BioSystems, 11 (1979)163--165 © Elsevier/North-Holland Scientific Publishers Ltd.
163
FUSION AS A POTENTIAL MECHANISM FOR GENERATING LYMPHOCYTE DIVERSITY*
HANS J. BREMERMANN
Department of Mathematics, University of California, Berkley 94720, U.S.A.
At birth there is a large reservoir of distinct l y m p h o c y t e sub-populations ( 1 0 6 - 1 0 7 ) each with a different antigenic specificity. For each of millions of different antigens the organism has a specific l y m p h o c y t e population ready to be stimulated and expanded rapidly during the immune response. How can the organism generate millions of l y m p h o c y t e varieties, each specialized for a distinct immunoglobulin, if the organism has only a limited number of genes (of the order of 10 s)? Since some genetic determination is required for all the numerous subsystems of an organism, only a fraction of the 10 s genes can be involved in organizing the several millions of differer t l y m p h o c y t e varieties. Watson (1976, p. 619) calls this "the major unresolved problem in i m m u n o l o g y " . Several theories have been proposed: 1. The somatic mutation theory (comp. Eisen, 1974; Watson, 1976): according to this theory a small number of germ line genes give rise to diversity by means of somatic mutations of the genes coding for the amino acids of the variable portions of the light chains. Such " h o t spots" next to the "cold spots" within the same gene are highly implausible and the theory has been largely discredited. An alternative theory is II. the germ line theory: this theory postulates a separate gene for each light end heavy chain. This would require from 2000 to 6000 genes, or some 1%-* This paper has been reprinted with the kind permission from J. Theor. Biol., (1979) 76,311--334, (Academic Press).
10% of the total (Watson, 1976, p. 619}. While the dedication of several thousand genes to the generation of lymphocyte diversity is not ruled out a priori it still seems excessive. Edelman and Galley (1970) proposed an alternative theory, III. Intracellular somatic recombination o f germ line genes: this theory requires only a small number of original germ line genes. Edelman and Gally ( 1 9 7 0 ) h y p o thesized that the recombinations occur through crossing over, between homologous chromatids during mitosis and through mispairing in regions of a family of repeated genes (which are identical except for the variable region of the immunoglobulin sequences). They made this suggestion within the larger c o n t e x t of an explanation for the phenomenon of repetitive DNA which occurs in eukaryotes. (For a mathematical analysis of the dymanics of unequal crossing over and for a recent bibliography see Perelson and Bell, 1977.) In the following we will suggest a variant of the somatic recombination theory which does not require the mechanism of unequal crossing over of mispaired sister chromatids. Instead, we postulate that crossing over occurs between chromosomes that are derived from different cells which have joined through cell fusion. IV. Intercellular recombination o f germ line genes: we envision the following mechanism: there are several germ line genes which code for identical sequences in the constant regions of the light and heavy chains. They differ in the variable regions. During embryonic development (and possibly throughout life) the l y m p h o c y t e precursor cells repeatedly
164 fuse with each other and subsequently divide again. In the course of these "matings" a great variety of different recombinations is generated through crossing over in the germ line genes and their recombinant descendants. This theory is analogous to the somatic recombination theory of Edelman and Gally except that the postulated mechanism of crossing over between mispaired regions of repeated genes is replaced by a postulated ordinary crossing over between chromosomes derived from different parent cells. The theory obviously can account for the observed variety. Since adjacent codons would have a smaller probability of segregating under crossing over there should be a linkage effect analogous to that which occurs between genes located on the same chromosome: the closer they are, the less likely they will segregate under crossing over. The same as the genetic distance on a chromosome can be mapped, so it should be possible to map genetic distances between amino acids in the variable region of the light and heavy chain. Eventually it should be possible to reconstruct the original germ line sequences ,vhich are the "ancestors" of the immunoglobulin population. Such a reconstruction would require knowledge of the amino-acid sequences of a representative sample of all immunoglobulins present. Watson (1976) points out (p. 620) that on the basis of the sequence data that are available the kappa immunoglobulins seem to fall into 3 distinct sub-groups, while the lambda immunoglobulins seem to fall into at least 5 sub-groups. Under our hypothesis this could be an indication that there are 3 and 5 (or more) germ line genes respectively. Thus l y m p h o c y t e mating would explain the lymphocyte variability puzzle. It might also play a role in some of the other unsolved problems of the immune response, like the transition from IgM to IgG immunoglobulins (comp. Watson, p. 623). In this transition the evolved variable part becomes " m a t e d " to a different constant part. Histocompatability: According to current
theories T-cells pass from the bone marrow to the thymus gland where they are screened for histo-compatability. Those that do not pass the histo-compatability test are eliminated in the thymus, the others pass on into the bloodstream (Burnet, 1974). An analogous process is envisioned for the B-cells. In each of the 4 theories lymphocytes are generated in a random or combinatorial fashion and the resulting immunoglobulin presumably would contain some that would bind to the organism's own antigens, Thus all 4 theories require a screening process. They also require that the generation of diversity stops when the lymphocytes have been released into the blood stream. Since the antigen stimulates small lymphocytes to proliferate (clonal selection theory) there are difficulties with theories I and III. In theory I hypermutability would have to stop after lymphocytes are released into the blood stream and in theory III crossing over among sister chromatids, or at least misalignment, would have to stop. This is difficult to imagine. In our theory histo-compatability requires that lymphocytes would have to cease mating once they have passed the thymus (or equivalent} and entered the bloodstream. A mechanism that terminates fusion at a certain stage in a lymphocyte's development (when it has passed the histo-compatibility screening process) poses no difficulty at all. Our theory avoids the difficulties that afflict theories I and III. L y m p h o c y t e hybridization in the bone marrow would be consistent with data on polyploidy. A survey of Hauschka (1961) of the ploidy of various tissues showed that in man, mouse and rat: testis and lung tissues are nearly 100% diploid, while the figures for bone marrow are 88% {man), 84% {mouse), 68% (rat). Hauschka's figures are indicative of 12%--32% tetraploid (and possibly higher polyploid) cells. The phenomenon of such a large percentage of polyploid cells would be hard to explain otherwise but it fits perfectly with our hypothesis.
165 R e - a s s o r t m e n t o f g e n e s t h r o u g h cell f u s i o n is n o t l i m i t e d t o l y m p h o c y t e s b u t o c c u r s i n cell c u l t u r e s a n d p r o b a b l y in vivo. A t h e o r y o f cell f u s i o n in v i t r o a n d in vivo as a p a r a s e x u a l p r o c e s s has b e e n d e v e l o p e d i n B r e m e r m a n n { 1 9 7 9 ) . T h e s a m e p a p e r c o n t a i n s also a s u m m a r y o f t h e f a c t s o f s p o n t a n e o u s cell f u s i o n , a survey of the relevant literature, a n d a discussion of possible means of detection. Lymphocyte h y b r i d i z a t i o n c o u l d possibly be d e t e c t e d through bone marrow transplants between i n b r e d mice, raSioactive labelling or genetic markers.
Note added in print A recent supley of the status of various t h e o r i e s o f a n t i b o d y d i v e r s i t y has a p p e a r e d in Kindt and Capra (1978). Our hypothesis would s e e m t o be c o n s i s t e n t w i t h r e c e n t d a t a o n g e n e i n t e r a c t i o n . A c o m p r e h e n s i v e review of facts a n d l i t e r a t u r e o t cell f u s i o n , i n c l u d i n g a c h a p -
ter on s p o n t a n e o u s fusion, m a y be f o u n d in R i n g e r t z a n d Savage ( 1 9 7 6 ) .
References Bremermann, H.J., 1979, Theory of spontaneous cell fusion. Sexuality in cell populations as an evolutionarily stable strategy. Applications to immunology and cancer, J. Theor. Biol. 76,311. Burnett, Sir Macfarlane, 1974, Intrinsic mutagenesis: A genetic approach to ageing (Wiley, New York). Edelmann, G.M. and J.A. Gaily, 1970, in: The neurosciences: Second study program, F.O. Schmidt (ed.) (Rockefeller University Press, New York). Eisen, H.N., 1974, Immunology (Harper and Row, New York). Hauschka, Th. S., 1961, Cancer Res. 2 1 , 9 5 7 . Kindt, Th. J. and J.D. Capra, 1978, Immunogenetics 6,309. Perelson, A.S. and G.I. Bell, 1977, Nature 265,305. Ringertz, N.R. and R.E. Savage, 1976, Cell hybrids (Academic Press, New York). Watson, J., 1976, Molecular biology of the gene, 3rd edn. (Benjamin, Menlo Park).
Commentary
F.H. Kirkpatrick It seems a bit p~:emature to rule to out the competing hypotheses, but this mechanism has several interesting aspects. The quoted data on the degree of polyploidy of marrow are old, but if correct give this mechanism plausibility. In addition, this model requires no specific mechanism to be "turned off" once lymphocytes leave the marrow, since in circulation they are very "dilute" and the probability of fusion would be much reduced. One of the few circumstances in which T lymphocytes are no longer dilute is in a concerted "attack" on an infection. In such circumstances, fusion among cells which have already been "screened" could lead to autoimmune disease. Note also that "screening" could occur in the marrow, or any other tissue: cells which recognize the tissue and attack cannot leave.
Otto Rossler There is one class of macromolecules which evolved an extreme functional diversification' perhaps like some molecules in the brain, but much more visible. For in this case the molecules are the "brain", so to speak, or at least more or less so. It is the set of immunological recognition (and attack-directing) molecules. Although we don't know whether this diversity reflects a functional necessity -- some "deductive biologists" tend to think so its factual existence in terms of the mechanisms that bring it about physiologically is still unexplained. Hans Bremermann shows that his recent favorite idea of cell fusion is capable of yielding a plausible, rounded-off picture, something earlier theories failed at.
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163
FUSION AS A POTENTIAL MECHANISM FOR GENERATING LYMPHOCYTE DIVERSITY*
HANS J. BREMERMANN
Department of Mathematics, University of California, Berkley 94720, U.S.A.
At birth there is a large reservoir of distinct l y m p h o c y t e sub-populations ( 1 0 6 - 1 0 7 ) each with a different antigenic specificity. For each of millions of different antigens the organism has a specific l y m p h o c y t e population ready to be stimulated and expanded rapidly during the immune response. How can the organism generate millions of l y m p h o c y t e varieties, each specialized for a distinct immunoglobulin, if the organism has only a limited number of genes (of the order of 10 s)? Since some genetic determination is required for all the numerous subsystems of an organism, only a fraction of the 10 s genes can be involved in organizing the several millions of differer t l y m p h o c y t e varieties. Watson (1976, p. 619) calls this "the major unresolved problem in i m m u n o l o g y " . Several theories have been proposed: 1. The somatic mutation theory (comp. Eisen, 1974; Watson, 1976): according to this theory a small number of germ line genes give rise to diversity by means of somatic mutations of the genes coding for the amino acids of the variable portions of the light chains. Such " h o t spots" next to the "cold spots" within the same gene are highly implausible and the theory has been largely discredited. An alternative theory is II. the germ line theory: this theory postulates a separate gene for each light end heavy chain. This would require from 2000 to 6000 genes, or some 1%-* This paper has been reprinted with the kind permission from J. Theor. Biol., (1979) 76,311--334, (Academic Press).
10% of the total (Watson, 1976, p. 619}. While the dedication of several thousand genes to the generation of lymphocyte diversity is not ruled out a priori it still seems excessive. Edelman and Galley (1970) proposed an alternative theory, III. Intracellular somatic recombination o f germ line genes: this theory requires only a small number of original germ line genes. Edelman and Gally ( 1 9 7 0 ) h y p o thesized that the recombinations occur through crossing over, between homologous chromatids during mitosis and through mispairing in regions of a family of repeated genes (which are identical except for the variable region of the immunoglobulin sequences). They made this suggestion within the larger c o n t e x t of an explanation for the phenomenon of repetitive DNA which occurs in eukaryotes. (For a mathematical analysis of the dymanics of unequal crossing over and for a recent bibliography see Perelson and Bell, 1977.) In the following we will suggest a variant of the somatic recombination theory which does not require the mechanism of unequal crossing over of mispaired sister chromatids. Instead, we postulate that crossing over occurs between chromosomes that are derived from different cells which have joined through cell fusion. IV. Intercellular recombination o f germ line genes: we envision the following mechanism: there are several germ line genes which code for identical sequences in the constant regions of the light and heavy chains. They differ in the variable regions. During embryonic development (and possibly throughout life) the l y m p h o c y t e precursor cells repeatedly
164 fuse with each other and subsequently divide again. In the course of these "matings" a great variety of different recombinations is generated through crossing over in the germ line genes and their recombinant descendants. This theory is analogous to the somatic recombination theory of Edelman and Gally except that the postulated mechanism of crossing over between mispaired regions of repeated genes is replaced by a postulated ordinary crossing over between chromosomes derived from different parent cells. The theory obviously can account for the observed variety. Since adjacent codons would have a smaller probability of segregating under crossing over there should be a linkage effect analogous to that which occurs between genes located on the same chromosome: the closer they are, the less likely they will segregate under crossing over. The same as the genetic distance on a chromosome can be mapped, so it should be possible to map genetic distances between amino acids in the variable region of the light and heavy chain. Eventually it should be possible to reconstruct the original germ line sequences ,vhich are the "ancestors" of the immunoglobulin population. Such a reconstruction would require knowledge of the amino-acid sequences of a representative sample of all immunoglobulins present. Watson (1976) points out (p. 620) that on the basis of the sequence data that are available the kappa immunoglobulins seem to fall into 3 distinct sub-groups, while the lambda immunoglobulins seem to fall into at least 5 sub-groups. Under our hypothesis this could be an indication that there are 3 and 5 (or more) germ line genes respectively. Thus l y m p h o c y t e mating would explain the lymphocyte variability puzzle. It might also play a role in some of the other unsolved problems of the immune response, like the transition from IgM to IgG immunoglobulins (comp. Watson, p. 623). In this transition the evolved variable part becomes " m a t e d " to a different constant part. Histocompatability: According to current
theories T-cells pass from the bone marrow to the thymus gland where they are screened for histo-compatability. Those that do not pass the histo-compatability test are eliminated in the thymus, the others pass on into the bloodstream (Burnet, 1974). An analogous process is envisioned for the B-cells. In each of the 4 theories lymphocytes are generated in a random or combinatorial fashion and the resulting immunoglobulin presumably would contain some that would bind to the organism's own antigens, Thus all 4 theories require a screening process. They also require that the generation of diversity stops when the lymphocytes have been released into the blood stream. Since the antigen stimulates small lymphocytes to proliferate (clonal selection theory) there are difficulties with theories I and III. In theory I hypermutability would have to stop after lymphocytes are released into the blood stream and in theory III crossing over among sister chromatids, or at least misalignment, would have to stop. This is difficult to imagine. In our theory histo-compatability requires that lymphocytes would have to cease mating once they have passed the thymus (or equivalent} and entered the bloodstream. A mechanism that terminates fusion at a certain stage in a lymphocyte's development (when it has passed the histo-compatibility screening process) poses no difficulty at all. Our theory avoids the difficulties that afflict theories I and III. L y m p h o c y t e hybridization in the bone marrow would be consistent with data on polyploidy. A survey of Hauschka (1961) of the ploidy of various tissues showed that in man, mouse and rat: testis and lung tissues are nearly 100% diploid, while the figures for bone marrow are 88% {man), 84% {mouse), 68% (rat). Hauschka's figures are indicative of 12%--32% tetraploid (and possibly higher polyploid) cells. The phenomenon of such a large percentage of polyploid cells would be hard to explain otherwise but it fits perfectly with our hypothesis.
165 R e - a s s o r t m e n t o f g e n e s t h r o u g h cell f u s i o n is n o t l i m i t e d t o l y m p h o c y t e s b u t o c c u r s i n cell c u l t u r e s a n d p r o b a b l y in vivo. A t h e o r y o f cell f u s i o n in v i t r o a n d in vivo as a p a r a s e x u a l p r o c e s s has b e e n d e v e l o p e d i n B r e m e r m a n n { 1 9 7 9 ) . T h e s a m e p a p e r c o n t a i n s also a s u m m a r y o f t h e f a c t s o f s p o n t a n e o u s cell f u s i o n , a survey of the relevant literature, a n d a discussion of possible means of detection. Lymphocyte h y b r i d i z a t i o n c o u l d possibly be d e t e c t e d through bone marrow transplants between i n b r e d mice, raSioactive labelling or genetic markers.
Note added in print A recent supley of the status of various t h e o r i e s o f a n t i b o d y d i v e r s i t y has a p p e a r e d in Kindt and Capra (1978). Our hypothesis would s e e m t o be c o n s i s t e n t w i t h r e c e n t d a t a o n g e n e i n t e r a c t i o n . A c o m p r e h e n s i v e review of facts a n d l i t e r a t u r e o t cell f u s i o n , i n c l u d i n g a c h a p -
ter on s p o n t a n e o u s fusion, m a y be f o u n d in R i n g e r t z a n d Savage ( 1 9 7 6 ) .
References Bremermann, H.J., 1979, Theory of spontaneous cell fusion. Sexuality in cell populations as an evolutionarily stable strategy. Applications to immunology and cancer, J. Theor. Biol. 76,311. Burnett, Sir Macfarlane, 1974, Intrinsic mutagenesis: A genetic approach to ageing (Wiley, New York). Edelmann, G.M. and J.A. Gaily, 1970, in: The neurosciences: Second study program, F.O. Schmidt (ed.) (Rockefeller University Press, New York). Eisen, H.N., 1974, Immunology (Harper and Row, New York). Hauschka, Th. S., 1961, Cancer Res. 2 1 , 9 5 7 . Kindt, Th. J. and J.D. Capra, 1978, Immunogenetics 6,309. Perelson, A.S. and G.I. Bell, 1977, Nature 265,305. Ringertz, N.R. and R.E. Savage, 1976, Cell hybrids (Academic Press, New York). Watson, J., 1976, Molecular biology of the gene, 3rd edn. (Benjamin, Menlo Park).
Commentary
F.H. Kirkpatrick It seems a bit p~:emature to rule to out the competing hypotheses, but this mechanism has several interesting aspects. The quoted data on the degree of polyploidy of marrow are old, but if correct give this mechanism plausibility. In addition, this model requires no specific mechanism to be "turned off" once lymphocytes leave the marrow, since in circulation they are very "dilute" and the probability of fusion would be much reduced. One of the few circumstances in which T lymphocytes are no longer dilute is in a concerted "attack" on an infection. In such circumstances, fusion among cells which have already been "screened" could lead to autoimmune disease. Note also that "screening" could occur in the marrow, or any other tissue: cells which recognize the tissue and attack cannot leave.
Otto Rossler There is one class of macromolecules which evolved an extreme functional diversification' perhaps like some molecules in the brain, but much more visible. For in this case the molecules are the "brain", so to speak, or at least more or less so. It is the set of immunological recognition (and attack-directing) molecules. Although we don't know whether this diversity reflects a functional necessity -- some "deductive biologists" tend to think so its factual existence in terms of the mechanisms that bring it about physiologically is still unexplained. Hans Bremermann shows that his recent favorite idea of cell fusion is capable of yielding a plausible, rounded-off picture, something earlier theories failed at.
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