Generation of immune specificity: A working hypothesis

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BioSysterns, 13 (1981) 259--266 © Elsevier/North-Holland Scientific Publishers Ltd.

GENERATION OF IMMUNE SPECIFICITY: A WORKING HYPOTHESIS

ANTOINE DANCHIN a'* and PIOTR P. SLONIMSKI b alnstitut de Biologic Physico-chimique, 13 rue Pierre Curie, F75005 Paris and bCentre de Gdndtique Moldculaire du C.N.R.S., F91190 Gif-sur-Yvette and Universitd Pierre et Marie Curie, Paris, France (Received February 2rid, 1981) A speculative model for epigenetic generation of immune specificity is presented. It is based on the idea that combinatorial splicing within the same pre-mRNA molecule could generate a variety of mRNAs leading to a diversity of immurLoglobulins. The model postulates a special role for the nuclear envelope where translation of intron sequences into messenger maturation proteins would take place. The postulates are justified and experimental implications of the model are discussed.

Higher vertebrates have evolved an exquisitely specialized system which is meant to respond to foreign substances (antigens) by the synthesis olF a class of proteins, the immunoglobulins, which exhibit strong selectivity for the antigen. One of the most puzzling features of this adaptive response is the apparent ability to synthesize new immunoglobulins for e~:h type of antigen even when the antigenic substance is entirely artificial. A number of theories have been put forward in order to explain this phenomenon. The most pertinent are selective theories. They assume a preexisting w~riability (real or virtual) in the cell types that are committed for immunoglobulin production. Essentially, two classes of selective theories exist: those that assume somatic mutations (cf. Jerne, 1975} and those that assume germ-line genetic recombination (cf. Hood and Talmage, 1970) as the major source of preexi.~ting variability. With the help of appropriate (but sometimes, ad hoc) parameters both theories can easily account for the extent of diversity which can be estimated in millions, if not billions, of different immunoglobulin molecules. In our opinion, however, these theories (in their purest form) do not readily explain how the immune *To whom all correspondance should be addressed.

response can be efficient enough if the antigenic substance has to meet a rare lymphocyte in order to elicit immunoglobulin production. Obviously, one could increase the probability of such an encounter but only at the cost of the decrease o f preexisting diversity. In order to dispose of this difficulty an epigenetic selective model has been recently proposed (Danchin, 1979). It involves two steps for specification. The first step, formally similar to the germ line hypothesis, provides a limited dictionary of germ cells which produce a label immunoglobulin molecule. This label can be selected after a weak interaction with the antigen, and triggers the second step of specification. This second step is an epigenetic process where variants of the label are produced without necessitating modifications o f the DNA sequence in the progeny of the triggered cell up to a point where a clone synthesizes a single specific immunoglobulin having a strong selectivity towards the antigen. We wish to present here a theoretical model based upon a molecular mechanism accounting for this epigenetic specification. Our model takes into account the well documented existence of split genes and splicing phenomena (cf. Gilbert, 1978; Crick, 1979; Abelson, 1979) and a recently advanced working hypothesis on "messenger maturation" pro-

260 teins or "maturases" involved in splicing and transport of messenger RNAs (Jacq et al., 1980; Lazowska et al., 1980; Slonimski, 1980). We wish to emphasize at this point that our model is highly speculative both in its immunological and molecular postulates. We have replaced the orthodox assumption that one immunocompetent cell line produces only one type of immunoglobulin, by a heterodox one. It postulates that such a cell line can transiently produce a spectrum of different immunoglobulins. Furthermore, it assumes that the antigen can penetrate into the nucleus where a specific RNA translation occurs. We believe, however, and we shall try to document this, that no convincing experimental (or logical) evidence exist against our assumptions, and that the predictions one can derive from our model justify its very existence be it only to be refuted by new types of experiments.

Postulates

Immunological postulates (0) The generation of immune specificity occurs exclusively through selective processes. (i) Two steps are involved in specification. The first provides gross specification. It involves recombination and/or mutation and produces numerous cell lines differing by their genotypes. Selection among cells carrying different genotypes results in elimination of the major production of antiself immunoglobulins (this will not be discussed here, see Danchin, 1979). The second provides fine specification. It selects epigenetically within a single cell among variants of gene products specified by DNA segments selected after gross specification. (ii) In the absence of a stimulating antigen, a given immunocompetent cell line produces a label single immunoglobulin which corresponds to a given expression state of its genotype. This antibody is synthesized ac-

cording to a molecular mechanism described below. It can interact with foreign antigen. Cell division and immunoglobulin production is triggered provided the interaction is stronger than a threshold value. (iii) Epigenetic specification comes from a process which involves selection through competition among antigen binding molecules, according to a mechanism explicited next.

Molecular postulates (iv) m R N A s coding for immunoglobulins (light and heavy chains) are derived from premRNA transcripts by splicing. Variable and constant regions (exons) of the immunoglobulin chains are separated by introns. Splicing occurs in (or at) the nuclear envelope and is concommitant with transport of m R N A across this envelope. (v) T w o translation machineries exist in eukaryotic cells. These machineries are located inside the nucleus (or in organelles) on the one hand, and in the cytoplasm on the other hand. Nuclear ribosomes translate pre-mRNA transcripts into messenger maturation proteins (m-proteins) with a code where UAA (and possibly UAG, but not UGA) are termination codons. Cytoplasmic ribosomes translate mRNA into eu-proteins and several codons act as terminators (UAA, UGA and probably UAG). (vi) m-proteins are nuclear translation products formed from in phase translation of continuous exon-intron sequences. They are made of, at least, two well defined domains. The amino terminal domain is similar to that found in the final immunoglobulin translated from the mRNA in the cytoplasm. The carboxy terminal domain (translated from the intron sequence) is highly hydrophobic. m-proteins are thus anchored into the nuclear envelope. Light and heavy chain m-proteins are constituted of a variable domain, denoted respectively VL and VH, at the N-terminus and a membrane anchored domain at their C-terminus.

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(vii) m-proteins are responsible for splicing specificity by triggering and directing the catalytic activity of a general splicing complex located in the nuclear envelope. In t h e case of immunoglobulins, the m-protein domain VL (or VH) directs splicing of the heavy chain pre-mRNA (or light chain premRNA) due to the quaternary interaction of the variable m-protein domain of one chain with the amino terminal polypeptide of the other chain. (viii) Combination of different splices withine the V-C segment of RNA transcribed from the same DNA sequence can generate a subset of m-proteins differing by their V domains.

Model for specification of immune diversity An i m m u n o c o m p e t e n t cell synthesizes a label antibody according to the following process. Light and heavy chain genes are transcribed into pre-mRNAs. These RNAs possess introns which separate sequences specifying variable (VL and VH) and constant (CL and CH) regions (Tonegawa et al., 1978; Max et al., 1979; Honjo et al., 1979). They are translated in the nucleus into m-proteins constituted of a variable domain translated from the V exons and a hydrophobic domain translated in phase from an intron sequence. Synthesis of light and heavy chains is coupled via reciprocal m-protein dependent splicing. m-protein VL attaches itself by the hydrophobic domain to the nuclear envelope; through quaternary interactions the VL domain selects the homologous VH polypeptide translated on nucleus ribosomes from the pre-mRNA H . As soon as the VL--VH interaction is estab][ished a splicing machinery splices off the pre-mRNAH intron and translocates a segment of the processed mRNAH into the cytoplasm. A symmetrical process involves the m-protein VH. mRNAH and mRNAL are consecutively translated into heavy and light chains. These constitute the label immunoglobulin molecule. W e would

like to emphasize two features of the mprotein activity: (i) a single m-protein molecule may catalyse the splicing of numerous pre-mRNA molecules; and (ii) the quantity of m-proteins molecules is auto-regulated by the very fact that it excises the intron RNA sequence which specifies its own Cterminal aminoacid sequence (Jacq et al., 1980; Lazowska et al., 1980; Slonimski, 1980). Thus, splicing process is regulated by the efficiency of pre-mRNA transcription together with the concentration of m-protein VL and m-protein VH in the nuclear envelope. Reciprocal interactions ensure maturation of both mRNAs in equivalent amounts. This process remains unperturbed provided there is no interference in the VL--VH interaction involved in the splicing mechanism. If an antigen comes to the nuclear envelope the process may be perturbed. The way we envisage this perturbation is based on the fact that the antigen binding site results from VL--VH interactions in the final immunogiobulin molecules (Edelman and Gall, 1969). By analogy, we assume that a similar type of binding site is formed by VL--VH interactions between .a preexisting m-protein and the nascent V domain. The antigen weakens this interaction and affects splicing in two ways: (a) splicing efficiency is decreased and thus m-protein synthesis is increased, (b) splicing specificity is altered and generates novel combinations of splices within the V--C RNA region with concommitant synthesis of a novel subset of m-proteins. The presence of antigen triggers cell division and therefore formation of new nuclei. Thus newly available nuclear envelope sites would be occupied by a variety of m-proteins (VL and VH) as cell multiplication proceeds in the presence of the antigen. Moreover, as the interaction with antigen becomes stronger, the newly synthesized set of m-proteins will tend to occupy all available sites. After a certain number of divisions the initial set of m-proteins VL1 and m-proteins VH1 will be replaced by a novel set of m-proteins VL2 and m-proteins VH2~ This new set directs splicing of pre-mRNAs

262 and therefore permits the production of an immunoglobulin molecule differing from the initial label and exhibiting a stronger selectivity for the antigen. Since the m-proteins alone direct splicing of pre-mRNA, the synthesis of the corresponding immunoglobulin can proceed now in the absence of the antigen. There has been epigenetic specification of an immunoglobulin structure. In summary four phases are observed: (1) an untriggered cell produces a single immunoglobulin type; (2) after contact with antigen the progeny of a triggered cell produces, for a few generations, a variety of immunoglobulin types; (3) a selection amongst m-proteins, through competition for available sites, takes place for that type of m-protein the synthesis of which has been most efficiently increased by the binding of antigen to the interacting V L - - V H domains; and (4) after a few days a clone has only one type of immunoglobulin m-proteins and therefore produces only one immunoglobulin type, endowed with a stronger selectivity for the antigen.

Justification of the postulates The essential feature of selective theories concerning the generation of immune specificity is the combinatorial use of various parts of genetic information. Until now most of this combinatorial use was restricted to the DNA level (Jerne, 1975; Hood, 1970; Smithies, 1973; Claflin and Rudikoff, 1976) emphasizing the importance of genetic recombination and or mutation (see also, Urbain, 1981, for recent developments of network theory). Recent advances in gene organization and expression of eucaryotes disclosed the phenomenon of splicing at the RNA level (Gilbert, 1978; Crick, 1979; Abelson, 1979). It brings the opportunity of another combinatorial use which would act at the epigenetic level. Gilbert (1978) has already pointed out briefly, that combination of different splices within the same pre-mRNA molecule could generate a

Variety of mRNA molecules leading therefore to a diversity of proteins specified by the same gene. The production of small t and large T proteins through different splices of SV40 RNA is a case in point (Crawford, 1980). Furthermore, various structurally related proteins are indeed generated by point mutations located in an intron of a single gene (Claisse et al., 1980; Lazowska et al., 1980). Although no experimental evidence, neither for nor against the involvement of RNA-splicing in the generation of immune specificity, is presently available (see discussions in Tonegawa et al., 1978; Rabbitts, 1978) it appears to us justifiable that such a possibility should be explored in all its logical consequences. One of the major justifications we can see in postulating involvement of epigenetic controls in this process is gene sparing. Let us consider that the combinatorial splicing of two pre-mRNA molecules (one for VH, one for VL) could produce between 102 and 1.03 different variants of mRNA molecules. This would reduce by the same order of magnitude the number of genes required for generating immune specificity. Furthermore, it provides means for increasing the rapidity of immune response (Cunningham, 1976). The reduction in the number of genes and their primary transcripts would automatically result in an increase of probability of a fast immune response elicited by an incoming antigen. Consequently, the classical "1 cell -- 1 immunoglobulin" postulate (Jerne, 1976), can be kept unmodified before and after the specification period. It has to be amended only during the transition which establishes the specification. Our postulates require a highly elaborated nuclear organization for splicing of premRNAs and translocation of mRNAs, as well as continuity of some essential features of this organization when cell divides. This can be justified as follows. Apart from its well known compact chromatine structure the eukaryotic cell nucleus displays several other highly organized structures the function of which is still poorly

263 understood (Fr~nke, 1974; Fry, 1977). We shall emphasize here only some of them, namely the network formed of ribonucleoprotein fibers (Miller et al., 1978; Herman et al., 1978} which organizes the HnRNA and may physically couple DNA transcription to mRNA maturation and transport, and the nuclear envelope made c,f a double lipoprotein bilayer internally coated with a protein matrix, the lamina densa (Gerace et al., 1978). The ribonucleoprotein fibers are connected to porecomplexes (Franke, 1974; Fry, 1977) in the nuclear envelope. These structures consist of a circular lumen (650--750 A in diameter) which connects interior of the nucleus to the cytoplasm. They are surrounded on the inner and outer face by two annulus made of eight ribonucleoprotein granules (,x, 200 /~ in diameter). Circumstantial evidence suggests that transport of ribonucleoproteins occurs through the pore-complexes (Franke, 1974). Protein synthesis in the nucleus is stillsubject to controversy (Goidl and Allen, 1978) but one frequently observes that fibrillar structures of ribonucleoprotein are connected to the inner annulus and that granules of the outer annulus are physically linked with polyribosomes (Franke, 1974). Whether these granules represent ribosomes is an open question although they appear significantly larger. Absent from this picture is the general splicing maturation and m R N A transport machinery(s) which we think, should represent a significant fraction of the nonhistene nuclear proteins. W e can tentatively interpret the lamina densa and the nuclear pore-complexes as the reservoir of m-proteins and the lieu for the machinery of H n R N A translation, splicing and translocation. Another argument suggesting a major function of nuclear pore-complexes in R N A proceasing is that in some differentiated states which are characterized by a decreased transcriptional activity one observes a decreased pore number. Conversely, there is an increase in nuclear cell surface and porecomplex number correlated with the activation of nuclear transcription in lymphocytes (Maul,

1971). That a translation process m a y be involved in splicing of H n R N A is also suggested by the fact that cells with deficient nucleoli fail to process the R N A (Goidl and Allen, 1978). The continuity of the above mentioned nuclear structures during cell division cycle has to be discussed. In most cells, nuclear membrane seems to disappear when the cell divides. However, it is admitted that the HnRNA does not diffuse to cytoplasm throughout cell divisions (Davidson and Britten, 1979). Thus, there must be ways and means to prevent it. Similarly, porecomplexes have been found bound to chromosomes during mitosis in an apparent absence of nuclear membrane (Maul, 1977). Although nuclear envelope constituants are found spread in the cytoplasm they are concentrated again in the envelope of the daughter nuclei (Ely et al., 1978). W e consider all these results as evidence for continuity of nuclear envelope structures from generation to generation. We must now attempt to justify m-protein existence for variable and heavy immunoglobulin chains. Firstly, the DNA organization of both chains has been shown to fit our assumptions: intron sequences separate exon sequences specifying variable and constant portions of each chain (Tonegawa et al., 1978; Max et al., 1979; Honjo et al., 1979). Moreover, it has been found (Max et al., 1979) that several short base-sequence segments, which could specify short polypeptides -- now called the J segments -- exist in the genomic DNA. In the immunoglobulin these J segments connect V to C regions and provide a major fraction of immunoglobulin diversity (Schilling et al., 1980). This has been interpreted in terms of DNA recombination processes, generating a whole set of germ lines differing by the sequence of J regions (Max et al., 1979; Schilling et al., 1980; Brack et al., 1978; Davis et al., 1980). It can, as well, be interpreted as we do, in terms of splicing processes in addition to recombination. The five J segments found in the Kappa light chain gene (Max et al., 1979) could thus easily

264 account for a variety of different mRNA structures all derived from a single DNA sequence. The second point Of justification concerns the specific nature of m-proteins, i.e. structuration into well defined polypeptide domains corresponding to exons. It is well known indeed that each immunoglobulin chain consist of several domains (Edelman and Gall, 1969; Segal et al., 1974) organized around intra-domain disulfide bridges and that the variable segments constitute a single individual domain encoded by a separate exon (Tonegawa et al., 1978; B r a c k e t al., 1978). Furthermore, it has been found (Segal et al., 1974) that quaternary interactions between the VL and VH domains are even stronger than the intra-chains inter-domain interactions VL--CL and VH--CH. This justifies therefore our postulate which assumes that a VL--VH interaction directs splicing of premRNAs (see also Gival et al., 1976). Finally, studies of the interaction between light and heavy chains have shown that presence of the antigen weakens the quaternary cohesion of the chains (Popova and Kositskaya, 1977) which is consistent with our model. Experimental implications of the model Our model has experimental implications at three levels at least: immune response of individual immunocompetent cells during the specification period, genetic control of the relationship between light and heavy chain synthesis, molecular mechanisms of mprotein synthesis and their function in premRNA splicing specificity. In the conceptual frame we have outlined here specification occurs through a competition process involving binding sites in the nuclear envelope. This process would result in the replacement of an initial set of mproteins by a new set, as the divisions proceed. This has immediate consequences: one should observe that a given cell, producing one species of immunoglobulin in its initial state, yields a progeny which produces another

species after sufficient interactions with an antigen. Moreover, during a transient intermediary period it should produce a variety of immunoglobulins having different specificities. Antoine et al. (1979) have indeed found such a response, which cannot easily be reconciled with the usual immunological postulate (1 cell -- 1 antibody). Besides, the time course needed for complete specification (8--10 generations) provides a rough estimate of the number of nuclear membrane binding sites required for specific antibody production, since the initial number of m-proteins bound to such sites is divided by 2 at each generation. The figure we find, l 0 s , is compatible with the pore number found on an average nucleus (200 #m 2, 1--60 pores/urn: ; Franke, 1974). A second set of implications is genetical. The model implies that syntheses of light and heavy chains are coupled via reciprocal mprotein dependent splicing. The three types of genetic defects found in immunocompetent cells: cells which do not produce either chains, cells which synthesize only heavy chains and cells which synthesize only light chains (Coffino and Scharff, 1971; Franklin and Frangione, 1975) are consistent with the model. In addition our hypothesis implies that appropriate deletions including the DNA sequence linking V and C sequences should result in the production of a shorter polypeptide chain (lacking at least the J region) and no synthesis of the other chain. A mutant, derived from a myeloma clone producing immunoglobulins harboring a deletion in the CH region (and producing normal L-chains), has been isolated which no longer produces light chains. It synthesizes new heavy chains having an additional small deletion probably in the V--C region (Morrison, 1978). This work shows that only certain types of deleiions of the heavy chain gene can affect synthesis of the light chains. This conclusion is further substantiated by the observation that "heavy chain diseases" corresponding to cell lines which synthesize heavy chains alone are usually deleted in the V--C region

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(Franklin and Frangione, 1975; Frangione and Franklin, 1979). Our model has even stronger implications: if, for instance, the light chain is n o t produced because of a defect in the m-protein synthesis of the heavy chain, one should nevertheless find the unspliced pre-mRNA corresponding to the light chaiTls. An interesting work by Cowan et al. (1974) has shown, by fingerprinting RNA found in a cell-line producing heavy chains but not light chains, that the RNA coding fo:r the latter was present within the cell as "unprocessed mRNA precursor". Another illusization of the model might be the immunoglobulin "heavy chain switch" in secondary response to antigen: our interpretation of available data would be somewhat different zFrom the most recent interpretation by Davis et al. {1980) and Rabbitts et al. (1980). We favor combinatorial splicing in addition to or instead of recombination. Finally a more biochemical approach would consist in attempts to modulate the putative intra-nuclear protein synthesizing machinery by various antibiotics. One could for instance predict that ambiguity generating protein synthesis inhibitors (cf. Singh et al., 1979) might be used to shift the immune response to new specificities. In this respect it is of interest to notice that inhibitors such as cycloheximide seem to inhibit less the nuclear translation machinery than the cytoplasmic one (Goidl and Allen, 1978). We would suggest therefore a systematic search for protein synthesis inhibitors in order to modify the specificity of the immune response.

Conclusions and perspectives We have s:hown how molecular hypotheses involving an explicit function of introns could account for an epigenetic specification o1[ immune diversity. In addition to its experimentally testable implications in the field of immunology, we believe that our model is a pm'adigmatic illustration of what could be the general p h e n o m e n o n of epigenesis

in eucaryotes. The first theoretical model of epigenesis, i.e. long-lasting, self-sustained, hereditary modifications without genotypic changes, was proposed by Delbriick (1949). Only a limited number of biological systems has been since amenable to comprehensive experimental analyses (Novick and Wiener, 1957; Cohn and Horibata, 1959; Thomas, 1979). These experiments demonstrate the crucial role played in some epigenetic phenomena by a special class of (real) proteins, the permeases, located in the bacterial cell envelope. We wish to point out that a competition in the nuclear envelope between a special class of (hypothetical) proteins, the mproteins, might be of importance in some epigenetic phenomena of eucaryotes.

Acknowledgements We thank S. Avrameas, M. Blanc, J. Brachet, J.C. Courvalin, F. Crick, F. Jacob, B. Maro, R. Thomas and A. Ullmann for discussions.

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