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Primary antibody response of rabbit blood lymphocytes in vitro
Journal of Immunological Methods, 47 (1981) 201--208
201
Elsevier/North-Holland Biomedical Press
PRIMARY A N T I B O D Y RESPONSE OF RABBIT BLOOD LYMPHOCYTES IN VITRO
ALMA L. L U Z Z A T I 1 and CARLO RAMONI
Laboratory of Cell Biology and Immunology, Istituto Superiore di SanitY, 00161 Rome, Italy, and Basel Institute for Immunology, Basel, Switzerland (Received 9 March 1981, accepted 8 July 1981)
This paper describes a method for in vitro induction of a primary response of rabbit peripheral blood lymphocytea to sheep red blood cells. The response is measured by visualizing and enumerating the plaque-forming cells (PFC). Removal of an adhering suppressor cell and use of a low cell concentration in culture are among the crucial requirements. Maximum response was usually reached after 10--15 days of culture. The number of PFC then decreased or stayed at roughly plateau level at least up to the fourth week of culture, when most of the experiments were terminated. In several instances the response had a cyclical character with repeating peaks of PFC. Only plaques of the direct type were found.
INTRODUCTION
Peripheral blood lymphocytes (PBL) from rabbits primed in vivo can be restimulated in vitro to give a secondary response. This was shown both for sheep red blood cells (SRBC) (Luzzati et al., 1973) and for streptococcal polysaccharide (Read and Braun, 1974; Braun et al., 1976) as antigen. The 'cell separation and culture conditions have been described in detail (Luzzati, 1979) and the role of different regulatory cells (Luzzati and Lafleur, 1976; Vaeck et al., 1980) was studied. Our first attempts to stimulate unprimed lymphocytes met with some difficulties (Luzzati et al., 1973). Because of its importance for studies of the events which occur during induction and development of a humoral immune response, we decided to investigate thoroughly the conditions required for a primary response to SRBC. The present paper gives the results of this study and describes some important and interesting aspects of the responses obtained.
1 Correspondence: Alma L. Luzzati, Laboratory of Cell Biology and Immunology, Istituto Superiore di SanitY, Viale Regina Elena 299, 00161 Rome, Italy. 0922-1759/81/0000--0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
202 MATERIALS AND METHODS
Animals Adult rabbits of both sexes were used. Some of the experiments were performed with animals quarantined in isolated rooms, others with animals kept in a less controlled environment. Cell separation and culture The blood was obtained from the central artery of the ear and collected into heparinized bottles. Leukocytes were isolated by the m e t h o d previously described (Luzzati, 1979). Briefly, whole blood was passed through a column of synthetic wool (Coop, Basel, Switzerland, no. 609002) and then mixed with 1% pig skin gelatin (Eastman Kodak Co., Rochester, NY, no. 5242). The red cells were allowed to settle and the l y m p h o c y t e rich supernatant was collected. The cells were extensively washed and suspended at an appropriate concentration in RPMI 1640 (Microbiological Associates, Bethesda, MD, no. 12-702) containing 10% fetal calf serum (FCS) and 50 U/ml of both penicillin and streptomycin. One ml aliquots were distributed in plastic tubes (Falcon Plastics, type 2003). Antigen was added in the form of 50 pl of 1% SRBC/ml. The tubes were placed in a vertical position in a humidified 5% CO2 incubator at 37°C. Harvesting o f cultures and assay o f PFC At different intervals after initiation, duplicate I ml cultures were pooled and assayed for PFC with a slide modification (Luzzati, 1979) of the hemolysis in gel m e t h o d (Jerne and Nordin, 1963). Tests for indirect plaques were done with sheep anti-rabbit Ig diluted I : 3000. The 2-mercaptoethanol sensitivity of the plaques was tested as described by Jerne et al. (1974). RESULTS
Cell separation and cell density As shown in Table 1 filtration of the blood through synthetic wool usually improved the o u t c o m e of the cultures. With some low responding animals PFC could be induced only following this treatment. With higher responding rabbits the effect was mostly evident at later stages of the cultures, resulting in a more persistent antibody response. The efficiency o f the response was highly dependent on the cell density (Table 2), with an o p t i m u m of 1 × 106 cells/ml. Higher concentrations resulted in considerable reduction of PFC, while 0.5 × 106 cells/ml yielded more variable results (not shown). Poor responding animals did n o t perform better with different cell doses.
203 TABLE 1 Effect of synthetic wool treatment. Rabbit
Wool
P l a q u e - f o r m i n g cells p e r c u l t u r e
no.
1672 080 020
+ __a
Day 12
Day 16
Day 20
Day 28
3 050 1 325
5650 32400
1 680 110
6 800 0
+
265
10
895
5
--
0
0
0
0
+
0
0
0
0
--
0
0
30
0
a T h e w o o l f i l t r a t i o n step was o m i t t e d f r o m t h e l y m p h o c y t e p r e p a r a t i o n p r o c e d u r e .
Antigen requirement As shown in Table 3, the response was entirely d e p e n d e n t on the presence o f antigen. As with the mouse system (Mishell and D u t t o n , 1967), SRBC obtained from different sheep varied in their capacity to stimulate a specific antibody response (Table 4).
Culture medium The medium used t h r o u g h o u t was RPMI 1640 supplemented with 10% FCS and antibiotics. Different batches of FCS varied greatly in their supportive capacity. Addition o f 5 X 10 -s M 2-mercaptoethanol did n o t produce any appreciable change in the results (data n o t shown).
Variability among different rabbits The results in Table 1 show that the magnitude o f the response varied from rabbit to rabbit. This difference seemed to be a reproducible characteristic of the animal: rabbit 020 never showed an appreciable response, while rabbit 1672 consistently gave high numbers of PFC and rabbit 080 TABLE 2 E f f e c t o f d i f f e r e n t cell d o s e s in c u l t u r e . Cells X 10 6
1 2 3
P l a q u e - f o r m i n g cells p e r c u l t u r e Day 13
Day 18
Day 2 2
Day 25
Day 28
4300 10 0
5200 1170 15
12000 3400 20
9700 10 15
9850 160 0
204 TABLE 3 Antigen dependence of the response. Antigen
P l a q u e - f o r m i n g cells p e r c u l t u r e Day 9
--
+
0
10300
Day 14
Day 18
100
0
16750
1925
responded in an intermediate range. Rabbit 1672 was used in 12 different experiments over a period of 4 months. Fig. 1 shows the results of 11 experiments: peak responses fell in a relatively narrow range. Only in 1 experiment, n o t reported in the figure, were very few PFC obtained. The rabbits used for the above experiments were housed in isolated rooms. Animals kept in a less controlled environment gave more variable results. One o f these rabbits (no. 256) was used in 9 experiments over a period of 8 m o n t h s : in 2 o f these very low numbers of PFC were induced, in the other 7 instances the results shown in Fig. 2 were obtained. It is clear t h a t peak responses vary over a large range. Time course o f appearance o f PFC Plaque-forming cells were rarely detectable before day 5. Thereafter their number increased, reaching a m a x i m u m at days 10--15. In some experiments the number of PFC decreased rapidly after day 20; in others no drop of response was observed up to 4 weeks (Fig. 1). In some cases the appearance o f PFC had a cyclical character, showing two or more peaks (Figs. 2 and 3). The plaques were always all of the direct type and could be completely inhibited by treatment with 2-mercaptoethanol.
TABLE 4 V a r i a b i l i t y in the in v i t r o r e s p o n s e t o e r y t h r o c y t e s o b t a i n e d f r o m d i f f e r e n t sheep. C u l t u r e s were a s s a y e d o n t h e r e d cells used as i m m u n i z i n g a n t i g e n . S h e e p no.
214 907 517 518 519 520
P l a q u e - f o r m i n g cells p e r c u l t u r e Day 11
Day 15
Day 18
Day 22
560 21050 7450 700 65 535
4700 28200 270 730 40 4 650
8750 4 500 24400 340 15 2600
4 300 15650 22900 2650 75 90
205
10 5
10 ~"
cc
.~
103:
L 10 2
5
10
15
20
25
30
bays in culfure
Fig. 1. P F C r e s p o n s e in v i t r o o f r a b b i t 1 6 7 2 in 11 d i f f e r e n t e x p e r i m e n t s .
~
10 +
'1 i,o~1 i
g ....
1'o'~'~'
' ' i2'o ....
"
2s=
Days in culture
Fig. 2. P F C r e s p o n s e in v i t r o o f r a b b i t 2 5 6 in 7 d i f f e r e n t e x p e r i m e n t s .
206
10 5
2 -~ 10 ~ c~
c
10 3
j - J
10 ~
5
10
15
20
25
30
Days in
35 culture
Fig. 3. PFC response in vitro: one experiment (o) was assayed from day 3 to day 17; a second experiment (o) with the same animal was assayed from day 11 to day 36.
When enough antibody had accumulated in the culture medium to allow a hemagglutination titration, complete inhibition of the activity was observed upon t r e a t m e n t with 2-mercaptoethanol (not shown). DISCUSSION
We describe a m e t h o d for the induction of a primary antibody response in cultures of rabbit blood lymphocytes. Optimal results required removal of a population of adhering inhibitor cells and the use of a low cell concentration in culture. These findings support the n o t i o n of the existence in the circulating blood of an unbalanced ratio of helper and suppressor cells (Luzzati and Lafleur, 1976; Luzzati et al., 1979). The o p t i m u m cell dose for induction of a primary response is 4 times lower than that for secondary responses to the same antigen (Luzzati et al., 1973), probably owing to a change in the ratio or in the nature o f the circulating cells following immunization. Under the conditions described significant differences in the capacity to m o u n t a response were observed among different rabbits. No correlation was f o u n d between the magnitude of the response induced in vitro and the titer of anti-SRBC agglutinin in the serum (not shown). Similar individual variations in capacity to respond to SRBC were observed in cultures of normal h u m a n PBL (Luzzati et al., 1979). In the mouse also the magnitude of the immune response in vitro is dependent u p o n the
207
strain used as spleen cell source (Schreier and Nordin, 1977). However, the data presented show that some environmental conditions m a y partially hide the individual characteristic of the animal: rabbits housed under conditions likely to expose them to different infections showed a more variable in vitro response. Since animals housed under different conditions were never directly compared in the same experiment, other reasons for these findings cannot be excluded. However, similar observations were made in mice b y Schreier and Nordin (1977). These authors were able in some cases to correlate failures to induce anti-SRBC PFC in cultures of mouse spleen cells with the existence of infection in the animal colony. It is thus clear that an uncontrollable interior environment created in the host b y infection may dramatically affect the in vitro immune response. The kinetics of the response in the in vitro system described here deserves some c o m m e n t . The onset of the response is rather slow and, once the m a x i m u m is reached, the cultures may. remain active for several weeks. In many experiments the response had a cyclical character, with repeating peaks of PFC at approximately weekly intervals. Similar kinetics in an in vitro system analogous to that described here have recently been observed by others (Demeur et al., 1981). In vivo, cyclical responses were reported in different species for both thymus
208
In conclusion, the in vitro system here described allows a vigorous primary antibody response to be induced and maintained. It shows some important and interesting features, which make it a unique tool for studies of regulatory mechanisms operating in the immune response. ACKNOWLEDGEMENT
We are grateful to Ms. Christine Werz for the skillful technical assistance. REFERENCES Braun, D.G., J. Quint~ns, A.L. Luzzati, I. Lefkovits and S.E. Read, 1976, J. Exp. Med. 143,360. Demeur, C., G. Urbain-Vansanten, M. Vaeck, C. Bruyns and J. Urbain, 1981, Immunol. Lett. 2, 297. Jerne, N.K. and A.A. Nordin, 1963, Science 1 4 0 , 4 0 5 . Jerne, N.K., C. Henry, A.A. Nordin, H. Fuji, A.M.C. Koros and I. Lefkovits, 1974, Transplant. Rev. 1 8 , 1 3 0 . Kelsoe, G. and J. Cerny, 1979, Nature 2 7 9 , 3 3 3 . Luzzati, A.L., 1979, in: Immunological Methods, eds. I. Lefkovits and B. Pernis (Academic Press, New York) p. 335. Luzzati, A.L. and L. Lafleur, 1976, Eur. J. Immunol. 6 , 1 2 5 . Luzzati, A.L., I. Lefkovits and B. Pernis, 1973, Eur. J. Immunol. 3 , 6 3 2 . Luzzati, A.L., I. Heinzer, H. Hengartner and M.H. Schreier, 1979, Clin. Exp. Immunol. 35,405. Mishell, R.I. and R.W. Dutton, 1967, J. Exp. Med. 1 2 6 , 4 2 3 . Read, S.E. and D.G. Braun, 1974, Eur. J. Immunol. 4 , 4 2 2 . Schreier, M. and A.A. Nordin, 1977, in: B and T cells in Immune Recognition, eds. F. Loor and G.E. Roelants (Wiley and Sons, London) p. 127. Vaeck, M., W. De Smet and P. De Baetselier, 1980, Eur. J. Immunol. 1 0 , 6 2 7 . Weigle, W.O., 1975, Adv. Immunol. 2 1 , 8 7 .
201
Elsevier/North-Holland Biomedical Press
PRIMARY A N T I B O D Y RESPONSE OF RABBIT BLOOD LYMPHOCYTES IN VITRO
ALMA L. L U Z Z A T I 1 and CARLO RAMONI
Laboratory of Cell Biology and Immunology, Istituto Superiore di SanitY, 00161 Rome, Italy, and Basel Institute for Immunology, Basel, Switzerland (Received 9 March 1981, accepted 8 July 1981)
This paper describes a method for in vitro induction of a primary response of rabbit peripheral blood lymphocytea to sheep red blood cells. The response is measured by visualizing and enumerating the plaque-forming cells (PFC). Removal of an adhering suppressor cell and use of a low cell concentration in culture are among the crucial requirements. Maximum response was usually reached after 10--15 days of culture. The number of PFC then decreased or stayed at roughly plateau level at least up to the fourth week of culture, when most of the experiments were terminated. In several instances the response had a cyclical character with repeating peaks of PFC. Only plaques of the direct type were found.
INTRODUCTION
Peripheral blood lymphocytes (PBL) from rabbits primed in vivo can be restimulated in vitro to give a secondary response. This was shown both for sheep red blood cells (SRBC) (Luzzati et al., 1973) and for streptococcal polysaccharide (Read and Braun, 1974; Braun et al., 1976) as antigen. The 'cell separation and culture conditions have been described in detail (Luzzati, 1979) and the role of different regulatory cells (Luzzati and Lafleur, 1976; Vaeck et al., 1980) was studied. Our first attempts to stimulate unprimed lymphocytes met with some difficulties (Luzzati et al., 1973). Because of its importance for studies of the events which occur during induction and development of a humoral immune response, we decided to investigate thoroughly the conditions required for a primary response to SRBC. The present paper gives the results of this study and describes some important and interesting aspects of the responses obtained.
1 Correspondence: Alma L. Luzzati, Laboratory of Cell Biology and Immunology, Istituto Superiore di SanitY, Viale Regina Elena 299, 00161 Rome, Italy. 0922-1759/81/0000--0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
202 MATERIALS AND METHODS
Animals Adult rabbits of both sexes were used. Some of the experiments were performed with animals quarantined in isolated rooms, others with animals kept in a less controlled environment. Cell separation and culture The blood was obtained from the central artery of the ear and collected into heparinized bottles. Leukocytes were isolated by the m e t h o d previously described (Luzzati, 1979). Briefly, whole blood was passed through a column of synthetic wool (Coop, Basel, Switzerland, no. 609002) and then mixed with 1% pig skin gelatin (Eastman Kodak Co., Rochester, NY, no. 5242). The red cells were allowed to settle and the l y m p h o c y t e rich supernatant was collected. The cells were extensively washed and suspended at an appropriate concentration in RPMI 1640 (Microbiological Associates, Bethesda, MD, no. 12-702) containing 10% fetal calf serum (FCS) and 50 U/ml of both penicillin and streptomycin. One ml aliquots were distributed in plastic tubes (Falcon Plastics, type 2003). Antigen was added in the form of 50 pl of 1% SRBC/ml. The tubes were placed in a vertical position in a humidified 5% CO2 incubator at 37°C. Harvesting o f cultures and assay o f PFC At different intervals after initiation, duplicate I ml cultures were pooled and assayed for PFC with a slide modification (Luzzati, 1979) of the hemolysis in gel m e t h o d (Jerne and Nordin, 1963). Tests for indirect plaques were done with sheep anti-rabbit Ig diluted I : 3000. The 2-mercaptoethanol sensitivity of the plaques was tested as described by Jerne et al. (1974). RESULTS
Cell separation and cell density As shown in Table 1 filtration of the blood through synthetic wool usually improved the o u t c o m e of the cultures. With some low responding animals PFC could be induced only following this treatment. With higher responding rabbits the effect was mostly evident at later stages of the cultures, resulting in a more persistent antibody response. The efficiency o f the response was highly dependent on the cell density (Table 2), with an o p t i m u m of 1 × 106 cells/ml. Higher concentrations resulted in considerable reduction of PFC, while 0.5 × 106 cells/ml yielded more variable results (not shown). Poor responding animals did n o t perform better with different cell doses.
203 TABLE 1 Effect of synthetic wool treatment. Rabbit
Wool
P l a q u e - f o r m i n g cells p e r c u l t u r e
no.
1672 080 020
+ __a
Day 12
Day 16
Day 20
Day 28
3 050 1 325
5650 32400
1 680 110
6 800 0
+
265
10
895
5
--
0
0
0
0
+
0
0
0
0
--
0
0
30
0
a T h e w o o l f i l t r a t i o n step was o m i t t e d f r o m t h e l y m p h o c y t e p r e p a r a t i o n p r o c e d u r e .
Antigen requirement As shown in Table 3, the response was entirely d e p e n d e n t on the presence o f antigen. As with the mouse system (Mishell and D u t t o n , 1967), SRBC obtained from different sheep varied in their capacity to stimulate a specific antibody response (Table 4).
Culture medium The medium used t h r o u g h o u t was RPMI 1640 supplemented with 10% FCS and antibiotics. Different batches of FCS varied greatly in their supportive capacity. Addition o f 5 X 10 -s M 2-mercaptoethanol did n o t produce any appreciable change in the results (data n o t shown).
Variability among different rabbits The results in Table 1 show that the magnitude o f the response varied from rabbit to rabbit. This difference seemed to be a reproducible characteristic of the animal: rabbit 020 never showed an appreciable response, while rabbit 1672 consistently gave high numbers of PFC and rabbit 080 TABLE 2 E f f e c t o f d i f f e r e n t cell d o s e s in c u l t u r e . Cells X 10 6
1 2 3
P l a q u e - f o r m i n g cells p e r c u l t u r e Day 13
Day 18
Day 2 2
Day 25
Day 28
4300 10 0
5200 1170 15
12000 3400 20
9700 10 15
9850 160 0
204 TABLE 3 Antigen dependence of the response. Antigen
P l a q u e - f o r m i n g cells p e r c u l t u r e Day 9
--
+
0
10300
Day 14
Day 18
100
0
16750
1925
responded in an intermediate range. Rabbit 1672 was used in 12 different experiments over a period of 4 months. Fig. 1 shows the results of 11 experiments: peak responses fell in a relatively narrow range. Only in 1 experiment, n o t reported in the figure, were very few PFC obtained. The rabbits used for the above experiments were housed in isolated rooms. Animals kept in a less controlled environment gave more variable results. One o f these rabbits (no. 256) was used in 9 experiments over a period of 8 m o n t h s : in 2 o f these very low numbers of PFC were induced, in the other 7 instances the results shown in Fig. 2 were obtained. It is clear t h a t peak responses vary over a large range. Time course o f appearance o f PFC Plaque-forming cells were rarely detectable before day 5. Thereafter their number increased, reaching a m a x i m u m at days 10--15. In some experiments the number of PFC decreased rapidly after day 20; in others no drop of response was observed up to 4 weeks (Fig. 1). In some cases the appearance o f PFC had a cyclical character, showing two or more peaks (Figs. 2 and 3). The plaques were always all of the direct type and could be completely inhibited by treatment with 2-mercaptoethanol.
TABLE 4 V a r i a b i l i t y in the in v i t r o r e s p o n s e t o e r y t h r o c y t e s o b t a i n e d f r o m d i f f e r e n t sheep. C u l t u r e s were a s s a y e d o n t h e r e d cells used as i m m u n i z i n g a n t i g e n . S h e e p no.
214 907 517 518 519 520
P l a q u e - f o r m i n g cells p e r c u l t u r e Day 11
Day 15
Day 18
Day 22
560 21050 7450 700 65 535
4700 28200 270 730 40 4 650
8750 4 500 24400 340 15 2600
4 300 15650 22900 2650 75 90
205
10 5
10 ~"
cc
.~
103:
L 10 2
5
10
15
20
25
30
bays in culfure
Fig. 1. P F C r e s p o n s e in v i t r o o f r a b b i t 1 6 7 2 in 11 d i f f e r e n t e x p e r i m e n t s .
~
10 +
'1 i,o~1 i
g ....
1'o'~'~'
' ' i2'o ....
"
2s=
Days in culture
Fig. 2. P F C r e s p o n s e in v i t r o o f r a b b i t 2 5 6 in 7 d i f f e r e n t e x p e r i m e n t s .
206
10 5
2 -~ 10 ~ c~
c
10 3
j - J
10 ~
5
10
15
20
25
30
Days in
35 culture
Fig. 3. PFC response in vitro: one experiment (o) was assayed from day 3 to day 17; a second experiment (o) with the same animal was assayed from day 11 to day 36.
When enough antibody had accumulated in the culture medium to allow a hemagglutination titration, complete inhibition of the activity was observed upon t r e a t m e n t with 2-mercaptoethanol (not shown). DISCUSSION
We describe a m e t h o d for the induction of a primary antibody response in cultures of rabbit blood lymphocytes. Optimal results required removal of a population of adhering inhibitor cells and the use of a low cell concentration in culture. These findings support the n o t i o n of the existence in the circulating blood of an unbalanced ratio of helper and suppressor cells (Luzzati and Lafleur, 1976; Luzzati et al., 1979). The o p t i m u m cell dose for induction of a primary response is 4 times lower than that for secondary responses to the same antigen (Luzzati et al., 1973), probably owing to a change in the ratio or in the nature o f the circulating cells following immunization. Under the conditions described significant differences in the capacity to m o u n t a response were observed among different rabbits. No correlation was f o u n d between the magnitude of the response induced in vitro and the titer of anti-SRBC agglutinin in the serum (not shown). Similar individual variations in capacity to respond to SRBC were observed in cultures of normal h u m a n PBL (Luzzati et al., 1979). In the mouse also the magnitude of the immune response in vitro is dependent u p o n the
207
strain used as spleen cell source (Schreier and Nordin, 1977). However, the data presented show that some environmental conditions m a y partially hide the individual characteristic of the animal: rabbits housed under conditions likely to expose them to different infections showed a more variable in vitro response. Since animals housed under different conditions were never directly compared in the same experiment, other reasons for these findings cannot be excluded. However, similar observations were made in mice b y Schreier and Nordin (1977). These authors were able in some cases to correlate failures to induce anti-SRBC PFC in cultures of mouse spleen cells with the existence of infection in the animal colony. It is thus clear that an uncontrollable interior environment created in the host b y infection may dramatically affect the in vitro immune response. The kinetics of the response in the in vitro system described here deserves some c o m m e n t . The onset of the response is rather slow and, once the m a x i m u m is reached, the cultures may. remain active for several weeks. In many experiments the response had a cyclical character, with repeating peaks of PFC at approximately weekly intervals. Similar kinetics in an in vitro system analogous to that described here have recently been observed by others (Demeur et al., 1981). In vivo, cyclical responses were reported in different species for both thymus
208
In conclusion, the in vitro system here described allows a vigorous primary antibody response to be induced and maintained. It shows some important and interesting features, which make it a unique tool for studies of regulatory mechanisms operating in the immune response. ACKNOWLEDGEMENT
We are grateful to Ms. Christine Werz for the skillful technical assistance. REFERENCES Braun, D.G., J. Quint~ns, A.L. Luzzati, I. Lefkovits and S.E. Read, 1976, J. Exp. Med. 143,360. Demeur, C., G. Urbain-Vansanten, M. Vaeck, C. Bruyns and J. Urbain, 1981, Immunol. Lett. 2, 297. Jerne, N.K. and A.A. Nordin, 1963, Science 1 4 0 , 4 0 5 . Jerne, N.K., C. Henry, A.A. Nordin, H. Fuji, A.M.C. Koros and I. Lefkovits, 1974, Transplant. Rev. 1 8 , 1 3 0 . Kelsoe, G. and J. Cerny, 1979, Nature 2 7 9 , 3 3 3 . Luzzati, A.L., 1979, in: Immunological Methods, eds. I. Lefkovits and B. Pernis (Academic Press, New York) p. 335. Luzzati, A.L. and L. Lafleur, 1976, Eur. J. Immunol. 6 , 1 2 5 . Luzzati, A.L., I. Lefkovits and B. Pernis, 1973, Eur. J. Immunol. 3 , 6 3 2 . Luzzati, A.L., I. Heinzer, H. Hengartner and M.H. Schreier, 1979, Clin. Exp. Immunol. 35,405. Mishell, R.I. and R.W. Dutton, 1967, J. Exp. Med. 1 2 6 , 4 2 3 . Read, S.E. and D.G. Braun, 1974, Eur. J. Immunol. 4 , 4 2 2 . Schreier, M. and A.A. Nordin, 1977, in: B and T cells in Immune Recognition, eds. F. Loor and G.E. Roelants (Wiley and Sons, London) p. 127. Vaeck, M., W. De Smet and P. De Baetselier, 1980, Eur. J. Immunol. 1 0 , 6 2 7 . Weigle, W.O., 1975, Adv. Immunol. 2 1 , 8 7 .