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Molecular immunology: growth into adolescence
TIBS - A p r i l 1 9 8 4
137
Molecular immunology: growth into adolescence David Baltimore Molecular immunology and TIBS are children of the same age. After many years of painful gestation, 1976 was the year that Hozumi and Tonegawa I published the paper which set molecular immunology on its course of post-natal development. Over eight years the field has grown rapidly, has learned how to solve problems and has expanded its vision to cover increasing territory. But, for all of its apparent maturity, it is still an adolescent. The experiment of Hozumi and Tonegawa was a fulfilment of the dreams of the field, which Dreyer and Bennett 2 had set down on paper in 1965. They had suggested that the immunoglobulin gene might be constructed from pieces rather than being inherited as a single unit. The direct demonstration that pieces of DNA are recombined to form an immunoglobulin gene came at a very opportune moment. Recombinant DNA methods were themselves climbing out of their infancy and the two children met, discovered their commonality of promise, and have grown up as loving playmates. No field of biology has benefited more from the recombinant DNA revolution than immunology. The years of gestational polemic discussions polarized immunology into two camps: those who believed that diversity was a somatic process and those who believed that diversity was an evolutionary process. The evolutionists expected to find in the genome large libraries of sequence ready to be mobilized as part of immunoglobulin genes. The strict somaticists expected to find one gene whose somatic mutation could generate the universe of possible antibodies. As is so frequently the case, nature was there first: it recognized the enormous potential of both types of generators of diversity and produced a mixed solution3. We now know that there is a large library of DNA that has been evolutionarily varied, but that also there is somatic mutation to further extend the range of that variation4. We also learned of an unexpected source of immunoglobulin gene diversity: microcombinational diversity. This comes about because the elements that make up the immunogiobulin gene, rather than being joined reproducibly at specific sites, are joined with a certain amount
of local variation of site 5. This generates innumerable local combinatorial variants and enormously increases the range of local variation that can occur at the joining site 6. Finally, there has recently come to light yet another mechanism of diversity, the apparent non-templated synthesis of DNA'. DNA is polymerized between the elements that are joined and adds a remarkable range of potential combinations to a system which already seems to have extraordinary possibilities of variation. Thus, the last eight years have seen one of the major controversies in biology settled with some finality. But, as many immunologists who grew up in the decades of serology continually remind us, we have a big challenge yet ahead: to understand the link between sequence diversity and immunologic specificity8. Although we know that certain loops on the surface of immunoglobulin molecules make the antigen combining site 9, we are quite ignorant about which sources of sequence diversity generate changes in specificity. One of the great deficiencies in the field is the lack of knowledge about immunoglobulin structure. Only a few immunoglobulin-related molecules have been crystallized and their structure solved to atomic resolution. Thus, in spite of the enormous knowledge about the primary structure of immunoglobulin molecules, the local tertiary structures which generate antibody combining sites can only be a guess. There is even a new model of immunoglobulin structure which suggests that some of the most variable residues in the immunoglobulin variable regions are actually at the ends of the beta sheets which provide rigidity to the molecule rather than being amino acids that contact antigen w. Our poverty of knowledge is shown by the fact that we David Baltimore, Whitehead Institute for Bio- do not know the structure of any antimedical Research, Cambridge, MA 02139; Center body molecule that is targeted to a for Cancer Research, and Department of Biology Massachusetts Institute of Technology, Cambridge, determinant on a protein rather than to a small hapten. An increased catalog of MA 02139, USA.
immunoglobulin structures is sure to be informative, but it is also probable that a deep understanding of the nature of antibody-antigen interactions will require knowledge about protein dynamics that is today well beyond the reach of even the most sophisticated protein analysts. Our need to understand antibody and antigen dynamics is shown by the observation that many anti-peptide monoclonal antibodies react with proteins containing the peptidell: the only reasonable model for this surprising lack of three-dimensional specificity of binding is that either the antibody or the antigen, or both, are quite flexible. Another refrain heard often from scientists who have grappled with the range of problems in immunology is that the mere determination of antibody structure only scratches the surface of the deep enigmas of cellular immunology. Leaving aside T-lymphocytes for a moment, the antibody-forming Blymphocytic series of cells goes through a long and complex differentiation before a plasma cell settles down to become an immunoglobulin factory. The control of antibody synthesis is the control of that differentiation pathway because antibodies are made on demand. Only when we understand the signals that control B-lymphoid cell differentiation will we have a clear understanding of how the immune system manages to respond to antigenic challenge. The recent identification of sequences in the J-C intron that control immunoglobulin gene expression is a beginning in that direction 12-14. The field of B-lymphoid cellular immunology has many practitioners but is still concerned with very fundamental debates. For instance, it is uncertain whether all B-lymphoid cells should be considered part of a single lineage or whether there are sub-fineages. In mature animals, a major type of Blymphoid cell is one that contains on its surface IgD molecules. In spite of many years of suggestive evidence, the reason that these cells have IgD molecules on their surface remains to be clarified 15. The immune system involves two fundamentally different types of cells: T-lymphocytes and B-lymphocytes. The revolution of the last eight years has brought us to some understanding of the properties of B-lymphoid cells, but understanding of T-lymphoid cells remains shrouded in perplexity. T cells recognize antigens that are on cell surfaces by an obscure process of joint recognition of a normal cell surface protein and the foreign antigenic moiety ~6. The nature of the T-cell receptor pro-
© 1984. Elsevier Soence Publishers B.V.. Amsterdam 0.376- 5~7/84/$02 (~0
138 tein that carries out the recognition is only just becoming clear ~7-19 and we still have no idea how many T-cell receptor molecules there might be. Thus, T-cell biology remains in a polemical phase because of two fundamental obscurities: we do not understand the antigen and we do not understand the receptor molecule. The adolescent field of molecular immunology appears just now poised to give birth to a child: at least two groups of scientists (led by T. Mak and M. Davis) are about to publish articles suggesting that the first glimmers of molecular knowledge about the T-cell receptor are at hand. Assuming that these preliminary reports generate the kind of progress that the initial reports about immunoglobulin genes generated, the next few years should see an explosion of knowledge about the Tcell receptor. The antigen recognized by that receptor, however, may be much harder to understand because the methods of molecular genetics are not obviously applicable to that problem. If I were to guess, I would suggest that the next few years will bring us to the point of understanding the T-cell receptor well enough for us to be able to make large amounts of it using recombinant D N A methods. Then we will be able to learn about the interaction of a pure protein with surface-bound antigens and perhaps to identify the areas of the Tcell receptor (or receptors) that interact with normal cell constituents (MHC products) and with foreign antigens. The maturity of molecular immunology will come when we can integrate our knowledge of B-lymphocytes and Tlymphocytes into a model that will generate three very fundamental results~ The model must first explain why we respond to an antigenic challenge with synthesis of a burst of highly specific recognition molecules. What role do T
TIBS - April 1984
cells play? How does somatic mutation interact with selection and is this the basis of the increasing avidity of the response? How is the system shut down? The second requirement of such a model is an explanation of memory. The immune system, like our brain, remembers each encounter with the external world and is able to generate a rapid recall when re-presented with the stimulus; how does the system manage to store some cells while others respond? The third, and most important, requirement of a complete model of the i m m u n e system is that it must generate tolerance. Tolerance is the word we use to describe the body's ability to differentiate between itself and foreign materials. Foreign molecules generate antibodies and T-cell recognition; selfmolecules are tolerated and do not generate such recognition molecules. Breakdown of tolerance leads to disease; therefore, the controlling processes that underlie the immune system must maintain tolerance. In spite of many imaginative proposals, notably those of Jerne TM, it seems likely that there are principles at work which we have yet to fathom. I speak about immunology from a peculiar perspective: although I had spent many years cognizant of the problems that immunologists cared about, it was only in 1976 that I began to take an experimental interest in the field. From my first childish steps through my own development into adolescent excitement, I have come to realize that the cloning and sequencing of D N A molecules is not the total interest in the field. It has been wonderful to explain the unfolding of the richness and diversity of problems that are subsumed under the rubric of immunology. These include questions of differentiation, questions of growth control, questions of D N A recombination, questions about the control of
gene expression and questions about the integration of different cellular systems. Only questions about the events of nucleic acid metabolism have been answered sufficiently well to be considered history. There is yet a lifetime in front of the adolescent field and one that promises to be rich, diverse and fascinating. References 1 Hozumi, N. and Tonegawa, S. (1976) Proc. Natl Acad. Sci. USA 73, 3628-3632
2 Dreyer, W. J. and Bennett, J. C. (1%5) 54, 864-869 3 Tonegawa, S. (1983) Nature 302, 575-581 4 Baltimore, D. (198l) Cell 26, 295-2% 5 Lewis, S., Gifford, A. and Baltimore, D. Nature (in press) 6 Weigert, M., Perry, R., Kelly, D., HunkapiUer, T., Schilling, J. and Hood, L. (1980) Nature 283, 497-500 7 Alt, F. W. and Baltimore, D. (1982) Proc. Natl Acad. Sci. USA 79, 4118--4122 8 Kabat, E. A. (1982) Pharm. Rev. 34, 23-38 9 Wu, T. T. and Kabat, E. A. (1970) J. Exp. Med. 132, 211-250 10 Novotny, J., Bruccoleri, R., Newell, J., Murphy, D., Haber E. and Karplus, M. (1983) J. Biol. Chem. 258, 14433-14437 11 Niman, H. L., Houghten, R. A., Walker, L. E., Reisfeld, R. A., Wilson, I. A., Hogle, J. M. and Lerner, R. A. (1983) Proc. Natl Acad. Sci. USA 80, 4949-4953 12 Queen, C. and Baltimore, D. (1983) Cell 33, 741-748 13 Banerji, J., Olson, L. and Schaffner, W. (1983) Cell 33, 729-740 14 Gillies, S. D., Morrison, S. L., Oi, V. T. and Tonegawa, S. (1983) Cell 33,717-728 15 Blanner, F. R. and Tucker, P. W. (1984) Nature 307, 417--422 16 Zinkernagel, R. M. and Doherty, P. C. (1974) Nature 248, 701-702 17 Acuto, O., Hussey, R. E., Fitzgerald, K. A., Protentis, J. P., Meuer, S.C., Schlossman, S.F. and Reinherz, E.L. (1983) Cell 34, 717-726 18 Kappler, J., Kubo, R., Haskins, K., White, J. and Manack, P. (1983) Cell 34, 727-737 19 Mclntyre, B. W. and Allison, J. P. (1983) Cell 34, 739-746 20 Jerne, N. K. (1971)Eur. J. Immunology 1, 1-9
137
Molecular immunology: growth into adolescence David Baltimore Molecular immunology and TIBS are children of the same age. After many years of painful gestation, 1976 was the year that Hozumi and Tonegawa I published the paper which set molecular immunology on its course of post-natal development. Over eight years the field has grown rapidly, has learned how to solve problems and has expanded its vision to cover increasing territory. But, for all of its apparent maturity, it is still an adolescent. The experiment of Hozumi and Tonegawa was a fulfilment of the dreams of the field, which Dreyer and Bennett 2 had set down on paper in 1965. They had suggested that the immunoglobulin gene might be constructed from pieces rather than being inherited as a single unit. The direct demonstration that pieces of DNA are recombined to form an immunoglobulin gene came at a very opportune moment. Recombinant DNA methods were themselves climbing out of their infancy and the two children met, discovered their commonality of promise, and have grown up as loving playmates. No field of biology has benefited more from the recombinant DNA revolution than immunology. The years of gestational polemic discussions polarized immunology into two camps: those who believed that diversity was a somatic process and those who believed that diversity was an evolutionary process. The evolutionists expected to find in the genome large libraries of sequence ready to be mobilized as part of immunoglobulin genes. The strict somaticists expected to find one gene whose somatic mutation could generate the universe of possible antibodies. As is so frequently the case, nature was there first: it recognized the enormous potential of both types of generators of diversity and produced a mixed solution3. We now know that there is a large library of DNA that has been evolutionarily varied, but that also there is somatic mutation to further extend the range of that variation4. We also learned of an unexpected source of immunoglobulin gene diversity: microcombinational diversity. This comes about because the elements that make up the immunogiobulin gene, rather than being joined reproducibly at specific sites, are joined with a certain amount
of local variation of site 5. This generates innumerable local combinatorial variants and enormously increases the range of local variation that can occur at the joining site 6. Finally, there has recently come to light yet another mechanism of diversity, the apparent non-templated synthesis of DNA'. DNA is polymerized between the elements that are joined and adds a remarkable range of potential combinations to a system which already seems to have extraordinary possibilities of variation. Thus, the last eight years have seen one of the major controversies in biology settled with some finality. But, as many immunologists who grew up in the decades of serology continually remind us, we have a big challenge yet ahead: to understand the link between sequence diversity and immunologic specificity8. Although we know that certain loops on the surface of immunoglobulin molecules make the antigen combining site 9, we are quite ignorant about which sources of sequence diversity generate changes in specificity. One of the great deficiencies in the field is the lack of knowledge about immunoglobulin structure. Only a few immunoglobulin-related molecules have been crystallized and their structure solved to atomic resolution. Thus, in spite of the enormous knowledge about the primary structure of immunoglobulin molecules, the local tertiary structures which generate antibody combining sites can only be a guess. There is even a new model of immunoglobulin structure which suggests that some of the most variable residues in the immunoglobulin variable regions are actually at the ends of the beta sheets which provide rigidity to the molecule rather than being amino acids that contact antigen w. Our poverty of knowledge is shown by the fact that we David Baltimore, Whitehead Institute for Bio- do not know the structure of any antimedical Research, Cambridge, MA 02139; Center body molecule that is targeted to a for Cancer Research, and Department of Biology Massachusetts Institute of Technology, Cambridge, determinant on a protein rather than to a small hapten. An increased catalog of MA 02139, USA.
immunoglobulin structures is sure to be informative, but it is also probable that a deep understanding of the nature of antibody-antigen interactions will require knowledge about protein dynamics that is today well beyond the reach of even the most sophisticated protein analysts. Our need to understand antibody and antigen dynamics is shown by the observation that many anti-peptide monoclonal antibodies react with proteins containing the peptidell: the only reasonable model for this surprising lack of three-dimensional specificity of binding is that either the antibody or the antigen, or both, are quite flexible. Another refrain heard often from scientists who have grappled with the range of problems in immunology is that the mere determination of antibody structure only scratches the surface of the deep enigmas of cellular immunology. Leaving aside T-lymphocytes for a moment, the antibody-forming Blymphocytic series of cells goes through a long and complex differentiation before a plasma cell settles down to become an immunoglobulin factory. The control of antibody synthesis is the control of that differentiation pathway because antibodies are made on demand. Only when we understand the signals that control B-lymphoid cell differentiation will we have a clear understanding of how the immune system manages to respond to antigenic challenge. The recent identification of sequences in the J-C intron that control immunoglobulin gene expression is a beginning in that direction 12-14. The field of B-lymphoid cellular immunology has many practitioners but is still concerned with very fundamental debates. For instance, it is uncertain whether all B-lymphoid cells should be considered part of a single lineage or whether there are sub-fineages. In mature animals, a major type of Blymphoid cell is one that contains on its surface IgD molecules. In spite of many years of suggestive evidence, the reason that these cells have IgD molecules on their surface remains to be clarified 15. The immune system involves two fundamentally different types of cells: T-lymphocytes and B-lymphocytes. The revolution of the last eight years has brought us to some understanding of the properties of B-lymphoid cells, but understanding of T-lymphoid cells remains shrouded in perplexity. T cells recognize antigens that are on cell surfaces by an obscure process of joint recognition of a normal cell surface protein and the foreign antigenic moiety ~6. The nature of the T-cell receptor pro-
© 1984. Elsevier Soence Publishers B.V.. Amsterdam 0.376- 5~7/84/$02 (~0
138 tein that carries out the recognition is only just becoming clear ~7-19 and we still have no idea how many T-cell receptor molecules there might be. Thus, T-cell biology remains in a polemical phase because of two fundamental obscurities: we do not understand the antigen and we do not understand the receptor molecule. The adolescent field of molecular immunology appears just now poised to give birth to a child: at least two groups of scientists (led by T. Mak and M. Davis) are about to publish articles suggesting that the first glimmers of molecular knowledge about the T-cell receptor are at hand. Assuming that these preliminary reports generate the kind of progress that the initial reports about immunoglobulin genes generated, the next few years should see an explosion of knowledge about the Tcell receptor. The antigen recognized by that receptor, however, may be much harder to understand because the methods of molecular genetics are not obviously applicable to that problem. If I were to guess, I would suggest that the next few years will bring us to the point of understanding the T-cell receptor well enough for us to be able to make large amounts of it using recombinant D N A methods. Then we will be able to learn about the interaction of a pure protein with surface-bound antigens and perhaps to identify the areas of the Tcell receptor (or receptors) that interact with normal cell constituents (MHC products) and with foreign antigens. The maturity of molecular immunology will come when we can integrate our knowledge of B-lymphocytes and Tlymphocytes into a model that will generate three very fundamental results~ The model must first explain why we respond to an antigenic challenge with synthesis of a burst of highly specific recognition molecules. What role do T
TIBS - April 1984
cells play? How does somatic mutation interact with selection and is this the basis of the increasing avidity of the response? How is the system shut down? The second requirement of such a model is an explanation of memory. The immune system, like our brain, remembers each encounter with the external world and is able to generate a rapid recall when re-presented with the stimulus; how does the system manage to store some cells while others respond? The third, and most important, requirement of a complete model of the i m m u n e system is that it must generate tolerance. Tolerance is the word we use to describe the body's ability to differentiate between itself and foreign materials. Foreign molecules generate antibodies and T-cell recognition; selfmolecules are tolerated and do not generate such recognition molecules. Breakdown of tolerance leads to disease; therefore, the controlling processes that underlie the immune system must maintain tolerance. In spite of many imaginative proposals, notably those of Jerne TM, it seems likely that there are principles at work which we have yet to fathom. I speak about immunology from a peculiar perspective: although I had spent many years cognizant of the problems that immunologists cared about, it was only in 1976 that I began to take an experimental interest in the field. From my first childish steps through my own development into adolescent excitement, I have come to realize that the cloning and sequencing of D N A molecules is not the total interest in the field. It has been wonderful to explain the unfolding of the richness and diversity of problems that are subsumed under the rubric of immunology. These include questions of differentiation, questions of growth control, questions of D N A recombination, questions about the control of
gene expression and questions about the integration of different cellular systems. Only questions about the events of nucleic acid metabolism have been answered sufficiently well to be considered history. There is yet a lifetime in front of the adolescent field and one that promises to be rich, diverse and fascinating. References 1 Hozumi, N. and Tonegawa, S. (1976) Proc. Natl Acad. Sci. USA 73, 3628-3632
2 Dreyer, W. J. and Bennett, J. C. (1%5) 54, 864-869 3 Tonegawa, S. (1983) Nature 302, 575-581 4 Baltimore, D. (198l) Cell 26, 295-2% 5 Lewis, S., Gifford, A. and Baltimore, D. Nature (in press) 6 Weigert, M., Perry, R., Kelly, D., HunkapiUer, T., Schilling, J. and Hood, L. (1980) Nature 283, 497-500 7 Alt, F. W. and Baltimore, D. (1982) Proc. Natl Acad. Sci. USA 79, 4118--4122 8 Kabat, E. A. (1982) Pharm. Rev. 34, 23-38 9 Wu, T. T. and Kabat, E. A. (1970) J. Exp. Med. 132, 211-250 10 Novotny, J., Bruccoleri, R., Newell, J., Murphy, D., Haber E. and Karplus, M. (1983) J. Biol. Chem. 258, 14433-14437 11 Niman, H. L., Houghten, R. A., Walker, L. E., Reisfeld, R. A., Wilson, I. A., Hogle, J. M. and Lerner, R. A. (1983) Proc. Natl Acad. Sci. USA 80, 4949-4953 12 Queen, C. and Baltimore, D. (1983) Cell 33, 741-748 13 Banerji, J., Olson, L. and Schaffner, W. (1983) Cell 33, 729-740 14 Gillies, S. D., Morrison, S. L., Oi, V. T. and Tonegawa, S. (1983) Cell 33,717-728 15 Blanner, F. R. and Tucker, P. W. (1984) Nature 307, 417--422 16 Zinkernagel, R. M. and Doherty, P. C. (1974) Nature 248, 701-702 17 Acuto, O., Hussey, R. E., Fitzgerald, K. A., Protentis, J. P., Meuer, S.C., Schlossman, S.F. and Reinherz, E.L. (1983) Cell 34, 717-726 18 Kappler, J., Kubo, R., Haskins, K., White, J. and Manack, P. (1983) Cell 34, 727-737 19 Mclntyre, B. W. and Allison, J. P. (1983) Cell 34, 739-746 20 Jerne, N. K. (1971)Eur. J. Immunology 1, 1-9