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response s. He has postulated that two types of helper mad suppressor T cells exist, one type which recognizes only antigen and a second type which recognizes both antigen and immunoglobulin (Ig) determinants. In this scheme, the first type of helper T cell, T h l , recognizes carrier determinants and provides help to B cells when an antigen bridge forms between them. This interaction affects the quantity but not the quality of the Ig produced. The second type of helper T cell, Th2, both requires and synergizes with Thl. Th2 recognizes both antigen and Ig, thus determining the qualitative composition of the Ig produced. The PP T-cell clones described here are difficult to fit into this scheme. The switch T cell is providing a unique signal to B cells which is dearly distinct from that provided by conventional helper T cells, whether T h l or Th2, and thus would not fit into the scheme. The PP T-cell clones described by Kiyono et al. have features of both T h l and Th2: they were able to promote the terminal differentiation of B cells by themselves, a Thl function, but also affected the isotype composition of the response, a Th2 function. The capacity of these clones to help IgA, and at the same time at least one other isotype, is puzzling. One possible explanation is that there is preferential expression ofisotype with certain idiotypes and that these PP T-cell clones are regulating idiotype primarily and isotype secondarily. All of the PP T-cell clones described above bear Fc receptors for IgA (IgA FcR). T cells bearing IgA FcR are attractive candidates for IgA-specific regulator cells because they fit the requirement ofbeingable to recognize both antigen and IgA simultaneously. The tissue distribution ofIgA FcR ÷ cells does not correlate well with
sites oflgA synthesis 6, but recent reports indicate that IgA FcR ÷ T cells can provide IgA-specific help 7 and suppression s. If IgA FcR ÷ T cells are isotype-specific regulators of IgA, then there is a good deal of functional complexity within this subset. The clones described by Kawanishi et al. are functionally quite different from the clones described by Kiyono et aL, although both bear the IgA Fc receptor. Identifying a cell-surface marker which distinguishes the T cells regulating IgA will be of tremendous advantage in defining the role that these T cells, as well as non-isotype-specific T cells, play in the IgA immune response in vivo, a goal which is now in view. C H A R L E S O. E L S O N
Departmentof Medicine, Medical Collegeof Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA.
References 1 Savage, D. C. (1977)Annu. Rev. Microbiol. 31, 107-133 2 Elson, C. O., Heck, J. A. and Strober, W. (1979)J. Exp. Med. 149, 632-643 3 Kawanishi, H., Saltzman, L. E. and Strober, W. (1983)J. Exp. Med. 157, 433-449 4 Kiyono, H., McGhee, J. R., Mostellar, L. M., Eldridge, J. H., Koopman, W. J., Kearney, J. F. and Michalek, S. M. (1982)J. Exp. Med. 156, 1115-1130 5 Janeway, C. A. (1981) inStrateglesoflmmuneRegulation(Sercarz, E. E. and Cunningham, A. J., eds), pp. 179-198, Academic Press, New York 6 Arnaud-Battandier, F., Hague, N. E., Lum, L. G., Elson, (3. O. and Strober, W. (1980) Cell. Immunol. 55, 106-113 7 Hoover, R. G. and Lynch, R. G. (1983)J. Immunol. 130, 521-523 8 Endoh, M., Sakai, H., Nomoto, Y., Tomino, Y. and Kaneshige, H. (1981).f Immunol. 127, 2612-2615
Synthetic peptides and their impact on immunology The use of synthetic peptides to raise antisera of predetermined specificity has immense implications for molecular and cellular immunology. Conceptually, little is new - indeed, it is twenty years since molecular immunologists began probing the antigenic domains of proteins by investigating the antigenicity of their peptide fragments ~. The modern adaptation is, however, considerably more elegant and its basis is simple. It requires (a) an amino acid sequence of interest, (b) the means to make enough peptide of sufficient purity, and (c) a procedure for coupling the peptide covalently to a carrier protein. It is possible to synthesize peptides of 10-20 amino acids rapidly, using a commercially available solid-phase peptide synthesizer, and the organic chemistry of peptide-carrier coupling is straightforward. It is thus the first requirement on which the usefulness of the approach depends. In principle, any peptide, even those not normally immunogenic (immunodominant) when part of an intact protein, can be used to make an antibody. The nature of the peptide sequences used as immunogens is limited only by the imagination of immunologists. Fortunately, this does not appear to be lacking and has been illustrated nicely by a series of recent articles. Perhaps the most original application of synthetic © 1983,ElsevierSciencePublishersB.V.,Amsterdam 0167- 4919/83/]t01.00
peptide technology, proposed by Lerner 2 and by Walter et aL 3, is to confirm the existence of proteins predicted by nucleic acid sequencing. The entire amino acid sequence of a protein can be determined from the DNA sequence of a cDNA clone. Short peptides synthesized from such a sequence can therefore be used to generate antibodies which recognize the native protein. This approach has identified new viral gene products 2'3and proteins encoded by retroviral oncogenes~'5. The potential of the use of antisera against synthetic peptides to detect new gene products is, however, far greater. For example, it is possible to isolate cDNA clones which show interesting properties, such as restriction in expression to a particular lymphocyte subset, but which have a protein product which cannot be identified by conventional means. Now there is the possibility of identifying the native protein specified by the clone, its location within the cell, and its functional role (by examining the effect of the antisera on functional assays). The ease with which synthetic peptides can be used to produce antisera reactive with intact viral products has obvious implications for vaccine production. Their eventual use for this purpose will depend partly upon cost effectiveness when compared with viral protein production through recombinant D N A techniques or with mass
191
Immunology Today, vol. 4, No. 7, 1983
culture of intact virus in vitro. Synthetic peptides, however, offer the advantage of possible selection of invariant regions of the viral protein which have functional activity or are crucial for protein folding, and which cannot, therefore, easily mutate in order to evade the immune response 6. One caveat is that antisera against short peptides have an intrinsically greater likelihood of cross-reactivity than antisera against intact proteins. Fortuitous cross-reactivity with cellular proteins could be a considerable technical problem. Synthetic peptides can also be employed to probe the domain structure of proteins, their functionally active regions, and their quaternary interactions. This approach, which has been reviewed in detail by Lerner 6 is of particular importance when the protein cannot be purified easily in large enough quantity to make conventional protein chemistry and X-ray analysis feasible, as in the case of class-I histocompatibility antigens. Reisfeld's group have attempted to make an antiserum against human class-I antigens using a dodecapeptide consisting of residues 39-50 of the HLA-B7 heavy-chain sequence as immunogen 7. Antibodies raised against this peptide bound specifically to free H L A A and B antigen heavy chains but failed to immunoprecipitate the heavy chain-/I2-microglobulin complex 7. Presumably this antiserum detected a conformational determinant on the heavy chain which is modified as a consequence of association with 02-microglobulin. It is interesting to recall that the successful cloning of human class-I antigen heavy-chain sequences necessitated an antiserum which reacted with free heavy chains, since antibodies which recognized the complex failed to be of use in the screening procedures 8. The use of synthetic peptides to generate reagents designed for specific purposes, such as cloning or purification, should find many applications. Synthetic peptides are also finding application in studies on the T-cell response to antigen. L a m b and colleagues have isolated long-term antigen-specific human T-lymphocyte clones which proliferate in response to purified haemagglutinin (HA) obtained from influenza type-A virus 9. The antigenic determinants recognized by these long-term T-lymphocyte lines have been investigated with chemically synthesized peptides corresponding to sequences of the haemagglutinin molecule '6. Haemagglutinin-specific T-cell populations have the repertoire necessary to respond to all the synthetic peptide analysed. Interestingly, however, when responses were analysed at the clonal level one peptide
(residues 306-330) located at the carboxyl terminus of the HA1 molecule and discrete from the proposed antibody sites appeared to be immunodominant 1°. The availability of cloned T-lymphocyte lines recognizing synthetic peptides derived from a protein whose tertiary structure has been determined has unparalleled potential in studies on the T-cell response to antigen. Recent work on the induction of tolerance in cloned human helper T cells with synthetic peptides of the H A molecule has, for example, shown that the clone could be rendered unresponsive by incubation with high concentrations of peptide in the absence of any accessory cells11. It should be possible, therefore, to elucidate in detail the surface events involved in the induction of tolerance in this system. Several surface antigens are modulated when these helper T-cell clones are incubated with the appropriate synthetic peptide ~2. In particular, the T3 antigen is lost in a dosedependent manner, providing further evidence for the involvement of T3 in T-cell triggering by antigen 13. The use of synthetic peptides coupled with advances in molecular and cellular cloning techniques will undoubtedly continue to yield exciting results. In the future, peptide synthesizers may well be as integral a part of an immunology department as cell sorters are today. MICHAEL J. OWEN ICRF Turnout Immunology Unit, Zoology Department, University College London, London WC1E 6BT, UK. References
1 Crumpton, M. J. (1974) in The Antigens (Sela, M., ed.) pp. 1-78, AcademicPress, New York 2 Sutcliffe,J. G., Shinnick,T. M., Green, N. et al. (1980)Nature (London) 287, 801-805 3 Walter,G., Scheidtmann,K.-H., Carbonek,A. et a/. (1980)Pro~.Nail Acad. Sci. USA 77, 5197-5200 4 Papkoff,J., Verma, I. M. and Hunter, T. (1982) Cell 29, 417-426 5 Wong,T.-W. and Goldberg,A. R. (1981)Proc.NatlAcad. Sci. USA 78, 7412-7416 6 Lerner, R. A. (1982)Nature (London) 299, 592-596 7 Church, W. R., Walker,L. E., Houghton, R. A. and Reisfeld,R. A. (1983) Proc.NatlAcad. Sci. USA 80, 255-258 8 Ploegh,H. L., Orr, H. T. and Strominger,J. L. (1980)Proc.NatlAcad. Sci. USA 77, 6081-6085 9 Lamb,J. R., Eckels,D. D., Phelan, M. et al. (1982)J. Immunol. 128, 1428-1432 10 Lamb,J. R., Eckels,D. D., Lake, P. et al. (1982)Nature (London) 300, 66-69 11 Lamb,J. R., Skidmore,B. J., Green, N. et al. J. Exp. Med. (in press) 12 Zanders,E. D., Lamb,J. R., Feldmann,M., Green, N. and Beverley, P. C. L. (1983) Nature (in press)
13 Reinherz,E. L., Meuer, S. C. and Schlossman,S. F. (1983) To~y 4, 5-8.
lmmunol.
Immunology Today, vol. 4, No. 7, 1983
response s. He has postulated that two types of helper mad suppressor T cells exist, one type which recognizes only antigen and a second type which recognizes both antigen and immunoglobulin (Ig) determinants. In this scheme, the first type of helper T cell, T h l , recognizes carrier determinants and provides help to B cells when an antigen bridge forms between them. This interaction affects the quantity but not the quality of the Ig produced. The second type of helper T cell, Th2, both requires and synergizes with Thl. Th2 recognizes both antigen and Ig, thus determining the qualitative composition of the Ig produced. The PP T-cell clones described here are difficult to fit into this scheme. The switch T cell is providing a unique signal to B cells which is dearly distinct from that provided by conventional helper T cells, whether T h l or Th2, and thus would not fit into the scheme. The PP T-cell clones described by Kiyono et al. have features of both T h l and Th2: they were able to promote the terminal differentiation of B cells by themselves, a Thl function, but also affected the isotype composition of the response, a Th2 function. The capacity of these clones to help IgA, and at the same time at least one other isotype, is puzzling. One possible explanation is that there is preferential expression ofisotype with certain idiotypes and that these PP T-cell clones are regulating idiotype primarily and isotype secondarily. All of the PP T-cell clones described above bear Fc receptors for IgA (IgA FcR). T cells bearing IgA FcR are attractive candidates for IgA-specific regulator cells because they fit the requirement ofbeingable to recognize both antigen and IgA simultaneously. The tissue distribution ofIgA FcR ÷ cells does not correlate well with
sites oflgA synthesis 6, but recent reports indicate that IgA FcR ÷ T cells can provide IgA-specific help 7 and suppression s. If IgA FcR ÷ T cells are isotype-specific regulators of IgA, then there is a good deal of functional complexity within this subset. The clones described by Kawanishi et al. are functionally quite different from the clones described by Kiyono et aL, although both bear the IgA Fc receptor. Identifying a cell-surface marker which distinguishes the T cells regulating IgA will be of tremendous advantage in defining the role that these T cells, as well as non-isotype-specific T cells, play in the IgA immune response in vivo, a goal which is now in view. C H A R L E S O. E L S O N
Departmentof Medicine, Medical Collegeof Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA.
References 1 Savage, D. C. (1977)Annu. Rev. Microbiol. 31, 107-133 2 Elson, C. O., Heck, J. A. and Strober, W. (1979)J. Exp. Med. 149, 632-643 3 Kawanishi, H., Saltzman, L. E. and Strober, W. (1983)J. Exp. Med. 157, 433-449 4 Kiyono, H., McGhee, J. R., Mostellar, L. M., Eldridge, J. H., Koopman, W. J., Kearney, J. F. and Michalek, S. M. (1982)J. Exp. Med. 156, 1115-1130 5 Janeway, C. A. (1981) inStrateglesoflmmuneRegulation(Sercarz, E. E. and Cunningham, A. J., eds), pp. 179-198, Academic Press, New York 6 Arnaud-Battandier, F., Hague, N. E., Lum, L. G., Elson, (3. O. and Strober, W. (1980) Cell. Immunol. 55, 106-113 7 Hoover, R. G. and Lynch, R. G. (1983)J. Immunol. 130, 521-523 8 Endoh, M., Sakai, H., Nomoto, Y., Tomino, Y. and Kaneshige, H. (1981).f Immunol. 127, 2612-2615
Synthetic peptides and their impact on immunology The use of synthetic peptides to raise antisera of predetermined specificity has immense implications for molecular and cellular immunology. Conceptually, little is new - indeed, it is twenty years since molecular immunologists began probing the antigenic domains of proteins by investigating the antigenicity of their peptide fragments ~. The modern adaptation is, however, considerably more elegant and its basis is simple. It requires (a) an amino acid sequence of interest, (b) the means to make enough peptide of sufficient purity, and (c) a procedure for coupling the peptide covalently to a carrier protein. It is possible to synthesize peptides of 10-20 amino acids rapidly, using a commercially available solid-phase peptide synthesizer, and the organic chemistry of peptide-carrier coupling is straightforward. It is thus the first requirement on which the usefulness of the approach depends. In principle, any peptide, even those not normally immunogenic (immunodominant) when part of an intact protein, can be used to make an antibody. The nature of the peptide sequences used as immunogens is limited only by the imagination of immunologists. Fortunately, this does not appear to be lacking and has been illustrated nicely by a series of recent articles. Perhaps the most original application of synthetic © 1983,ElsevierSciencePublishersB.V.,Amsterdam 0167- 4919/83/]t01.00
peptide technology, proposed by Lerner 2 and by Walter et aL 3, is to confirm the existence of proteins predicted by nucleic acid sequencing. The entire amino acid sequence of a protein can be determined from the DNA sequence of a cDNA clone. Short peptides synthesized from such a sequence can therefore be used to generate antibodies which recognize the native protein. This approach has identified new viral gene products 2'3and proteins encoded by retroviral oncogenes~'5. The potential of the use of antisera against synthetic peptides to detect new gene products is, however, far greater. For example, it is possible to isolate cDNA clones which show interesting properties, such as restriction in expression to a particular lymphocyte subset, but which have a protein product which cannot be identified by conventional means. Now there is the possibility of identifying the native protein specified by the clone, its location within the cell, and its functional role (by examining the effect of the antisera on functional assays). The ease with which synthetic peptides can be used to produce antisera reactive with intact viral products has obvious implications for vaccine production. Their eventual use for this purpose will depend partly upon cost effectiveness when compared with viral protein production through recombinant D N A techniques or with mass
191
Immunology Today, vol. 4, No. 7, 1983
culture of intact virus in vitro. Synthetic peptides, however, offer the advantage of possible selection of invariant regions of the viral protein which have functional activity or are crucial for protein folding, and which cannot, therefore, easily mutate in order to evade the immune response 6. One caveat is that antisera against short peptides have an intrinsically greater likelihood of cross-reactivity than antisera against intact proteins. Fortuitous cross-reactivity with cellular proteins could be a considerable technical problem. Synthetic peptides can also be employed to probe the domain structure of proteins, their functionally active regions, and their quaternary interactions. This approach, which has been reviewed in detail by Lerner 6 is of particular importance when the protein cannot be purified easily in large enough quantity to make conventional protein chemistry and X-ray analysis feasible, as in the case of class-I histocompatibility antigens. Reisfeld's group have attempted to make an antiserum against human class-I antigens using a dodecapeptide consisting of residues 39-50 of the HLA-B7 heavy-chain sequence as immunogen 7. Antibodies raised against this peptide bound specifically to free H L A A and B antigen heavy chains but failed to immunoprecipitate the heavy chain-/I2-microglobulin complex 7. Presumably this antiserum detected a conformational determinant on the heavy chain which is modified as a consequence of association with 02-microglobulin. It is interesting to recall that the successful cloning of human class-I antigen heavy-chain sequences necessitated an antiserum which reacted with free heavy chains, since antibodies which recognized the complex failed to be of use in the screening procedures 8. The use of synthetic peptides to generate reagents designed for specific purposes, such as cloning or purification, should find many applications. Synthetic peptides are also finding application in studies on the T-cell response to antigen. L a m b and colleagues have isolated long-term antigen-specific human T-lymphocyte clones which proliferate in response to purified haemagglutinin (HA) obtained from influenza type-A virus 9. The antigenic determinants recognized by these long-term T-lymphocyte lines have been investigated with chemically synthesized peptides corresponding to sequences of the haemagglutinin molecule '6. Haemagglutinin-specific T-cell populations have the repertoire necessary to respond to all the synthetic peptide analysed. Interestingly, however, when responses were analysed at the clonal level one peptide
(residues 306-330) located at the carboxyl terminus of the HA1 molecule and discrete from the proposed antibody sites appeared to be immunodominant 1°. The availability of cloned T-lymphocyte lines recognizing synthetic peptides derived from a protein whose tertiary structure has been determined has unparalleled potential in studies on the T-cell response to antigen. Recent work on the induction of tolerance in cloned human helper T cells with synthetic peptides of the H A molecule has, for example, shown that the clone could be rendered unresponsive by incubation with high concentrations of peptide in the absence of any accessory cells11. It should be possible, therefore, to elucidate in detail the surface events involved in the induction of tolerance in this system. Several surface antigens are modulated when these helper T-cell clones are incubated with the appropriate synthetic peptide ~2. In particular, the T3 antigen is lost in a dosedependent manner, providing further evidence for the involvement of T3 in T-cell triggering by antigen 13. The use of synthetic peptides coupled with advances in molecular and cellular cloning techniques will undoubtedly continue to yield exciting results. In the future, peptide synthesizers may well be as integral a part of an immunology department as cell sorters are today. MICHAEL J. OWEN ICRF Turnout Immunology Unit, Zoology Department, University College London, London WC1E 6BT, UK. References
1 Crumpton, M. J. (1974) in The Antigens (Sela, M., ed.) pp. 1-78, AcademicPress, New York 2 Sutcliffe,J. G., Shinnick,T. M., Green, N. et al. (1980)Nature (London) 287, 801-805 3 Walter,G., Scheidtmann,K.-H., Carbonek,A. et a/. (1980)Pro~.Nail Acad. Sci. USA 77, 5197-5200 4 Papkoff,J., Verma, I. M. and Hunter, T. (1982) Cell 29, 417-426 5 Wong,T.-W. and Goldberg,A. R. (1981)Proc.NatlAcad. Sci. USA 78, 7412-7416 6 Lerner, R. A. (1982)Nature (London) 299, 592-596 7 Church, W. R., Walker,L. E., Houghton, R. A. and Reisfeld,R. A. (1983) Proc.NatlAcad. Sci. USA 80, 255-258 8 Ploegh,H. L., Orr, H. T. and Strominger,J. L. (1980)Proc.NatlAcad. Sci. USA 77, 6081-6085 9 Lamb,J. R., Eckels,D. D., Phelan, M. et al. (1982)J. Immunol. 128, 1428-1432 10 Lamb,J. R., Eckels,D. D., Lake, P. et al. (1982)Nature (London) 300, 66-69 11 Lamb,J. R., Skidmore,B. J., Green, N. et al. J. Exp. Med. (in press) 12 Zanders,E. D., Lamb,J. R., Feldmann,M., Green, N. and Beverley, P. C. L. (1983) Nature (in press)
13 Reinherz,E. L., Meuer, S. C. and Schlossman,S. F. (1983) To~y 4, 5-8.
lmmunol.
