CELLULAR
IMMUNOLOGY
Cellular
38,
193-197
(1978)
Immune Response toward MS-2 Phage and a Synthetic Fragment of Its Coat Protein HARRY LANGBEHEIM, Department
DVORA TEITELBAUM,
AND RUTH
of ChemicalImntunology,The Weizmann of Scielzce,Rehovot, Israel Received
November
ARNON
Irzstit~~te
7, 1977
The cellular immune responses toward MS-Z phage and toward a synthetic fragment of its coat protein conjugated to synthetic carrier (PaA--L) were evaluated in guinea pigs, both in vivo by the delayed-type hypersensitivity skin test and in vitro by lymphocyte transformation assay. Both MS-Z phage and PrA--L elicited high homologous cellular reaction, but no significant cross-reaction was observed between them.
INTRODUCTION The system studied in this investigation involves a synthetic conjugate, denoted Ps-A-L ( 1) , consisting of a synthetic fragment (Pz) of the coat protein of MS-2 coliphage attached to a synthetic carrier (multi-poly-uL-alanyl-poly-L-lysine), and its immunological comparison with the intact phage. In a previous report from this laboratory we have shown that the synthetic fragment Pz is involved in the neutralization of the phage, by antiphage serum, in the sensethat it is capable of inhibiting this inactivation. Furthermore, when attached to a carrier, the completely synthetic conjugate elicited, in rabbits and guinea pigs, antibodies capable of neutralizing intact MS-2 phage (1, 2). These findings clearly indicate a cross-reaction between the MS-2 phage and the conjugate Pz-A-L, on the humoral level. It has been shown that both the specificity and the manifestation of humoral and cellular responsesare not always parallel (3). There are several examples that illustrate this point : Lysozyme and its unfolded polypeptide chain do not cross-react humorally but show cross-reaction on the cellular level (4). A similar phenomenon was observed in the interaction between mammalian and Ascaris collagen (5) or between lysozyme and cr-lactalbumin (6) and between myelin basic protein and Fza histone (7). In the case of glucagon it has been demonstrated that the amino-terminal part of the molecule binds with antibodies while another region in the C terminus induces lymphocyte transformation (8). In this study we have investigated the cellular immune response towartl the intact MS-2 phage and the synthetic P,-A-I. conjugate and the cross-reaction between them on the cellular level. 193 000%8749/78/0381-0193%02.00/(1 All
Copyright 0 1978 by Academic Press, Inr rights of reproduction in any form reserved.
194
SHORT
MATERIALS
COMMUNICATIONS
AND
METHODS
Phge and reagents. Bacteriophage strain MS-2 was grown on Escherichia coli C3OOO according to the method of Gesteland and Spahr (9). Poly-DL-alanyl-poly+lysine (A-L) was prepared as described by Sela et al. (10). Animals. Random-bred DH albino guinea pigs weighing 350-500 g were obtained from the Experimental Animal Center of the Weizmann Institute of Science. Isolation of phage MS-2 coat protein. The procedure we used is based on that of Hohn (11). 0 ne volume of a purified suspension of infectious phage (10 mg) suspended in buffer (0.1 M NaCI, 0.01 M Tris, pH 7.6) was added to 2 ~01 of precooled glacial acetic acid. This treatment leads to a precipitate of A protein and RNA, whereas the major coat protein remains soluble in the supernatant. After 15 min, the precipitated RNA-A protein complex was removed by centrifugation for 10 min at 10,000 rpm (Sorvall SS-34 rotor) ; the protein was decanted and dialyzed against three changes of distilled water resulting in a protein precipitate. This precipitate was sedimented for 15 min at 10,000 rpm (Sorvall SS-34 rotor) and lyophilized. Preparation of Pz peptide and Ps-A-L conjugate. The Pz fragment corresponds to region 89-108 in the amino acid sequence of the MS-2 coat protein, namely, GluLeu-Thr-Ile-Pr~Ile-Phe-Ala-Thr-Asn-Ser-AspCys-Glu-Leu-Ile-Val-LysAla-(Met). For the synthesis of fragment Pg the Merrifield solid-phase technique was used (12). Pz-A-L was prepared as described previously by linking the Pz fragment to the synthetic carrier A -L via carbodiimide reagent (1). Delayed hypersensitivity reactions. Guinea pigs were sensitized by injection into the hind footpads of 100 pg of the antigen in complete Freund’s adjuvant (Difco Laboratories, Detroit, Mich.). They were tested 10 days later by intradermal injection of 20 or 50 ,,,g of the test antigen in 0.1 ml of physiological saline (0.15 M NaCl). The appearance of erythema and induration at the site of injection was observed after 3, 24, and 48 hr and the extent of the delayed hypersensitivity was quantitated by measuring the diameter of the skin reaction. Only reactions larger than 8 mm in diameter were considered positive. Lymphocyte stimulation. Lymphocyte stimulation was studied by [3H] thymidine incorporation in vitro. Draining lymph nodes were excised 12-20 days after sensitization. They were trimmed of fat and teased apart under sterile conditions in RPM1 1640 medium with added antibiotics (penicillin, 100 mg/ml; streptomycin, 100 units/ml ; (BioLab, Laboratories, Ltd., Jerusalem, Israel). Cells were washed three times in the same medium and plated on a petri dish for 1 hr at 37°C. The nonadherent cells were suspended in medium supplemented with 3% fetal calf serum and glutamine (2 mM>. The lymphocytes (1 x lo6 cells/culture) were incubated in microtiter plates (Falcon, Oxnard, Calif.) for 72 hr with varying concentrations of the test antigens, followed by 5-16 hr of incubation with 1 &i of [methyZ-8H]The cultures were then thymidine (Nuclear Research Centre, Negev, Israel). harvested in an automatic harvesting machine. The filters were dried and the radioactivity was measured in a Tri-Carb liquid scintillation spectrometer (Packard). The results are expressed as stimulation indexes namely, the ratio of the radioactivity (in counts per minute) in cultures containing antigen to the radioactivity in antigen-free cultures (control), and these represent the averages of values obtained in triplicate cultures.
SHORT
TABLE Skin
Test
Reactions
of Guinea
Sensitization with
Skin Immediate
195
COMMUNICATIONS
1
Pigs to MS-2 test
(mean
phagc
and
diameter
in mm) Delayed
response
I’m-A-L
response
3 hr 48 hr
24 hr P2-A-L
Phage
Phage Phage P,-A-L Adjuvant ” Numbers
19.2 16.0 -
(4/4)‘= (3/4) (O/4)
in parentheses
14.0 15.0 represent
12.0 (4/4) 9.0 (2/4) (O/4)
(4/4) (4/4) (O/4)
the proportion
P,pA-L 14.7 (4/4) 9.0 (l/4) -
of animals
(O/4)
Phage 9.2 (4/4) - (O/4) - (O/4)
P,pA-L 9.7 (4/4) - (O/4) - (O/S)
reacting.
RESULTS Delayed Hypersensitivity
Reactions
to MS-2 Phage and P2-A-L
The cellular immune responses to the MS-2 phage and to the synthetic conjugate P2-A-L were studied by the in viva delayed hypersensitivity skin test. Guinea pigs sensitized with MS-2 phage or Pz-A-L were skin tested with these antigens, and immediate (3 hr) and delayed reactions (24 and 48 hr) were followed. These were compared with the reactions obtained in control animals sensitized with adjuvant alone. The results summarized in Table 1 demonstrate that guinea pigs sensitized with phage or P2-A-L exhibit strong immediate reactions to the homologous test antigens. In addition, a high degree of cross-reactivity between the MS-2 phage and the PB--A-L was observed. Delayed reactions to these antigens were also manifested, especially in animals sensitized with MS-2 phage, 24 hr and even 48 hr after the test injections. These animals gave positive delayed responses with both phage and Pz-A-L. However, since the immediate reactions were very strong, it is not absolutely certain whether the late response is a pure delayed type or a trailing of the immediate response, or a combination of the two. Further studies to establish this point were therefore performed using the lymphocyte transformation technique.
Lymphocyte
Transformation
Response
to MS-2 Phage and P2-A-L
The cellular immune response to MS-2 phage and Ps-A-L was evaluated in vitro by means of lymphocyte transformation, as measured by stimulation of incorporation of labeled thymidine. In lymphocyte cultures from six guinea pigs sensitized to the intact MS-2, high indexes of stimulation were obtained with the homologous antigen, but no significant cross stimulation was detectable with either the Pz peptide or the P2-A-L conjugate (Fig. 1). When P2-A-L was used for sensitization, a high cellular response was demonstrated with the homologous conjugate in all animals tested (seven guinea pigs), but only a weak reaction was observed with free Pz peptide, and no significant cellular response against the intact virus was observed (Fig. 2).
196
SHORT
COMMUNICATIONS
ANTlGENpglml FIG. 1. In vitro stimulation of lymph node cells by phage (A-A) P2 peptide (O-O) and PTA-L (U-W) in guinea pigs immunized with phage. Each dose-response curve was established in the same animal from triplicate cultures.
DISCUSSION The immunological system studied here serves as a model for a synthetic vaccine in the sensethat it has demonstrated the capacity of a completely synthetic conjugate to elicit neutralizing antibodies toward a living virus, the MS-2 coliphage. One of the crucial points in the host defense mechanism following vaccination is the cellular response it elicits, since in many cases cell-mediated phenomena are known to be a prerequisite for efficient protection and in others suppressor T cells may interfere with the efficacy of the process. The results of the present study demonstrate a strong cellular response elicited by both the intact phage and the synthetic conjugate. In the case of guinea pigs sensitized with Pa-A-L high indexes of lymphocyte stimulation were obtained in vitro in all the guinea pigs tested, though the delayed hypersensitivity response was very weak. This may be due to the functional distinction between T cell mediating delayed hypersensitivity and T helper cell (13). We have previously demonstrated that guinea pigs immunized with phage and the synthetic conjugate developed the antibody response (2). However, the specificity of this cellular reactivity is not parallel to that observed previously in the humoral response, namely, no significant
cross-reaction
between
the two antigens
was observed.
These
findings
are
in accord with results obtained with other systems showing differences in specificity between humoral and cellular responses(3-8). They are also in agreement with the
ANTIGEN
pglml
FIG. 2. In V&O stimulation of lymph node cells in Guinea pigs immunized with PrA--L, hy phage (A-A), Pz peptide ( O-O) and PTA--L (W-W). Each dose-response curve was established in the same animal from triplicate cultures.
SHORT
197
COMMUNICATIONS
known phenomenon that, in response to hapten- carrier conjugates, the resultant antibodies will react with the same hapten on a different carrier, but the sensitized T cell will recognize only the homologous hapten-carrier combination (14). The conclusion from this study is that, if indeed cellular immunity is important in vaccination, then in each case in which a synthetic material is used for vaccination against a particular organism, an evaluation of its capacity to elicit cell-mediated response is necessary. In particular it is essential to test for the cross-reactivity and similarity in specificity between the antigenic determinant used in the immunogen and the intact organism, on both humoral and cellular levels. ACKNOWLEDGMENT We wish to thank Mrs. Edna Kalef
for her skillful1
technical
assistance.
REFERENCES 1. Langbeheitn, H., Arnon, R., and Sela, M., Proc. Nat. Acad. Sci. US,4 73, 4636, 1976. 2. Langbeheim, H., Arnon, R., and Sela, M., Immunology, in press. 3. Senyk, G., Williams, E. B., Nitecki, D. E., and Goodman, J. W., 1. Ex#. Med. 133, 1294, 1971. 4. Thompson, K., Harris, M., Benyamin, E., Mitchell, G., and Noble, M., Nature New Biol. 238, 20, 1972. 5. Michaeli, D., Senyk, G., Maoz, A., and Fuchs, S., Inzmunolo~y 109, 103, 1972. 6. Maron, E., Webb, C., Teitelbaum, D., and Arnon, R., Eur. J. Inzmu~ol. 2, 294, 1972. 7. Bustin, M., Teitelbaum, D., and Webb, C., Eur. J. Biochem. 53, 615, 1975. 8. Senyk, G., Williams, E. B., Nitecki, D. E., and Goodman, J. W., J. Exp. Med. 133, 1294, 1971. 9. Gesteland, R., and Sphar, P., Biochem. Biophys. Res. Comnzun. 41, 1267, 1970. 10. Sela, M., Katchalski, E., and Gehatia, M., J. Amer. Chem. Sot. 78, 746, 1956. 11. Hohn, T., Biochem. Biophys. Res. Commun. 36, 7, 1969. 12. Merrifield, B. B., Science 150, 178, 1965. 13. Beverly, P. C. L., In “B and T cells in Immune Recognition” (F. Loor and G. E. Roelants, Eds.), p. 35. Wiley, New York, 1977. 14. Benacerraf, B., and Levine, B. B., J. Exp. Med. 115, 1023, 1962.
IMMUNOLOGY
Cellular
38,
193-197
(1978)
Immune Response toward MS-2 Phage and a Synthetic Fragment of Its Coat Protein HARRY LANGBEHEIM, Department
DVORA TEITELBAUM,
AND RUTH
of ChemicalImntunology,The Weizmann of Scielzce,Rehovot, Israel Received
November
ARNON
Irzstit~~te
7, 1977
The cellular immune responses toward MS-Z phage and toward a synthetic fragment of its coat protein conjugated to synthetic carrier (PaA--L) were evaluated in guinea pigs, both in vivo by the delayed-type hypersensitivity skin test and in vitro by lymphocyte transformation assay. Both MS-Z phage and PrA--L elicited high homologous cellular reaction, but no significant cross-reaction was observed between them.
INTRODUCTION The system studied in this investigation involves a synthetic conjugate, denoted Ps-A-L ( 1) , consisting of a synthetic fragment (Pz) of the coat protein of MS-2 coliphage attached to a synthetic carrier (multi-poly-uL-alanyl-poly-L-lysine), and its immunological comparison with the intact phage. In a previous report from this laboratory we have shown that the synthetic fragment Pz is involved in the neutralization of the phage, by antiphage serum, in the sensethat it is capable of inhibiting this inactivation. Furthermore, when attached to a carrier, the completely synthetic conjugate elicited, in rabbits and guinea pigs, antibodies capable of neutralizing intact MS-2 phage (1, 2). These findings clearly indicate a cross-reaction between the MS-2 phage and the conjugate Pz-A-L, on the humoral level. It has been shown that both the specificity and the manifestation of humoral and cellular responsesare not always parallel (3). There are several examples that illustrate this point : Lysozyme and its unfolded polypeptide chain do not cross-react humorally but show cross-reaction on the cellular level (4). A similar phenomenon was observed in the interaction between mammalian and Ascaris collagen (5) or between lysozyme and cr-lactalbumin (6) and between myelin basic protein and Fza histone (7). In the case of glucagon it has been demonstrated that the amino-terminal part of the molecule binds with antibodies while another region in the C terminus induces lymphocyte transformation (8). In this study we have investigated the cellular immune response towartl the intact MS-2 phage and the synthetic P,-A-I. conjugate and the cross-reaction between them on the cellular level. 193 000%8749/78/0381-0193%02.00/(1 All
Copyright 0 1978 by Academic Press, Inr rights of reproduction in any form reserved.
194
SHORT
MATERIALS
COMMUNICATIONS
AND
METHODS
Phge and reagents. Bacteriophage strain MS-2 was grown on Escherichia coli C3OOO according to the method of Gesteland and Spahr (9). Poly-DL-alanyl-poly+lysine (A-L) was prepared as described by Sela et al. (10). Animals. Random-bred DH albino guinea pigs weighing 350-500 g were obtained from the Experimental Animal Center of the Weizmann Institute of Science. Isolation of phage MS-2 coat protein. The procedure we used is based on that of Hohn (11). 0 ne volume of a purified suspension of infectious phage (10 mg) suspended in buffer (0.1 M NaCI, 0.01 M Tris, pH 7.6) was added to 2 ~01 of precooled glacial acetic acid. This treatment leads to a precipitate of A protein and RNA, whereas the major coat protein remains soluble in the supernatant. After 15 min, the precipitated RNA-A protein complex was removed by centrifugation for 10 min at 10,000 rpm (Sorvall SS-34 rotor) ; the protein was decanted and dialyzed against three changes of distilled water resulting in a protein precipitate. This precipitate was sedimented for 15 min at 10,000 rpm (Sorvall SS-34 rotor) and lyophilized. Preparation of Pz peptide and Ps-A-L conjugate. The Pz fragment corresponds to region 89-108 in the amino acid sequence of the MS-2 coat protein, namely, GluLeu-Thr-Ile-Pr~Ile-Phe-Ala-Thr-Asn-Ser-AspCys-Glu-Leu-Ile-Val-LysAla-(Met). For the synthesis of fragment Pg the Merrifield solid-phase technique was used (12). Pz-A-L was prepared as described previously by linking the Pz fragment to the synthetic carrier A -L via carbodiimide reagent (1). Delayed hypersensitivity reactions. Guinea pigs were sensitized by injection into the hind footpads of 100 pg of the antigen in complete Freund’s adjuvant (Difco Laboratories, Detroit, Mich.). They were tested 10 days later by intradermal injection of 20 or 50 ,,,g of the test antigen in 0.1 ml of physiological saline (0.15 M NaCl). The appearance of erythema and induration at the site of injection was observed after 3, 24, and 48 hr and the extent of the delayed hypersensitivity was quantitated by measuring the diameter of the skin reaction. Only reactions larger than 8 mm in diameter were considered positive. Lymphocyte stimulation. Lymphocyte stimulation was studied by [3H] thymidine incorporation in vitro. Draining lymph nodes were excised 12-20 days after sensitization. They were trimmed of fat and teased apart under sterile conditions in RPM1 1640 medium with added antibiotics (penicillin, 100 mg/ml; streptomycin, 100 units/ml ; (BioLab, Laboratories, Ltd., Jerusalem, Israel). Cells were washed three times in the same medium and plated on a petri dish for 1 hr at 37°C. The nonadherent cells were suspended in medium supplemented with 3% fetal calf serum and glutamine (2 mM>. The lymphocytes (1 x lo6 cells/culture) were incubated in microtiter plates (Falcon, Oxnard, Calif.) for 72 hr with varying concentrations of the test antigens, followed by 5-16 hr of incubation with 1 &i of [methyZ-8H]The cultures were then thymidine (Nuclear Research Centre, Negev, Israel). harvested in an automatic harvesting machine. The filters were dried and the radioactivity was measured in a Tri-Carb liquid scintillation spectrometer (Packard). The results are expressed as stimulation indexes namely, the ratio of the radioactivity (in counts per minute) in cultures containing antigen to the radioactivity in antigen-free cultures (control), and these represent the averages of values obtained in triplicate cultures.
SHORT
TABLE Skin
Test
Reactions
of Guinea
Sensitization with
Skin Immediate
195
COMMUNICATIONS
1
Pigs to MS-2 test
(mean
phagc
and
diameter
in mm) Delayed
response
I’m-A-L
response
3 hr 48 hr
24 hr P2-A-L
Phage
Phage Phage P,-A-L Adjuvant ” Numbers
19.2 16.0 -
(4/4)‘= (3/4) (O/4)
in parentheses
14.0 15.0 represent
12.0 (4/4) 9.0 (2/4) (O/4)
(4/4) (4/4) (O/4)
the proportion
P,pA-L 14.7 (4/4) 9.0 (l/4) -
of animals
(O/4)
Phage 9.2 (4/4) - (O/4) - (O/4)
P,pA-L 9.7 (4/4) - (O/4) - (O/S)
reacting.
RESULTS Delayed Hypersensitivity
Reactions
to MS-2 Phage and P2-A-L
The cellular immune responses to the MS-2 phage and to the synthetic conjugate P2-A-L were studied by the in viva delayed hypersensitivity skin test. Guinea pigs sensitized with MS-2 phage or Pz-A-L were skin tested with these antigens, and immediate (3 hr) and delayed reactions (24 and 48 hr) were followed. These were compared with the reactions obtained in control animals sensitized with adjuvant alone. The results summarized in Table 1 demonstrate that guinea pigs sensitized with phage or P2-A-L exhibit strong immediate reactions to the homologous test antigens. In addition, a high degree of cross-reactivity between the MS-2 phage and the PB--A-L was observed. Delayed reactions to these antigens were also manifested, especially in animals sensitized with MS-2 phage, 24 hr and even 48 hr after the test injections. These animals gave positive delayed responses with both phage and Pz-A-L. However, since the immediate reactions were very strong, it is not absolutely certain whether the late response is a pure delayed type or a trailing of the immediate response, or a combination of the two. Further studies to establish this point were therefore performed using the lymphocyte transformation technique.
Lymphocyte
Transformation
Response
to MS-2 Phage and P2-A-L
The cellular immune response to MS-2 phage and Ps-A-L was evaluated in vitro by means of lymphocyte transformation, as measured by stimulation of incorporation of labeled thymidine. In lymphocyte cultures from six guinea pigs sensitized to the intact MS-2, high indexes of stimulation were obtained with the homologous antigen, but no significant cross stimulation was detectable with either the Pz peptide or the P2-A-L conjugate (Fig. 1). When P2-A-L was used for sensitization, a high cellular response was demonstrated with the homologous conjugate in all animals tested (seven guinea pigs), but only a weak reaction was observed with free Pz peptide, and no significant cellular response against the intact virus was observed (Fig. 2).
196
SHORT
COMMUNICATIONS
ANTlGENpglml FIG. 1. In vitro stimulation of lymph node cells by phage (A-A) P2 peptide (O-O) and PTA-L (U-W) in guinea pigs immunized with phage. Each dose-response curve was established in the same animal from triplicate cultures.
DISCUSSION The immunological system studied here serves as a model for a synthetic vaccine in the sensethat it has demonstrated the capacity of a completely synthetic conjugate to elicit neutralizing antibodies toward a living virus, the MS-2 coliphage. One of the crucial points in the host defense mechanism following vaccination is the cellular response it elicits, since in many cases cell-mediated phenomena are known to be a prerequisite for efficient protection and in others suppressor T cells may interfere with the efficacy of the process. The results of the present study demonstrate a strong cellular response elicited by both the intact phage and the synthetic conjugate. In the case of guinea pigs sensitized with Pa-A-L high indexes of lymphocyte stimulation were obtained in vitro in all the guinea pigs tested, though the delayed hypersensitivity response was very weak. This may be due to the functional distinction between T cell mediating delayed hypersensitivity and T helper cell (13). We have previously demonstrated that guinea pigs immunized with phage and the synthetic conjugate developed the antibody response (2). However, the specificity of this cellular reactivity is not parallel to that observed previously in the humoral response, namely, no significant
cross-reaction
between
the two antigens
was observed.
These
findings
are
in accord with results obtained with other systems showing differences in specificity between humoral and cellular responses(3-8). They are also in agreement with the
ANTIGEN
pglml
FIG. 2. In V&O stimulation of lymph node cells in Guinea pigs immunized with PrA--L, hy phage (A-A), Pz peptide ( O-O) and PTA--L (W-W). Each dose-response curve was established in the same animal from triplicate cultures.
SHORT
197
COMMUNICATIONS
known phenomenon that, in response to hapten- carrier conjugates, the resultant antibodies will react with the same hapten on a different carrier, but the sensitized T cell will recognize only the homologous hapten-carrier combination (14). The conclusion from this study is that, if indeed cellular immunity is important in vaccination, then in each case in which a synthetic material is used for vaccination against a particular organism, an evaluation of its capacity to elicit cell-mediated response is necessary. In particular it is essential to test for the cross-reactivity and similarity in specificity between the antigenic determinant used in the immunogen and the intact organism, on both humoral and cellular levels. ACKNOWLEDGMENT We wish to thank Mrs. Edna Kalef
for her skillful1
technical
assistance.
REFERENCES 1. Langbeheitn, H., Arnon, R., and Sela, M., Proc. Nat. Acad. Sci. US,4 73, 4636, 1976. 2. Langbeheim, H., Arnon, R., and Sela, M., Immunology, in press. 3. Senyk, G., Williams, E. B., Nitecki, D. E., and Goodman, J. W., 1. Ex#. Med. 133, 1294, 1971. 4. Thompson, K., Harris, M., Benyamin, E., Mitchell, G., and Noble, M., Nature New Biol. 238, 20, 1972. 5. Michaeli, D., Senyk, G., Maoz, A., and Fuchs, S., Inzmunolo~y 109, 103, 1972. 6. Maron, E., Webb, C., Teitelbaum, D., and Arnon, R., Eur. J. Inzmu~ol. 2, 294, 1972. 7. Bustin, M., Teitelbaum, D., and Webb, C., Eur. J. Biochem. 53, 615, 1975. 8. Senyk, G., Williams, E. B., Nitecki, D. E., and Goodman, J. W., J. Exp. Med. 133, 1294, 1971. 9. Gesteland, R., and Sphar, P., Biochem. Biophys. Res. Comnzun. 41, 1267, 1970. 10. Sela, M., Katchalski, E., and Gehatia, M., J. Amer. Chem. Sot. 78, 746, 1956. 11. Hohn, T., Biochem. Biophys. Res. Commun. 36, 7, 1969. 12. Merrifield, B. B., Science 150, 178, 1965. 13. Beverly, P. C. L., In “B and T cells in Immune Recognition” (F. Loor and G. E. Roelants, Eds.), p. 35. Wiley, New York, 1977. 14. Benacerraf, B., and Levine, B. B., J. Exp. Med. 115, 1023, 1962.
