chosen because it has proven to be simple, rapid and reproducible in earlier studies (Leise et al., 1970}.
162 METIIODS AND MATERIAI.S
Preparation of cells Peripheral lymphocytes were isolated by the method of B~byum (BCyum, 1968) from approximately 10 ml of blood obtained by cardiac puncture of 300--400 mg male Sprague--Dawley rats (Flow Laboratories, Rockville, Md.). The cells were diluted in culture medium to give a suspension of 1----5 X 107 cells/ml. The culture medium consisted of RPMI 1640 (Gibco, Grand Island, N.Y.), 10% heat inactivated fetal calf serum (Gibco), 100 units penicillin and 100 #g streptomycin (Gibco). The diluted cell suspension was divided among 10 culture tubes (12 X 75 mm), each containing 1.85 ml of RPMi 1640, 0.1 ml of tile cell suspension (1--5 X 10 ~ cells/tube) and in those cultures receiving phytohemagglutinin (PHA-P, Difco, Detroit, Mich.), the lectin was added to give a final concentration of 2.5 pg/ml. Tile cell cultures were incubated for 72 h at 37°C in air containing 5% CO2. At the end of this time period, the 10 cultures were recombined, centrifuged and the cells resuspended in 1.0 ml of a 1.020 g/ml dextran (see below) solution for placement on a discontinuous density gradient. No clumping of cells was seen.
Preparation of density gradient Discontinuous density gradients were prepared using dextran (m.w. 40,000, Nutritional Biochemicals Corporation, Cleveland, Ohio) dissolved in Hanks Balanced Salt Solution (BSS). Six dextran solutions were made, as previously described by Leise (1970) to give the following specific gravities: 1.0600, 1.0620, 1.0640, 1.0670, 1.0700 and 1.0750. The gradient was prepared by layering 1 ml of each of the appropriate solutions into culture tubes (17 X 100 mm). The top, seventh, layer on the gradient was 1--2 ml of cell suspension (d = 1.0200) described above. The tubes were centrifuged for 15 rain at 1000 rpm (International, Model PR-B with head No. 269), approximately 140 g. Seven fractions of cells were collected with a Pasteur pipette by removing each density layer plus the cells accumulated at its lower interface. Each of the fractions collected was placed in a glass tube (12 X 75 mm). To facilitate settling of the cells, the specific gravity of each fraction was reduced by diluting to 5 ml with BBS. The diluted suspensions were centrifuged at 2500 rpm for 10 min. The cell pellet was resuspended in 0.5 ml of a 5% dextran solution. The fractions collected from the gradient were analyzed for 1) total cells per fraction, determined using a Neubauer h e m o c y t o m e t e r , and 2) distribution of cell type within each fraction, determined oil the basis of morphology. Slides for the latter determination were prepared by applying two to three drops of the cell suspension to a glass microscope slide. The cells were
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Fig. 1. D i s t r i b u t i o n o f cells as a f u n c t i o n o f the p e r c e n t o f c~,ll ~vpe f o r ~'aeh f r a c t i o n . Solid bars r e p r e s e n t s m a l l n o n - t r a n s f o r m e d l y m p l m c y t e s a n d strip(,d bars r e l ) r o s e n t transf o r m e d l y m p h o c y t c s . E a c h bar r e p r e s e n t s t h e m e a n o f 5 e x p ~ . r i m e n t s . ' T ' - b a r ref)r(~senls the standard deviation.
164
spread, air dried, fixed in 95% e t h a n o l and again allowed to air dry. T h e slides were stained with H e m a l B l o o d Stain ( H e m a l Stain Co., D a n b u r y , Ct.). T h e cells were c o u n t e d u n d e r oil i m m e r s i o n , at least 100 per slide. Cell viability was d e t e r m i n e d using t r y p a n blue exclusion as p r e v i o u s l y r e p o r t e d (Leise and LeSane, 1974}. I{FSULTS
Distribution o f cells on gradient T h e results o f the s e p a r a t i o n o f t r a n s f o r m e d l y m p h o c y t e s are s h o w n in fig. 1. It is clearly e v i d e n t t h a t f r a c t i o n 3 (d = 1 . 0 6 2 0 g/ml) is e n r i c h e d with t r a n s f o r m e d l y m p h o c y t e s to a p p r o x i m a t e l y 85%, w h e r e a s the cell p o p u l a tion placed on t h e g r a d i e n t c o n t a i n e d 30% t r a n s f o r m e d l y m p h o c y t e s . T h e d e n s e r f r a c t i o n s c o n t a i n a p r e d o m i n a n c e o f small n o n t r a n s f o r m e d l y m p h o cytes. Fig. 2 s h o w s the d i s t r i b u t i o n o f cell t y p e s on tile g r a d i e n t as a p e r c e n t a g e o f t o t a l cells r e c o v e r e d . A t o t a l o f 44% o f the cells r e c o v e r e d were transf o r m e d , o f w h i c h 54% a p p e a r in f r a c t i o n 3. T h e t o t a l o f the cells r e c o v e r e d is 70% o f t h a t p u t on the gradient. T h e d i s t r i b u t i o n o f cell t y p e s p u t on the gradient, was 70% n o n - t r a n s f o r m e d a n d 30% t r a n s f o r m e d l y m p h o c y t e s . A f t e r c e n t r i f u g a t i o n the d i s t r i b u t i o n in the t o t a l o f cells r e c o v e r e d was 56% and
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Fig. 2. Distribution of non-transformed lymphocytes (0) and transformed lymphocytes (o) as a function of the total cells r~,covered.
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Fig. 3. Distribution of the total lymphocyte population under three different conditions: without any incubation period (0); after 72 h of culture without PHA (O);after 72 h of culture in the presence of PHA (0). This data is based on 3 experiments.
44% respectively. It w o u l d appear then that there is a selective loss o f the n o n t r a n s f o r m e d cells. When the viability o f each fraction was assessed, n o adverse e f f e c t o f the gradient centrifugation was seen. Before centrifugation the viability was 8 8 - 92%, after centrifugation it was 90--92%.
Changes in cell distribution Fig. 3 s h o w s the distribution o f cells on the gradient under three different c o n d i t i o n s ; prior to culture, after 72 h culture with PHA, and after 72 h culture w i t h o u t PHA. These curves take intQ a c c o u n t all the l y m p h o c y t e s present and in the case o f those cultured with PHA includes the transformed
166 cells. This c o n t r a s t s with fig. 2 w h i c h differentiates b e t w e e n t r a n s f o r m e d and n o n - t r a n s f o r m e d cells. Cells prior to c u l t u r e are primarily f o u n d in the first t h r e e fractions with a peak in f r a c t i o n 2 {d = 1 . 0 6 0 0 g/ml), r e p r e s e n t i n g 35% o f the total recovered cells. Cells c u l t u r e d with P H A s h o w a shift t o w a r d s the layers less dense than t h o s e i n c u b a t e d w i t h o u t the lectin, with a peak o f 27% in f r a c t i o n 3. Cells c u l t u r e d for 72 h w i t h o u t lectin, s h o w a shift to the denser layers, fractions 4--6 (d = 1 . 0 6 4 0 - - 1 . 0 7 0 0 g/ml). SUMMARY AND CONCLUSION It has been previously d e m o n s t r a t e d (Sell et al., 1 9 7 5 ) t h a t when l y m p h o c y t e s are c u l t u r e d in the presence o f p h y t o h e m a g g l u t i n i n t w o m o r p h o l o gically d i f f e r e n t cell p o p u l a t i o n s result. Using a d e x t r a n d i s c o n t i n u o u s d e n s i t y gradient, we have separated this m i x e d cell p o p u l a t i o n into several s u b p o p u l a t i o n s . F r o m fig. 1 it is a p p a r e n t t h a t the t r a n s f o r m e d cells were c o n c e n t r a t e d in the layer having a d e n s i t y o f 1 . 0 6 2 0 g/ml o f d e x t r a n . The d i s t r i b u t i o n o f cells in this fraction was a p p r o x i m a t e l y 85% t r a n s f o r m e d cells and 15% n o n - t r a n s f o r m e d l y m p h o c y t e s . O f all the t r a n s f o r m e d cells recovered, 54% were f o u n d in the 1 . 0 6 2 0 g/ml f r a c t i o n (fig. 2). F o l l o w i n g 72 h o f culture, cells b e c o m e m o r e dense as c o m p a r e d to the cell d i s t r i b u t i o n i m m e d i a t e l y after isolation (fig. 3). This change in the d i s t r i b u t i o n m i g h t be due to the repair o f injury incurred at the time o f cell isolation ( L o o s and R o o s , 1 9 7 4 ; S t e w a r t et al., 1975). Cells c u l t u r e d with p h y t o h e m a g g l u t i n i n also increase in density, b u t n o t to the same c x t e n t as those c u l t u r e d w i t h o u t lectin. REFERENCES B~yum, A., 1968, Scan. J. Clin. Lab. Invest. Suppl. 97, 9. Coulson, A., 1964, Lancet 1,468. Faguet, G., 1974, J. Retic. Soc. 16(2), 114. Hilal, S., D. Messer, M. Lohen and R. Johnson, 1964, Ann. N.Y. Acad. Sei. 11,1,661. Knight, S., J. Farrant and G. Morris, 1972, Nature New Biol. 239, 88. Leise, E., T. Morita, I. Gray and F. LeSane, 1970, Biochem. Med. ,1, 327. Leise, E. and F. LeSane, 197,1, Prep. Biochem. 4(5), 395. Ling, N. and J. Kay, 1975, Lymphocyte Stimulation (North-Holland Publishing Co., Amsterdam, Netherlands). Loos, J. and D. Roos, 1974, Exp. Cell. Res. 86, 3.12. Newlin, C., 1973, Cell. Immunol. 8, 198. Parker, C., T. Sullivan and J. Wedner, 1974, Adv. Cyclic Nucl. Res. 4, I. Sasaki, M. and A. Norman, 1966, Nature, 210,913. Sell, S., H. Sheppard and D. Redelman, 1975, Exp. Cell Res. 90, 309. Serler, F., R. Johannsen, H. Sedlacck and K. Zeiller, 1974, Transplant. Proc. 6(2), 173. Stewart, C., S. Cramer and P. Steard, 1975, (Jell lmmunol. 16, 237. Turner, R., 1967, J. Cell Physiol. 69, 73. Uwlas, B. and 1. Thon, 1959, Exp. Cell Res. 18, 512.
161
E NR I C HMENT OF PHA T R A N S F O R M E D LYMPHOCYTES IN SAMPLES CONTAINING MIXED POPULATIONS
BRUCE SHENKER and IRVING GRAY Department of Biology, Georgetown University, Washington, D.C. 20057. U.S.A.
(Received 5 February 1976, accepted 27 April ]976) A dextran density gradient centrifugation method has been used to separate PHA transformed lymphocytes from nontransformed lymphocytes obtained from rat blood. Fifty-four percent of the transformed lymphocytes were recovered in the fraction at a density of 1.0620 g/ml. Eighty-five percent of the cells in this fraction were transformed cells while the remainder were non-transformed lymphocytes. INTRODUCTION Stimulation of l y m p h o c y t e s by plant lectins such as phytohemagglutinin results in a n u m b e r of biochemical and morphological changes. These alterations include: increased turnover of m e m b r a n e phospholipids, increased protein, RNA and DNA synthesis as well as an increase in cell size (Sasaki et al., 1966; Parker et al., 1974). The action o f mitogens on l y m p h o c y t e s has been used for man y purposes, in particular, the study of the immune response as a correlate to antigen stimulation (Faguet et al., 1974). Since mitogen stimulation of l y m p h o c y t e s usually results in activation of a portion of the total cell population, depending on the experimental conditions (Faguet et al., 1974; Ling and Kay, 1975; Sell et al., 1975), this presents a problem for b o t h biochemical and physiological studies. In studies dealing with a heterogeneous population of cells, only a fraction of which has been transf o rmed , the resulting data are at best qualitative. Therefore, it was our purpose to develop a rapid technique for the c o n c e n t r a t i o n of transformed lymphocytes. Many m e t h o d s have been used for separating l y m p h o c y t e s into subpopulations, for example, electrophoresis (Serler et al., 1974), freezing and thawing (Knight et al., 1972), density gradient centrifugation (Newlin, 1973; Loos and Roos, 1974) and more recently, isoelectric focusing (Leise and LeSane, 1974). Density gradient centrifugation m e t h o d s seem to be the most rapid and require the least a m o u n t of elaborate e q u i p m e n t (Leise et al., 1970). Many types of gradients have been used to separate cells on the basis of small differences in specific gravity, for example, gelatin (Coulson, 1964), albumin (Turner, 1967), Ficoll (Uvnas and T hon, 1959) and dextran (Hilal et al., 1964). Dextran gradient centrifugation was chosen because it has proven to be simple, rapid and reproducible in earlier studies (Leise et al., 1970}.
162 METIIODS AND MATERIAI.S
Preparation of cells Peripheral lymphocytes were isolated by the method of B~byum (BCyum, 1968) from approximately 10 ml of blood obtained by cardiac puncture of 300--400 mg male Sprague--Dawley rats (Flow Laboratories, Rockville, Md.). The cells were diluted in culture medium to give a suspension of 1----5 X 107 cells/ml. The culture medium consisted of RPMI 1640 (Gibco, Grand Island, N.Y.), 10% heat inactivated fetal calf serum (Gibco), 100 units penicillin and 100 #g streptomycin (Gibco). The diluted cell suspension was divided among 10 culture tubes (12 X 75 mm), each containing 1.85 ml of RPMi 1640, 0.1 ml of tile cell suspension (1--5 X 10 ~ cells/tube) and in those cultures receiving phytohemagglutinin (PHA-P, Difco, Detroit, Mich.), the lectin was added to give a final concentration of 2.5 pg/ml. Tile cell cultures were incubated for 72 h at 37°C in air containing 5% CO2. At the end of this time period, the 10 cultures were recombined, centrifuged and the cells resuspended in 1.0 ml of a 1.020 g/ml dextran (see below) solution for placement on a discontinuous density gradient. No clumping of cells was seen.
Preparation of density gradient Discontinuous density gradients were prepared using dextran (m.w. 40,000, Nutritional Biochemicals Corporation, Cleveland, Ohio) dissolved in Hanks Balanced Salt Solution (BSS). Six dextran solutions were made, as previously described by Leise (1970) to give the following specific gravities: 1.0600, 1.0620, 1.0640, 1.0670, 1.0700 and 1.0750. The gradient was prepared by layering 1 ml of each of the appropriate solutions into culture tubes (17 X 100 mm). The top, seventh, layer on the gradient was 1--2 ml of cell suspension (d = 1.0200) described above. The tubes were centrifuged for 15 rain at 1000 rpm (International, Model PR-B with head No. 269), approximately 140 g. Seven fractions of cells were collected with a Pasteur pipette by removing each density layer plus the cells accumulated at its lower interface. Each of the fractions collected was placed in a glass tube (12 X 75 mm). To facilitate settling of the cells, the specific gravity of each fraction was reduced by diluting to 5 ml with BBS. The diluted suspensions were centrifuged at 2500 rpm for 10 min. The cell pellet was resuspended in 0.5 ml of a 5% dextran solution. The fractions collected from the gradient were analyzed for 1) total cells per fraction, determined using a Neubauer h e m o c y t o m e t e r , and 2) distribution of cell type within each fraction, determined oil the basis of morphology. Slides for the latter determination were prepared by applying two to three drops of the cell suspension to a glass microscope slide. The cells were
163
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7
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Fig. 1. D i s t r i b u t i o n o f cells as a f u n c t i o n o f the p e r c e n t o f c~,ll ~vpe f o r ~'aeh f r a c t i o n . Solid bars r e p r e s e n t s m a l l n o n - t r a n s f o r m e d l y m p l m c y t e s a n d strip(,d bars r e l ) r o s e n t transf o r m e d l y m p h o c y t c s . E a c h bar r e p r e s e n t s t h e m e a n o f 5 e x p ~ . r i m e n t s . ' T ' - b a r ref)r(~senls the standard deviation.
164
spread, air dried, fixed in 95% e t h a n o l and again allowed to air dry. T h e slides were stained with H e m a l B l o o d Stain ( H e m a l Stain Co., D a n b u r y , Ct.). T h e cells were c o u n t e d u n d e r oil i m m e r s i o n , at least 100 per slide. Cell viability was d e t e r m i n e d using t r y p a n blue exclusion as p r e v i o u s l y r e p o r t e d (Leise and LeSane, 1974}. I{FSULTS
Distribution o f cells on gradient T h e results o f the s e p a r a t i o n o f t r a n s f o r m e d l y m p h o c y t e s are s h o w n in fig. 1. It is clearly e v i d e n t t h a t f r a c t i o n 3 (d = 1 . 0 6 2 0 g/ml) is e n r i c h e d with t r a n s f o r m e d l y m p h o c y t e s to a p p r o x i m a t e l y 85%, w h e r e a s the cell p o p u l a tion placed on t h e g r a d i e n t c o n t a i n e d 30% t r a n s f o r m e d l y m p h o c y t e s . T h e d e n s e r f r a c t i o n s c o n t a i n a p r e d o m i n a n c e o f small n o n t r a n s f o r m e d l y m p h o cytes. Fig. 2 s h o w s the d i s t r i b u t i o n o f cell t y p e s on tile g r a d i e n t as a p e r c e n t a g e o f t o t a l cells r e c o v e r e d . A t o t a l o f 44% o f the cells r e c o v e r e d were transf o r m e d , o f w h i c h 54% a p p e a r in f r a c t i o n 3. T h e t o t a l o f the cells r e c o v e r e d is 70% o f t h a t p u t on the gradient. T h e d i s t r i b u t i o n o f cell t y p e s p u t on the gradient, was 70% n o n - t r a n s f o r m e d a n d 30% t r a n s f o r m e d l y m p h o c y t e s . A f t e r c e n t r i f u g a t i o n the d i s t r i b u t i o n in the t o t a l o f cells r e c o v e r e d was 56% and
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Fig. 2. Distribution of non-transformed lymphocytes (0) and transformed lymphocytes (o) as a function of the total cells r~,covered.
165
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Fig. 3. Distribution of the total lymphocyte population under three different conditions: without any incubation period (0); after 72 h of culture without PHA (O);after 72 h of culture in the presence of PHA (0). This data is based on 3 experiments.
44% respectively. It w o u l d appear then that there is a selective loss o f the n o n t r a n s f o r m e d cells. When the viability o f each fraction was assessed, n o adverse e f f e c t o f the gradient centrifugation was seen. Before centrifugation the viability was 8 8 - 92%, after centrifugation it was 90--92%.
Changes in cell distribution Fig. 3 s h o w s the distribution o f cells on the gradient under three different c o n d i t i o n s ; prior to culture, after 72 h culture with PHA, and after 72 h culture w i t h o u t PHA. These curves take intQ a c c o u n t all the l y m p h o c y t e s present and in the case o f those cultured with PHA includes the transformed
166 cells. This c o n t r a s t s with fig. 2 w h i c h differentiates b e t w e e n t r a n s f o r m e d and n o n - t r a n s f o r m e d cells. Cells prior to c u l t u r e are primarily f o u n d in the first t h r e e fractions with a peak in f r a c t i o n 2 {d = 1 . 0 6 0 0 g/ml), r e p r e s e n t i n g 35% o f the total recovered cells. Cells c u l t u r e d with P H A s h o w a shift t o w a r d s the layers less dense than t h o s e i n c u b a t e d w i t h o u t the lectin, with a peak o f 27% in f r a c t i o n 3. Cells c u l t u r e d for 72 h w i t h o u t lectin, s h o w a shift to the denser layers, fractions 4--6 (d = 1 . 0 6 4 0 - - 1 . 0 7 0 0 g/ml). SUMMARY AND CONCLUSION It has been previously d e m o n s t r a t e d (Sell et al., 1 9 7 5 ) t h a t when l y m p h o c y t e s are c u l t u r e d in the presence o f p h y t o h e m a g g l u t i n i n t w o m o r p h o l o gically d i f f e r e n t cell p o p u l a t i o n s result. Using a d e x t r a n d i s c o n t i n u o u s d e n s i t y gradient, we have separated this m i x e d cell p o p u l a t i o n into several s u b p o p u l a t i o n s . F r o m fig. 1 it is a p p a r e n t t h a t the t r a n s f o r m e d cells were c o n c e n t r a t e d in the layer having a d e n s i t y o f 1 . 0 6 2 0 g/ml o f d e x t r a n . The d i s t r i b u t i o n o f cells in this fraction was a p p r o x i m a t e l y 85% t r a n s f o r m e d cells and 15% n o n - t r a n s f o r m e d l y m p h o c y t e s . O f all the t r a n s f o r m e d cells recovered, 54% were f o u n d in the 1 . 0 6 2 0 g/ml f r a c t i o n (fig. 2). F o l l o w i n g 72 h o f culture, cells b e c o m e m o r e dense as c o m p a r e d to the cell d i s t r i b u t i o n i m m e d i a t e l y after isolation (fig. 3). This change in the d i s t r i b u t i o n m i g h t be due to the repair o f injury incurred at the time o f cell isolation ( L o o s and R o o s , 1 9 7 4 ; S t e w a r t et al., 1975). Cells c u l t u r e d with p h y t o h e m a g g l u t i n i n also increase in density, b u t n o t to the same c x t e n t as those c u l t u r e d w i t h o u t lectin. REFERENCES B~yum, A., 1968, Scan. J. Clin. Lab. Invest. Suppl. 97, 9. Coulson, A., 1964, Lancet 1,468. Faguet, G., 1974, J. Retic. Soc. 16(2), 114. Hilal, S., D. Messer, M. Lohen and R. Johnson, 1964, Ann. N.Y. Acad. Sei. 11,1,661. Knight, S., J. Farrant and G. Morris, 1972, Nature New Biol. 239, 88. Leise, E., T. Morita, I. Gray and F. LeSane, 1970, Biochem. Med. ,1, 327. Leise, E. and F. LeSane, 197,1, Prep. Biochem. 4(5), 395. Ling, N. and J. Kay, 1975, Lymphocyte Stimulation (North-Holland Publishing Co., Amsterdam, Netherlands). Loos, J. and D. Roos, 1974, Exp. Cell. Res. 86, 3.12. Newlin, C., 1973, Cell. Immunol. 8, 198. Parker, C., T. Sullivan and J. Wedner, 1974, Adv. Cyclic Nucl. Res. 4, I. Sasaki, M. and A. Norman, 1966, Nature, 210,913. Sell, S., H. Sheppard and D. Redelman, 1975, Exp. Cell Res. 90, 309. Serler, F., R. Johannsen, H. Sedlacck and K. Zeiller, 1974, Transplant. Proc. 6(2), 173. Stewart, C., S. Cramer and P. Steard, 1975, (Jell lmmunol. 16, 237. Turner, R., 1967, J. Cell Physiol. 69, 73. Uwlas, B. and 1. Thon, 1959, Exp. Cell Res. 18, 512.