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Haplotype exclusion: the solution to a problem in natural selection
seminars in IMMUNOLOGY, Vol. 14, 2002: pp. 153–162 doi:10.1016/S1044–5323(02)00039-8, available online at http://www.idealibrary.com on
Haplotype exclusion: the solution to a problem in natural selection Rodney Langman and Melvin Cohn∗
asitism. These mechanisms are conveniently divided into ‘innate’ defense mechanisms and ‘adaptive’ immune systems. As an aside we would like to avoid referring to all protective defense mechanisms as immune systems. The term immune system is best reserved for the adaptive defense mechanism. Innate defense mechanisms (e.g. lysis, phagocytosis, chemokine warfare, etc.) are alone sufficient in invertebrates. Vertebrates require both innate and adaptive defense mechanisms. The term ‘adaptive’ is used to convey the notion of a defense mechanism that allows the host to adapt during its lifetime to an ever changing environment of parasites. Both the innate and adaptive systems require recognitive elements that trigger the innate ridding reactions. Germline selection on the specificity and number of innate recognitive elements is limited if the host lives for a long time relative to its parasites because the parasites are able to evolve vastly more rapidly than the hosts. This selection pressure resulted in a vertebrate mechanism for somatically generating a random repertoire of paratopes that divided the antigenic universe into combinatorially distributed epitopes making it much more difficult for pathogens to escape recognition. This somatic repertoire, which includes the recognition of both host and parasite epitopes, is coupled to the numerous innate effector mechanisms to result in the vertebrate immune system. Other characteristics of the immune system derive from the requirements imposed by the coupling of this random recognitive repertoire to the relevant biodestructive ridding reactions. The somatically generated random repertoire of paratopes defines a corresponding complementary set of epitopes that are randomly distributed on antigens of the host and the parasite. The randomness of this repertoire is independent of its size. However, the selection pressure that makes a random repertoire advantageous also selects for a large and rapidly diversifying array of epitopes on parasites capable of killing the host. There is a trade-off between the degree of
Antibody that possesses two identical paratopes (bivalent) is aggregated by antigen to trigger effector function. Antibody that possesses two different paratopes behaves as functionally monovalent. If these two antibodies interact with a given epitope, the monovalent antibody will block the aggregation of the bivalent antibody thereby inhibiting effector activation. We advance the hypothesis that haplotype exclusion is driven by the necessity to reduce the level of monovalent antibody. This assumption is compared to previous suggestions and quantitated. Further, several mechanisms of haplotype exclusion used by various species are analyzed in the light of this hypothesis. Key words: selection for haplotype exclusion / mechanism of haplotype exclusion / Ig-receptor signaling / Ig-mediated effector function / gene rearrangement / role of D-gene segment © 2002 Elsevier Science Ltd. All rights reserved.
Orientation Molecular immunology makes sense only when viewed in the light of natural selection. Living units produce copies, some of which vary in a way that makes them more or less likely to survive in successive generations. As selective pressures change over time they force the germline evolution of populations, which become lineages that compete with each other for resources. The competition created by parasitism of one lineage by another has resulted in organisms from bacteria to man having mechanisms that protect against parFrom the Conceptual Immunology Group, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. * Corresponding author. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1044–5323 / 02 / $– see front matter
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specificity and the size of the repertoire. On the one hand an increase in specificity requires a larger repertoire, while on the other hand the number of copies of each specificity per unit protected decreases as the size of the repertoire increases. Consequently, rapidly dividing parasites can out-grow an immune response that starts with too few copies of a required specificity because it takes too long to reach an effective ridding concentration. The host–parasite relationship is one in which either the host or the parasite is eliminated. Thus, natural selection requires that we consider selection on the immune system to be at the level of ridding parasites that would otherwise rid the host. Any interaction between the host and parasite that does not affect the ridding reaction cannot be considered as a selective pressure on the adaptive immune system. In this essay we are concerned with the recognitive structures that function in solution as protective antibodies and mediate the ridding of pathogens as well as their harmful components.
nase, etc.). The paratope itself is constructed as a heterodimer (LH). In order to participate in an aggregation reaction with monomers, the antibody must be a homodimer of the form (LH)2 and each paratope has to be identical (i.e. it is a homo-specific homo-dimer). The term bivalency is often used to describe the homodimer but this term fails to emphasize the fact that both binding sites are the same. An antibody that is a homo-specific homodimer can not be polymerized or aggregated by a monomeric antigen. A mixture of two different antibodies, specific for two different epitopes on the monomer are required to act in concert when they form linear chains of antibody and antigen. Only when three different antibodies that react with three different epitopes on the monomer can there be a three dimensional polymer that contains a high local concentration of antibody. Given that most pathogens are in fact polymeric viruses or bacteria, a monoclonal homo-specific antibody alone would be sufficient to cause the cross-linking of viral or bacterial particles. However, it is not the aggregate of antigen that the ridding mechanism is concerned with but the aggregate of antibody Fc constant regions. In order to obtain a high local density of antibody on the surface of a polymeric virus or bacterium the concentration of antibody would need to be quite high in order ensure that the scattered binding of antibody is, by chance, in sufficiently close proximity to trigger the ridding mechanisms. Thus, haplotype exclusion is unlikely to have been under significant selection via IgG-polymeric pathogen interactions. Although we would probably never have predicted it on a priori grounds, the IgM and IgA antibody classes are pre-polymerized and as such they introduces a new insight into antibody effector function. Although pentameric IgM is in principle an aggregate, even when not bound to antigen, as best as we can tell the Fc’s in an IgM pentamer all point towards the center of a disc-like arrangement of (LH)2 molecules that form the five spokes of a wheel. When IgM binds to two or more epitopes on the surface of a bacterium or virus we presume that the disk is disrupted so that the spokes of (LH)2 now are held together by the J chain and are oriented vertical to the antigenic surface where they present a high local concentration of Fc detectable by the appropriate effector ridding mechanism. In the case of already aggregated IgM there is some advantage to having all of the paratopic sites with the same specificity in order to ensure the maximum likelihood of binding the repetitive epitope. However, it is difficult to quantitate the effects of haplotype
Background The coupling of antibody binding events to those triggering ridding effector functions For antibody to perform a ridding function, there has to be a way for the effector mechanism to detect the paratope–epitope binding event. There are two possibilities—either it detects a change in conformation, or in concentration (e.g. aggregates of antibodies versus single antibody molecules). The interaction between a given antibody and a pathogen takes place in the presence of a vast excess of unbound antibody in serum. We know today that the triggering of effector function by antibody requires that it be aggregated by antigen. This is understandable on a priori grounds because the detection of a conformational change in single antibody molecules that are bound to antigen would require identifying single molecules present in a 107 -fold excess of unbound antibody; and, in the face of thermodynamic equilibria that allow the bound conformation to occur in the absence of antigen, this level of discrimination is beyond the capability of normal biological systems. The limiting selection pressure both on the size of the paratopic repertoire and on its coupling to an effector function is exerted by monomers (e.g. diphtheria or tetanus toxin, Streptolysins, α-hemolysins, β-hemolysins, lytic enzymes like the C. welchi lecithi154
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to cause receptor aggregation, and this would mean that receptors with two different paratopic sites (i.e. effectively monovalent) would actively interfere with signaling.
inclusion that would result in relatively small amounts of non-functional IgM. Only if both paratopes of the (LH)2 spokes have to be bound before undergoing the conformational change would haplotype inclusion be deleterious. Taken together the interactions between antibodies and whole pathogens do not seem to be a source of selection that would make haplotype exclusion highly advantageous. Thus, the role of monomeric antigens in the attack of pathogens on vertebrate hosts was probably the major selection pressure for haplotype exclusion, and given the obvious extreme measures taken by the immune system to achieve a high degree of haplotype exclusion, the immune system cannot, as some have argued, remain largely disinterested in responding to monomeric antigens.
(IV) Triggering of effector function requires that secreted antibody form aggregates (three or more Ig per aggregate) which, in the case of monomeric antigens, requires that all the paratopes per Ig to be identical so that any Ig with different paratopes would actively inhibit aggregation. If mechanism IV applied, then the other possible selection pressures would not be operative. For example, if the output after induction were ineffective in ridding nonself due to mechanism IV, then this would also be true for self, and mechanism II could not be a selection pressure. Mechanism I is quite unlikely to have been much of a selection pressure anytime during evolution. Mechanism II was considered in detail as part of Protecton Theory.1, 2 While the selection pressure due to production of unselected anti-self might have acted to drive haplotype exclusion to the 95% level, we were not convinced that it could account for haplotype exclusion to the >99% level, eventually leading us to consider mechanism IV. Mechanism III acting alone at the level of receptor signaling is considered unlikely on the grounds that evolution could not have decided a priori to rule out single binding per Ig receptor as an ineffective signal. It is, however, conceivable that selection against a single binding Ig under mechanism IV includes having a mechanism of B cell signaling that makes single-binding Ig receptors inefficient and/or deleterious. Consider the response of the B cell interaction with a monomer. In order for monomers to signal, the receptor must undergo a conformational change that results in some degree of polymerization in order to deliver signal [1]. If each (LH) subunit in the homodimer undergoes a conformational transition independently on binding ligand then at low occupancy levels, signaling dimers will dominate. If low occupancy level is the important physiological situation, then signaling dimers should function. If higher order polymers are required for signaling, then both LH subunits would have to be bound, and any hetero-specific receptors would actively block signaling. As an aside, we note that only linear polymers can be formed by this type of reaction. Thus, the notion that ‘normal’ antigen-driven receptor cross-linking can be mimicked by anti-Ig reagents, most of which have many different specificities, is not well founded.
What is nature and magnitude of the problem that haplotype exclusion attempts to solve? The first question in looking at this problem is to consider the relationships between paratopes, antibodies and cells. To begin, let us simplify the problem. As an example, a mouse possesses 2H-loci and 4L-loci (two each, kappa, lambda1, and lambda2). In the absence of any exclusion rules, one cell would express 12 paratopes (2H × 6L). These would be distributed two at a time in antibody to yield 78 antibody molecules—clearly a non-functional situation. If the cell expressed four paratopes, 2H and 2L, then it would express 10 antibodies, four homo-specific and six hetero-specific. For any given specificity, the ratio homo-specific/hetero-specific would be 1/6. A still simpler case would be 1L + 2H or two paratopes, a and b, distributed in three antibodies aa, ab, bb (0.25:0.50:0.25). The ratio homodimer to heterodimer for a given specificity would be 1/2. In the end, the problem simplifies to this latter case. There are four levels at which multiparatopic expression per B cell could be envisioned to be selected against. (I) A cell that expressed too many paratopes would upon induction by an antigen produce an insufficiency of the selected specific antibody and an excess of unselected antibody. (II) Some of the specificities in a multiparatopic cell could be directed at the host so that induction via a second specificity for a pathogen would entrain an anti-self response. (III) Signaling via the receptor that has bound ligand requires both paratopes to be occupied in order 155
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and hence the name D-segment. This interpretation of the role of D can be questioned on the grounds that most of the potential sequence diversity in the D segment is apparently unused. Of the three possible reading frames only one appears in functional H chains, a second reading frame is not used for some reason, and the third reading frame has a STOP codon. Various proposals have been advanced to explain why one DH reading frame is readable, but not present in functional H chains. The only explanation that applies to all species known to date is that this reading frame is in some way nonfunctional, or as we called it, D-disaster. These views have been explained in detail elsewhere1–3 but are in need of updating in the light of our new emphasis on selection at the level of effector function D-dimerization?). When a gene fusion event occurs at any of the linker-mediated joints the fusion process occurs without any regard for the reading frame. Because the joints are all in exons and because there is no way to ensure that the reading frame will be preserved across the junction, there is only a 1/3 chance that a full length protein will be produced. It is not as if evolution was given no choice when making 2/3 of joints un-readable because there are well-documented examples of equivalent fusion events at the level of isotype switching among the different CH gene segments. In isotype switching the objective is to preserve the VDJC sequence functional while merely changing the H chain isotype. To achieve this, the so-called switch regions allow VDJ to connect in cis with any other CH gene segment via a corresponding switch site just 3 of the first CH exon. The point is that the class switching sites are located within introns so that the inevitable randomization of the reading frame at the joint does not end up being expressed in the immunoglobulin product thanks to the accuracy of RNA processing. Thus, it would seem that there is a strong selection for errors that are created during gene fusion events as they make a significant contribution to achieving haplotype exclusion (i.e. by reducing the number of functional loci per cell from 2 to 1). At the H chain locus the number of such joining errors has been maximized by the insertion of the additional D segment which has stop codons in one of the three reading frames (a feature of all immunoglobulin D segments, but it is worth noting that TCRB D segments do not have any stop codons). It would not be possible to incorporate additional segments beyond the three of the H chain locus without introducing an entirely new class of linker sites.
A standard view of gene rearrangements at the Ig loci (i.e. from the human/mouse perspective) The immune system has gone to extraordinary lengths to ensure that the vast majority of B cells express a single functional paratope or LH pair. At the genetic level V and C segments are not only maintained in separate gene clusters on a particular chromosome (the kappa, lambda and heavy chain loci are all on different chromosomes), but, at each locus, the separation of V and C clusters is essentially in the same configuration that places a set of J gene segments between V and C such that V–J joints occur by fusion of the gene segments and the J–C region is an intron. There is a set of orientation-specific linkers (termed the 12/24 or 1-turn/2-turn pairs) that can only be joined in opposites, such as 1-turn → 2-turn → 1-turn. The VH , Vlambda , TCRVB, and TCRVA loci all have 3 2-turn linkers; only Vkappa has a 1-turn linker. In the case of the H locus there are, in addition, D gene segments that are inserted between VH and JH . The D segments have 1-turn linkers on both ends, thereby limiting concatenation of D segments while still allowing VH → DH joints. The JH segments have 2-turn linkers that allow DH → JH but not VH → JH joints. The Jlambda segments have 1-turn linkers whereas the Jkappa segments have 2-turn linkers. An obvious question is why are D and J segments both needed when the C segment could have been constructed to include a single J-like sequence? We have no definitive answers, only more or less popular rationalizations. In the case of multiple J segments, one strong suspicion has been that it is a needle-in-the-haystack problem and one V linker finding one C linker would be just too slow, so a family of linkers in the form of J segments increases the effective concentration of the reacting species and makes the formation of joints more rapid. This guess is plausible. However, recently it has been argued under a phenomenon termed ‘receptor editing’ that multiple Js are required at the kappa locus in mice in order to ‘correct’ unwanted joinings. This will be commented on in greater detail later. The problem of why D and why only in the H locus has been obscured by the context in which the D segment was discovered. A D region was first recognized as a short sequence of diverse amino acids at the VH –JH junction that could not be accounted for by the known VH or JH gene segments. Later, when the D gene segments between VH and JH were discovered, the two observations were linked as D-diversity, 156
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population of H+ were greater than 0.54 as may well be the case (see Reference 5, p. 578).
Analysis A minimum stochastic model for human/mouse
(ii) DN effects (the fit factor): the D-reading frame preference and length matching have been pooled to result in a fit factor that describes the probability that a translated H chain will be functional in Ig.2 While a significant reduction in functional doubles contributing to the ineffective mixed molecules of Ig can be achieved, there are consequences of this assumption that remain to be tested. This argument that the DN region of the BCR (not the TCR) plays its major role in haplotype exclusion as D-disaster, not in the generation of diversity, is being surprisingly ignored by the immunological community. The problem arises because amino acid diversity when translated directly into combining site diversity, is too much of a good thing.1, 2 Rather than argue that point here (see References 2, 6–8 for discussion), let’s consider how D-disaster might function in haplotype exclusion.
As a minimum we consider three steps: First, a branching ratio that describes the order in which the H- and L-loci become targets for gene fusion events. Typical values for mouse are H: kappa = 10, kappa:lambda1 = 100, lambda1:lambda2 = 1. Second, a fusion efficiency ( f ) describes the probability that a given fusion event will result in an arrangement of gene segments that allows in frame translation of an entire H or L chain. Reasonable values for fH = 0.17, fkappa = 0.2, flambda = 0.3 for mouse. Third, a STOP signal that results in the cessation of gene fusion events: there is an agreed upon LH-STOP. The branching ratio, H:kappa = 10, places the burden of reducing the doubles on the H-locus. As an example, given fH = 0.17 then: H +/− = 2(1 − 0.17)(0.17) = 0.2822 H +/+ = (0.17)2 = 0.0289.
and
First, D in the non-preferred frame might block a conformational change rendering the BCR nonsignaling. This implies that a large proportion of B cells would be non-functional although they might still bind antigen. This interpretation is testable by determining the distribution of D reading frames expressed in antigen-unselected populations of B cells. The presence of a high proportion of the non-preferred frame in B cells, and its absence in secreted Ig, would confirm this assumption.2 Second, D in the non-preferred frame might inactivate the H-chain so that it never appears in the BCR. This assumption would be confirmed by the above experiment if the non-preferred frame was simply absent from the normal antigen-unselected B cell population. This latter explanation has been ruled out in the case of the avian immune system (see next). In any case, no competing interpretation of the preferred D reading frame has surfaced. Third, if selection is operating at the level of triggering effector function, then so long as the DN-fit effects do not stop the formation of paratopes, then they would not change the proportion of molecules with mixed specificities and, we predict, would not contribute to the selection on haplotype exclusion. In other words, the DN-fit would apply only if selection operates at the level of B cell induction. As an aside we note that the finding in mice of promoter and an initiation codon 5 to the DH cluster can result in a DJC transcript when the DJ joint is in the
H+/+
The proportion of doubles is 0.0289/0.3111 = 9.3%. If this level is to be reduced, what mechanisms are there and how effective are they? Two have been proposed: (i) H-STOP-H: the haplotype that produces the first H-chain peptide turns off rearrangement at the sister locus. The efficacy of such a mechanism depends on the ratio of the time it takes to rearrange to the time it takes to STOP. If rearrangement is long compared to STOP, then this mechanism would be efficient. However, this seems unlikely because transcription, RNA processing, export from nucleus, translation on ribosomes, complementation with surrogate light chain, insertion in outer membrane and then signaling back to the nucleus to cease H-locus rearrangement can hardly take less time than joining alone. As an aside we should point out that arrests in B cell development caused by the lack of surrogate light chains is not sufficient to conclude the surrogate light chain combined with H chain forms a STOP signal. If the formation of a membrane Ig receptor complex is required in order for B cell differentiation to proceed, then the ‘STOP’ signal is not simply one of stopping further H locus rearrangements, but B cell development as a whole. In any case, H-STOP-H would be disproved if the proportion of H+/− in the 157
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non-preferred frame,9 is as far as we can tell unique to mice. In other words, the explanation in mice for the missing D reading frame is the production of a toxic DJC peptide, but this explanation does not apply to other species. In fact in the rabbit it has been found that the various D segments have a preferred reading frame, but it differs among different D segments. All of the other known examples of D reading frames and their usage points to all D’s in the cluster have the same preferred frame. One of the difficulties in this area is the arbitrary naming of reading frames. We suggested3 some time ago that a logical reading frame determined by the boundary of the conserved nonamer/heptamer region would help simplify the comparison of species.
haplotype exclusion. A strictly random R:S is 2.8:1.The observed R:S for framework regions of NAR is 1.6:1, and this implies that 57% of replacements are lost. Rounding this down to 50% gives us the probability that an otherwise functional gene will be inactivated. We calculated next the effects of various mechanisms that contribute to haplotype exclusion, starting with 1/3 for VJ fusions, assuming no effects due to D, we then added a 50% mutational inactivation factor. This set of assumptions was then compared with adding in the D-disaster effect (i.e. a 2/9 fusion efficiency) and again adding the 1/2 for mutational inactivation. We have assumed that after mutation, the probability of two functional V segments having the same specificity is vanishingly small. To illustrate the calculation we name the two binding sites a (the paratope under selection) and b (any paratope not under selection) and calculate the fraction of gene rearrangements that are a+b+ (i.e. hetero-specific doubles) versus the a+b− (i.e. homo-specific singles). In cells that express two functional V-segments there is a random pairing of the chains so that in an a+b+ cell, half of the paratopes are a, and half are b, and the probability of producing aa is 0.25, ab is 0.5 and bb, 0.25. Thus, the probability of any secreted dimer containing an a binding site is 0.75, but the fraction of a binding sites present in mixed ab molecules is half that (i.e. the probability that an a chain will end up in a mixed (heterodimer, monovalent) molecule is 0.375). Applying these probabilities to the NAR system we obtain the results shown in Table 1. These calculations show that under the best conditions, the combined effects of D-disaster plus mutational inactivation result in about 4% of the total anti-a produced being present as mixed ab hetero-specific heterodimers. The next step is to evaluate the effects of different concentrations of ab in a system of polymerization that requires aa. We calculated the theoretical effects of hetero-specific heterodimers that comprise between 3 and 15% of the total dimers containing an anti-a site. Assuming different lengths of polymerization starting with those that are at least 3 long, then 4, 5, and 10, we calculated the probability of obtaining a chain of the desired length before a chain-terminating hetero-specific ab molecule attaches. The calculation depends on the probability that there will not be a mixed molecule in 3–5, or 10 consecutive binding events in the presence of 3–15% of mixed molecules. The results are summarized in the Table 2.
A new lesson from the shark The simplest informative system we found to analyze was the antibody-like molecule termed NAR that is found in the Nurse shark.10–13 This antibody is a homodimer of two heavy-like chains and this allows us to put aside the effects of any STOP signals. A reasonable assumption is that NAR has two identical binding sites per molecule as would be required for it to drive aggregation reactions with antigen in solution. There are two apparently similar gene clusters that encode separate V, D, J, and C segments. A single V is present at each locus along with three D segments, a single J, and C. The D segments can be joined in all three frames. In cDNA libraries, which represent presumptive functional NAR, only two reading frames appear to be used. The C region is characterized by classical exons, including separate membrane and secretory C-terminal exons. Extensive sequencing of cDNA clones shows that the membrane-bound NAR predominantly uses V in the germline form whereas, the secreted form of NAR is extensively mutated with an average of five replacements per V segment, with an overall replacement to silent ratio of 1.6:1 in the framework regions. From these data some rough calculations have been made using gene fusion efficiencies of 2/3 for D (based on two reading frames) and 1/3 for V–J, making for an overall fusion efficiency of 2/9. Based on the assumption that the two NAR loci do not form mixed molecules, we were able to estimate the frequency of B cells that might produce two different specificities via the expression of two different genes at a given NAR locus. The replacement to silent ratio (R:S) allows us to calculate the contribution of mutational inactivation to 158
Haplotype exclusion: the solution to a problem in natural selection
Table 1. The proportion of a given specificity being present as a hetero-specific dimer 1/3 No D effect
1/6 Add mut (D−)
2/9 Add D effect
2/18 Add mut (D+)
a+b+
(a+ab) 0.75 (ab) 0.375
0.0833 (1/9) 0.0417
0.02083 (1/36) 0.01042
0.03704 (4/81) 0.01852
0.0926 (4/324) 0.00463
a+b−
(a+ab) 1.0 (ab) 0.0
0.2222 (2/9)
0.13889 (5/36)
0.1728 (14/81)
0.9876 (32/324)
Sum
(ab) (a+ab) Percentage of mixed ab
0.01042 0.015972 6.5%
0.01852 0.20984 8.8%
0.00463 0.10802 4.2%
0.0417 0.3055 13.7%
to somatically generate a repertoire.14 Gene conversion as a generator of diversity poses a problem for haplotype exclusion that is distinct from that posed by somatic mutation. Gene conversion can correct an out-of-frame joint restoring it to functional. This means that haplotype exclusion becomes dependent on cells that express the configuration L+/0 H+/0 , one haplotype functional, L+ H+ the other in the germline configuration, L0 H0 . In order to accomplish this, a mechanism to select and sequester the very first rearranging pre-B cells that express membrane bound Ig (mIg) is required. The first B cells to express mIg would be L+/0 H+/0 in which an LH-STOP operates. To optimize the proportion of L+/0 H+/0 that appear initially, simultaneous expression of L and H (the branching ratio must be 1:1) is required. Unlike mouse where the burden of reducing doubles is placed on the H-chain locus by having an order of expression H then L, in chicken the order of expression of the two loci is random. Under conditions where the fusion efficiency at the H locus is 0.22 and at the L-locus 0.33, about 20% of a population rearranged to exhaustion would be H+/0 L+/0 . Of course, the initial mIg+ population after two rearrangements would be all H+/0 L+/0 . Due to our postulated role of the D gene segment, only 1/2 of these are targets for follicular entry.
The results illustrate the sharp drop in the percentage of antigen that is in complexes as the proportion of mixed antibody molecules increases. Even when 3% of the NAR antibody is mixed, only 91% of the antigen (a potential pathogen) is in complexes of three or more antibodies. In order to have >99% of antigen in complexes of three or more the mixed molecules have to be less than 0.3% of the total (i.e. 0.9973 = 0.991). The shark NAR which produces under our calculations around 4% of doubles under the best conditions would be sufficient to have 90% of the antigen in polymers of three or more. These calculations are only illustrative because we simply have no way to determine from the available data how many antibody molecules need to be in a minimum-sized aggregate in order trigger ridding. An aggregate of three is a theoretical lower limit if three different antibodies have to bind, but this seems intuitively too small, and as the calculations in Table 2 show, as the minimum size of the polymer increases, the mixed molecules have to be reduced to very low levels. An old lesson from the chicken (gene conversion) The chicken (as well as several other species) use gene conversion from a donor pool of V gene segments
Table 2. The effect of chain-terminating hetero-specific heterodimers on polymerization by homo-specific homodimers Percent mixed
Percentage in 3 or more
Percentage in 4 or more
Percentage in 5 or more
Percentage in 10 or more
15 10 8 6 4 3
61 73 79 83 89 91
52 66 72 78 85 89
44 59 66 73 81 86
20 35 43 54 67 74
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Next a mechanism to capture the very first cells expressing membrane bound Ig (mIg) is required. This is accomplished by encapsulating a minimum number of these mIg+ cells into bursal follicles where they divide and undergo conversion. How might mIg+ be detected? It can be assumed that the germline BCR expressed by these cells recognizes a follicular component the interaction with which triggers encapsulation. Or, it can be assumed that the follicle recognizes mIg or something associated with its expression and this interaction triggers encapsulation of the initial L+/0 H+/0 cells. The two suggestions are not equally explicative because the first one explains what maintains the germline recipient genes functional, whereas the second explanation leaves this question open. In either case, haplotype exclusion in a system that gene converts requires a selectively sequestering mechanism for L+/0 H+/0 cells and this is the postulated role of the follicular structure of the bursa. The chicken immune system has highlighted the role of the D gene segment.15 In the absence of a bursa, the germline VL and VH genes rearrange to produce a population of B-cells that can be induced to secrete IgM, IgG and IgA. This serum Ig is essentially monoclonal by physical criteria presumably encoded by the germline recipient VL and VH genes. It does not react with a large variety of randomly chosen antigens, its ligand being unknown. The key here is that, in the absence of a bursa, a proportion of the mIg+ B cells resulting solely from gene rearrangement are functional antigen-responsive iB cells, even if their ligand is unknown. The cells are born without N-additions and expressing two of three D reading frames, the third being chain terminating. If their ligand is a follicular component, those iB cells with D in the preferred frame would be deleted as anti-self; only those in the non-preferred frame could initiate follicular formation. This non-preferred frame must then be corrected to the preferred frame by gene conversion from a set of donor VH DH ‘JH ’ gene segments with D in the preferred frame, thus making the converted B-cells functionally responsive to antigen. If it is argued that no signal via the mIg is required to seed follicles then it is an open question as to why the donor gene segments are not just VH rather than VH DH ‘JH ’. Further, if the recipient germline VL VH has not been selected by ligand for function as a paratope, what is the source of the serum Ig in the embryonically bursectomized animals? Lastly, the D-reading frame expressed in the secreted Ig would
predictably be in the preferred frame, a formal test of D-disaster. To this might be added that some B cells enter the follicle with two to three concatenated D segments, presumably a mixture of reading frames, but they leave with the concatenation of Ds in the preferred frame corrected by conversion. This, too, implies a role in BCR function for D (D-disaster), not one of D-diversity. Receptor editing or run-on kappa rearrangements In a murine B-cell expressing an H-chain, the expression of a kappa-chain results in an LH-STOP to further rearrangement. It has been argued that if such a cell expresses an anti-self specificity, rather than waste the cell by deletion, it overwrites its expressed kappa-chain to result in a new LH-pair that is now anti-nonself. For such a cell saving device to be selectable, it must be a high frequency event and this translates into the assumption that a large part (surmised to be 63%) of the somatically generated random repertoire must be anti-self. This assumption alone is not sufficient to justify this interpretation of receptor editing. The probability that the overwriting by one rearrangement will successfully replace the anti-self specificity, is only 0.037. The probability of rearranging the kappa+ haplotype is 0.5, the probability that the rearrangement will be in-frame is 0.2 and the surmised probability that it will be anti-nonself is 0.37 (0.5 × 0.2 × 0.37 = 0.037). Correcting only 4% of anti-self is hardly a waste-saving device when a small increase in total B cell output would have much the same effect. Further, the postulate that 63% of the unselected repertoire is anti-self is open to question12 not to mention whether or not an anti-self signal1 normally overrides the LH-STOP to any significant extent. If it did, in an animal expressing an anti-self transgenic BCR, the endogenous Ig-loci should rearrange to exhaustion, clearly contrary to fact. The cells are deleted.16, 17 Finally, if our estimate is correct that the probability of being anti-self is actually ∼0.01, receptor editing to correct anti-self-ness becomes unselectably rare. Repeated rearrangement of the kappa-locus that is sequential from J1 to J4 and the eventual exhaustion of Jkappa , has been proposed18 to explain findings of kappa:lambda ratios >10 in the apparent absence of antigenic selection (see below). The fundamental difference between ‘receptor editing’ and this ‘iterative rearrangement’ is that the former, initiated by signal,1 overrides an LH-STOP whereas the latter continues to rearrange over an out-of-frame joint in a kappa−/− 160
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Table 3. The virgin kappa:lambda ratios as a function of the apparent fusion efficiency at the kappa locus fkappa
kappa:lambda1
kappa:lambda2
kappa:lambda(1+2)
0.6 0.5 0.4 0.3 0.2
10:1 6:1 3:1 2:1 1:1
21:1 12:1 7:1 4:1 2:1
7:1 4:1 2:1 1:1 0.7:1
sion efficiency is by sequential repeat joining of Vkappa from Jkappa1 → Jkappa4 . Random repeat joining does not change the apparent fusion efficiency. The reason to introduce the unlikely assumption of sequential rearrangement is the dispute at the experimental level as to what is the virgin kappa:lambda ratio (reviewed in Reference 19). If the value is less than 2–3 as the Minimum Stochastic Model predicts, then sequential rearrangement to exhaustion at the kappa locus is ruled out (albeit independently testable). If the virgin kappa:lambda ratio is >10, then the apparent fusion efficiency, fkappa , must be at least 0.6 and this requires sequential rearrangement to exhaustion of the Jkappa -gene segments, the prediction being that Jkappa1 will be used at higher frequency than Jkappa4 in functional kappa chains. By comparison random repeat rearrangements results in Jkappa1 being present less frequently than Jkappa4 in the same population of functional kappa chains. Even were we to entertain the fustian of sequential rearrangement to exhaustion uniquely at the kappa locus in mice, we would be left with the question, ‘what would be the selection pressure to go to such lengths just to achieve an antigen-unselected kappa:lambda expression ratio >10 in the first place?’ Our argument has been that kappa:lambda ratios >10 are due to antigen-selection.20 The virgin ratio is <10, closer to 2–3, and with good reason! It allows evolution to encode in the minor locus, lambda in this case, a critical specificity that would be expressed in very high copy number, thus allowing an exceedingly rapid response to the target pathogen that appears soon after birth. As an illustration, a kappa:lambda of 1:1 implies the 1 Vlambda -gene segment is expressed antigen-independently in mice at a 50-fold higher frequency than any Vkappa -gene segment. Nothing seems to have changed since 1992 when we wrote,19 ‘there is an irreconcilable inconsistency in the data that must be faced. Either the kappa:lambda ratios in antigen-unselected B cells are <10:1 (more correctly ∼1:1), the proportions of doubly and singly rearranged kappa and lambda haplotypes are correct as measured, and kappa:lambda ratios of >10 are the result of antigenic selection, or the kappa:lambda ratio in antigen-unselected B cells is >10 and the kappa- and lambda-gene rearrangement data are faulty’.
cell until a STOP is produced or there are no more V’s or J’s left to rearrange. Any role for receptor editing has to be restricted to those loci which lack a D segment and have clusters of multiple V and J segments, thus ruling out editing at the H chain locus and the lambda loci. If multiple rearrangements occur at kappa because there is no way to stop them in the absence of an LH-STOP signal, then any examples of productive joints at kappa being lost in a second rearrangement must be the result of a leakiness in the LH-STOP signal. The kappa:lambda ratio To begin with, there are two or more light chain loci in most species. The oft advanced argument is weak that in the mouse for example the one or two functional Vlambda gene segments encode unique antibodies that could not have been encoded in kappa V segments. Making matters worse is the observation that in many species, one of the two loci carries the vast majority of V segments; why not, for example, spread them evenly as in the exceptional case found in humans? These and other considerations led us to analyze the consequences of isotype exclusion, that is embodied in the kappa:lambda ratio. The antigen-unselected or virgin kappa:lambda ratio is a fundamental test of the proposed pathways of expression of the light chain Ig loci. In the light of the Minimum Standard Stochastic Model, only the fusion efficiency contributes in a significant way to the virgin kappa:lambda ratio. In Table 3 the kappa:lambda ratios for mice as a function of the fusion efficiency at the kappa locus is calculated ( f lambda is 0.3). Most measurements involve the kappa:lambda1 ratio and, as is obvious, to attain a kappa:lambda1 ratio greater than 10 the apparent fusion efficiency at the kappa locus must be at least 0.6. The fusion efficiency at the kappa locus per joint is 0.2 due to nonfunctional Vkappa gene segments that have STOP codons, yet join. The only way to increase the apparent fu-
Hapex: a computer model We have put on the web http://www.cig.salk.edua program ‘Hapex’ under the link ‘Ig Haplotype Exclusion’ on the main page that allows one to deter161
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mine the output of any set of assumptions concerning the gene rearrangements that result in BCR (Ig) expression in cells.
References 1. Langman RE, Cohn M (1987) The E–T (elephant–tadpole) paradox necessitates the concept of a unit of B-cell function: the Protecton. Mol Immunol 24:675–697 2. Cohn M, Langman RE (1990) The Protecton: the evolutionarily selected unit of humoral immunity. Immunol Rev 115:1–131 3. Langman RE, Cohn M (1993) A theory of the ontogeny of the chicken humoral immune system: the consequences of diversification by gene hyperconversion and its extension to rabbit. Res Immunol 144:421–446 4. Claverie J-M, Langman R (1984) Models for the rearrangements of immunoglobulin genes: a computer view. TIBS 9:293–296 5. Ehlich A, Martia V, Muller W, Rajewsky K (1994) Analysis of the B-cell progenitor compartment at the level of single cells. Curr Biol 4:573–583 6. Langman RE, Cohn M (1992) What is the selective pressure that maintains the gene loci encoding the antigen-receptors of T- and B-cells? Immunol Cell Biol 70:397–404 7. Cohn M (1997) A new concept of immune specificity emerges from a consideration of the self-nonself discrimination. Cell Immunol 181:103–108 8. Langman RE (2001) The specificity of immunological reactions. Mol Immunol 37:555–561 9. Kitamura D, Rajewsky K (1992) Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion. Nature 356:154–156 10. Diaz M, Flajnik MF (1998) Evolution of somatic hypermutation and gene conversion in adaptive immunity. Immunol Rev 162:13–24 11. Diaz M, Greenberg AS, Flajnik MF (1998) Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: possible role in antigen-driven reactions in the absence of germinal centers. Proc Natl Acad Sci USA 95:14343–14348 12. Roux DH, Greenberg AS, Greene L, Strelets L, Avila D, McKinney EC, Flajnik MF (1998) Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins. Proc Natl Acad Sci USA 95:11804–11809 13. Diaz M, Velez J, Singh M, Cerny J, Flajnik MF (1999) Mutational pattern of the nurse shark antigen receptor gene (NAR) is similar to that of mammalian Ig genes and to spontaneous mutations in evolution: the translesion synthesis model of somatic hypermutation. Int Immunol 11:825–833 14. Langman RE, Cohn M, eds (1993) The challenge of chickens and rabbits to immunology, in 52nd Forum in Immunology, vol. 144 pp. 421–520 15. Reynaud CA, Dahan A, Weill JC (1991) The chicken D locus and its contribution to the immunoglobulin heavy chain repertoire. Eur J Immunol 21:2661–2670 16. Goodnow CC, Adelstein S, Basten A (1990) The need for central and peripheral tolerance in the B cell repertoire. Science 248:1373–1379 17. Goodnow CC (1992) Transgenic mice and analysis of B cell tolerance. Annu Rev Immunol 10:489–518 18. Takeda S, Sonoda E, Arakawa H (1996) The k/l ratio of immature B cells. Immunol Today 17:200–201 19. Langman RE, Cohn M (1992) What determines k/l ratio? Res Immunol 143:803–811 20. Langman RE, Cohn M (1995) The proportion of B cell subsets expressing kappa and lambda light chains changes following antigenic selection. Immunol Today 16:141–144
Conclusions While details of mechanism are important, they are easily misinterpreted in the absence of a consistent framework based on selectable function. Our knowledge of gene fusion events, initiation factors, and STOP conditions far outstrips our ability to map them onto their roles in explaining physiological behavior. The goal of this paper was to formulate as consistent a position as possible in a way that highlights the data that do not fit. We have explored the assumption that haplotype exclusion is driven by selection on the requirements for coupling antibody binding to effector function. A Minimum Stochastic Model ties the mechanisms of initiation, fusion, and STOP together in an attempt to rationalize the role of D and of the kappa:lambda ratio. We have not resolved the paradox that, if haplotype exclusion is driven by the need to limit the level of hetero-specific Ig, then D must operate at the level of signaling. Yet we do not have a convincing model as to how D might act. A cell producing two paratopes, one with a functional D and one with a nonfunctional D must be selected against at the level of signaling, and this implies a role for D in the antigen-induced formation of dimers (D-dimerization?) and higher order polymers. While this is not ruled out by the data, it would have to be regarded as a totally surprising conclusion. The striking differences in mechanism used by sharks (inactivation by hypermutation), birds (selective sequestering of the first Ig+ L+/0 H+/0 cells in follicles), and human/mouse (ordered initiation, a low fusion efficiency, and STOP) illustrate the various tacks taken by evolution to achieve the same end of haplotype exclusion. In all cases the role of D remains to be settled. We do not have a consensus view on the most fundamental question, ‘why haplotype exclusion in B cells?’ Further, we have not successfully explained: 1. Why D, why a preferred reading frame in functional Ig, and why only at the H locus 2. Why multiple nearly homologous J’s? 3. Why have two homologous L chain loci with grossly unequal numbers of V gene segments? 162
Haplotype exclusion: the solution to a problem in natural selection Rodney Langman and Melvin Cohn∗
asitism. These mechanisms are conveniently divided into ‘innate’ defense mechanisms and ‘adaptive’ immune systems. As an aside we would like to avoid referring to all protective defense mechanisms as immune systems. The term immune system is best reserved for the adaptive defense mechanism. Innate defense mechanisms (e.g. lysis, phagocytosis, chemokine warfare, etc.) are alone sufficient in invertebrates. Vertebrates require both innate and adaptive defense mechanisms. The term ‘adaptive’ is used to convey the notion of a defense mechanism that allows the host to adapt during its lifetime to an ever changing environment of parasites. Both the innate and adaptive systems require recognitive elements that trigger the innate ridding reactions. Germline selection on the specificity and number of innate recognitive elements is limited if the host lives for a long time relative to its parasites because the parasites are able to evolve vastly more rapidly than the hosts. This selection pressure resulted in a vertebrate mechanism for somatically generating a random repertoire of paratopes that divided the antigenic universe into combinatorially distributed epitopes making it much more difficult for pathogens to escape recognition. This somatic repertoire, which includes the recognition of both host and parasite epitopes, is coupled to the numerous innate effector mechanisms to result in the vertebrate immune system. Other characteristics of the immune system derive from the requirements imposed by the coupling of this random recognitive repertoire to the relevant biodestructive ridding reactions. The somatically generated random repertoire of paratopes defines a corresponding complementary set of epitopes that are randomly distributed on antigens of the host and the parasite. The randomness of this repertoire is independent of its size. However, the selection pressure that makes a random repertoire advantageous also selects for a large and rapidly diversifying array of epitopes on parasites capable of killing the host. There is a trade-off between the degree of
Antibody that possesses two identical paratopes (bivalent) is aggregated by antigen to trigger effector function. Antibody that possesses two different paratopes behaves as functionally monovalent. If these two antibodies interact with a given epitope, the monovalent antibody will block the aggregation of the bivalent antibody thereby inhibiting effector activation. We advance the hypothesis that haplotype exclusion is driven by the necessity to reduce the level of monovalent antibody. This assumption is compared to previous suggestions and quantitated. Further, several mechanisms of haplotype exclusion used by various species are analyzed in the light of this hypothesis. Key words: selection for haplotype exclusion / mechanism of haplotype exclusion / Ig-receptor signaling / Ig-mediated effector function / gene rearrangement / role of D-gene segment © 2002 Elsevier Science Ltd. All rights reserved.
Orientation Molecular immunology makes sense only when viewed in the light of natural selection. Living units produce copies, some of which vary in a way that makes them more or less likely to survive in successive generations. As selective pressures change over time they force the germline evolution of populations, which become lineages that compete with each other for resources. The competition created by parasitism of one lineage by another has resulted in organisms from bacteria to man having mechanisms that protect against parFrom the Conceptual Immunology Group, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. * Corresponding author. E-mail: [email protected] © 2002 Elsevier Science Ltd. All rights reserved. 1044–5323 / 02 / $– see front matter
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specificity and the size of the repertoire. On the one hand an increase in specificity requires a larger repertoire, while on the other hand the number of copies of each specificity per unit protected decreases as the size of the repertoire increases. Consequently, rapidly dividing parasites can out-grow an immune response that starts with too few copies of a required specificity because it takes too long to reach an effective ridding concentration. The host–parasite relationship is one in which either the host or the parasite is eliminated. Thus, natural selection requires that we consider selection on the immune system to be at the level of ridding parasites that would otherwise rid the host. Any interaction between the host and parasite that does not affect the ridding reaction cannot be considered as a selective pressure on the adaptive immune system. In this essay we are concerned with the recognitive structures that function in solution as protective antibodies and mediate the ridding of pathogens as well as their harmful components.
nase, etc.). The paratope itself is constructed as a heterodimer (LH). In order to participate in an aggregation reaction with monomers, the antibody must be a homodimer of the form (LH)2 and each paratope has to be identical (i.e. it is a homo-specific homo-dimer). The term bivalency is often used to describe the homodimer but this term fails to emphasize the fact that both binding sites are the same. An antibody that is a homo-specific homodimer can not be polymerized or aggregated by a monomeric antigen. A mixture of two different antibodies, specific for two different epitopes on the monomer are required to act in concert when they form linear chains of antibody and antigen. Only when three different antibodies that react with three different epitopes on the monomer can there be a three dimensional polymer that contains a high local concentration of antibody. Given that most pathogens are in fact polymeric viruses or bacteria, a monoclonal homo-specific antibody alone would be sufficient to cause the cross-linking of viral or bacterial particles. However, it is not the aggregate of antigen that the ridding mechanism is concerned with but the aggregate of antibody Fc constant regions. In order to obtain a high local density of antibody on the surface of a polymeric virus or bacterium the concentration of antibody would need to be quite high in order ensure that the scattered binding of antibody is, by chance, in sufficiently close proximity to trigger the ridding mechanisms. Thus, haplotype exclusion is unlikely to have been under significant selection via IgG-polymeric pathogen interactions. Although we would probably never have predicted it on a priori grounds, the IgM and IgA antibody classes are pre-polymerized and as such they introduces a new insight into antibody effector function. Although pentameric IgM is in principle an aggregate, even when not bound to antigen, as best as we can tell the Fc’s in an IgM pentamer all point towards the center of a disc-like arrangement of (LH)2 molecules that form the five spokes of a wheel. When IgM binds to two or more epitopes on the surface of a bacterium or virus we presume that the disk is disrupted so that the spokes of (LH)2 now are held together by the J chain and are oriented vertical to the antigenic surface where they present a high local concentration of Fc detectable by the appropriate effector ridding mechanism. In the case of already aggregated IgM there is some advantage to having all of the paratopic sites with the same specificity in order to ensure the maximum likelihood of binding the repetitive epitope. However, it is difficult to quantitate the effects of haplotype
Background The coupling of antibody binding events to those triggering ridding effector functions For antibody to perform a ridding function, there has to be a way for the effector mechanism to detect the paratope–epitope binding event. There are two possibilities—either it detects a change in conformation, or in concentration (e.g. aggregates of antibodies versus single antibody molecules). The interaction between a given antibody and a pathogen takes place in the presence of a vast excess of unbound antibody in serum. We know today that the triggering of effector function by antibody requires that it be aggregated by antigen. This is understandable on a priori grounds because the detection of a conformational change in single antibody molecules that are bound to antigen would require identifying single molecules present in a 107 -fold excess of unbound antibody; and, in the face of thermodynamic equilibria that allow the bound conformation to occur in the absence of antigen, this level of discrimination is beyond the capability of normal biological systems. The limiting selection pressure both on the size of the paratopic repertoire and on its coupling to an effector function is exerted by monomers (e.g. diphtheria or tetanus toxin, Streptolysins, α-hemolysins, β-hemolysins, lytic enzymes like the C. welchi lecithi154
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to cause receptor aggregation, and this would mean that receptors with two different paratopic sites (i.e. effectively monovalent) would actively interfere with signaling.
inclusion that would result in relatively small amounts of non-functional IgM. Only if both paratopes of the (LH)2 spokes have to be bound before undergoing the conformational change would haplotype inclusion be deleterious. Taken together the interactions between antibodies and whole pathogens do not seem to be a source of selection that would make haplotype exclusion highly advantageous. Thus, the role of monomeric antigens in the attack of pathogens on vertebrate hosts was probably the major selection pressure for haplotype exclusion, and given the obvious extreme measures taken by the immune system to achieve a high degree of haplotype exclusion, the immune system cannot, as some have argued, remain largely disinterested in responding to monomeric antigens.
(IV) Triggering of effector function requires that secreted antibody form aggregates (three or more Ig per aggregate) which, in the case of monomeric antigens, requires that all the paratopes per Ig to be identical so that any Ig with different paratopes would actively inhibit aggregation. If mechanism IV applied, then the other possible selection pressures would not be operative. For example, if the output after induction were ineffective in ridding nonself due to mechanism IV, then this would also be true for self, and mechanism II could not be a selection pressure. Mechanism I is quite unlikely to have been much of a selection pressure anytime during evolution. Mechanism II was considered in detail as part of Protecton Theory.1, 2 While the selection pressure due to production of unselected anti-self might have acted to drive haplotype exclusion to the 95% level, we were not convinced that it could account for haplotype exclusion to the >99% level, eventually leading us to consider mechanism IV. Mechanism III acting alone at the level of receptor signaling is considered unlikely on the grounds that evolution could not have decided a priori to rule out single binding per Ig receptor as an ineffective signal. It is, however, conceivable that selection against a single binding Ig under mechanism IV includes having a mechanism of B cell signaling that makes single-binding Ig receptors inefficient and/or deleterious. Consider the response of the B cell interaction with a monomer. In order for monomers to signal, the receptor must undergo a conformational change that results in some degree of polymerization in order to deliver signal [1]. If each (LH) subunit in the homodimer undergoes a conformational transition independently on binding ligand then at low occupancy levels, signaling dimers will dominate. If low occupancy level is the important physiological situation, then signaling dimers should function. If higher order polymers are required for signaling, then both LH subunits would have to be bound, and any hetero-specific receptors would actively block signaling. As an aside, we note that only linear polymers can be formed by this type of reaction. Thus, the notion that ‘normal’ antigen-driven receptor cross-linking can be mimicked by anti-Ig reagents, most of which have many different specificities, is not well founded.
What is nature and magnitude of the problem that haplotype exclusion attempts to solve? The first question in looking at this problem is to consider the relationships between paratopes, antibodies and cells. To begin, let us simplify the problem. As an example, a mouse possesses 2H-loci and 4L-loci (two each, kappa, lambda1, and lambda2). In the absence of any exclusion rules, one cell would express 12 paratopes (2H × 6L). These would be distributed two at a time in antibody to yield 78 antibody molecules—clearly a non-functional situation. If the cell expressed four paratopes, 2H and 2L, then it would express 10 antibodies, four homo-specific and six hetero-specific. For any given specificity, the ratio homo-specific/hetero-specific would be 1/6. A still simpler case would be 1L + 2H or two paratopes, a and b, distributed in three antibodies aa, ab, bb (0.25:0.50:0.25). The ratio homodimer to heterodimer for a given specificity would be 1/2. In the end, the problem simplifies to this latter case. There are four levels at which multiparatopic expression per B cell could be envisioned to be selected against. (I) A cell that expressed too many paratopes would upon induction by an antigen produce an insufficiency of the selected specific antibody and an excess of unselected antibody. (II) Some of the specificities in a multiparatopic cell could be directed at the host so that induction via a second specificity for a pathogen would entrain an anti-self response. (III) Signaling via the receptor that has bound ligand requires both paratopes to be occupied in order 155
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and hence the name D-segment. This interpretation of the role of D can be questioned on the grounds that most of the potential sequence diversity in the D segment is apparently unused. Of the three possible reading frames only one appears in functional H chains, a second reading frame is not used for some reason, and the third reading frame has a STOP codon. Various proposals have been advanced to explain why one DH reading frame is readable, but not present in functional H chains. The only explanation that applies to all species known to date is that this reading frame is in some way nonfunctional, or as we called it, D-disaster. These views have been explained in detail elsewhere1–3 but are in need of updating in the light of our new emphasis on selection at the level of effector function D-dimerization?). When a gene fusion event occurs at any of the linker-mediated joints the fusion process occurs without any regard for the reading frame. Because the joints are all in exons and because there is no way to ensure that the reading frame will be preserved across the junction, there is only a 1/3 chance that a full length protein will be produced. It is not as if evolution was given no choice when making 2/3 of joints un-readable because there are well-documented examples of equivalent fusion events at the level of isotype switching among the different CH gene segments. In isotype switching the objective is to preserve the VDJC sequence functional while merely changing the H chain isotype. To achieve this, the so-called switch regions allow VDJ to connect in cis with any other CH gene segment via a corresponding switch site just 3 of the first CH exon. The point is that the class switching sites are located within introns so that the inevitable randomization of the reading frame at the joint does not end up being expressed in the immunoglobulin product thanks to the accuracy of RNA processing. Thus, it would seem that there is a strong selection for errors that are created during gene fusion events as they make a significant contribution to achieving haplotype exclusion (i.e. by reducing the number of functional loci per cell from 2 to 1). At the H chain locus the number of such joining errors has been maximized by the insertion of the additional D segment which has stop codons in one of the three reading frames (a feature of all immunoglobulin D segments, but it is worth noting that TCRB D segments do not have any stop codons). It would not be possible to incorporate additional segments beyond the three of the H chain locus without introducing an entirely new class of linker sites.
A standard view of gene rearrangements at the Ig loci (i.e. from the human/mouse perspective) The immune system has gone to extraordinary lengths to ensure that the vast majority of B cells express a single functional paratope or LH pair. At the genetic level V and C segments are not only maintained in separate gene clusters on a particular chromosome (the kappa, lambda and heavy chain loci are all on different chromosomes), but, at each locus, the separation of V and C clusters is essentially in the same configuration that places a set of J gene segments between V and C such that V–J joints occur by fusion of the gene segments and the J–C region is an intron. There is a set of orientation-specific linkers (termed the 12/24 or 1-turn/2-turn pairs) that can only be joined in opposites, such as 1-turn → 2-turn → 1-turn. The VH , Vlambda , TCRVB, and TCRVA loci all have 3 2-turn linkers; only Vkappa has a 1-turn linker. In the case of the H locus there are, in addition, D gene segments that are inserted between VH and JH . The D segments have 1-turn linkers on both ends, thereby limiting concatenation of D segments while still allowing VH → DH joints. The JH segments have 2-turn linkers that allow DH → JH but not VH → JH joints. The Jlambda segments have 1-turn linkers whereas the Jkappa segments have 2-turn linkers. An obvious question is why are D and J segments both needed when the C segment could have been constructed to include a single J-like sequence? We have no definitive answers, only more or less popular rationalizations. In the case of multiple J segments, one strong suspicion has been that it is a needle-in-the-haystack problem and one V linker finding one C linker would be just too slow, so a family of linkers in the form of J segments increases the effective concentration of the reacting species and makes the formation of joints more rapid. This guess is plausible. However, recently it has been argued under a phenomenon termed ‘receptor editing’ that multiple Js are required at the kappa locus in mice in order to ‘correct’ unwanted joinings. This will be commented on in greater detail later. The problem of why D and why only in the H locus has been obscured by the context in which the D segment was discovered. A D region was first recognized as a short sequence of diverse amino acids at the VH –JH junction that could not be accounted for by the known VH or JH gene segments. Later, when the D gene segments between VH and JH were discovered, the two observations were linked as D-diversity, 156
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population of H+ were greater than 0.54 as may well be the case (see Reference 5, p. 578).
Analysis A minimum stochastic model for human/mouse
(ii) DN effects (the fit factor): the D-reading frame preference and length matching have been pooled to result in a fit factor that describes the probability that a translated H chain will be functional in Ig.2 While a significant reduction in functional doubles contributing to the ineffective mixed molecules of Ig can be achieved, there are consequences of this assumption that remain to be tested. This argument that the DN region of the BCR (not the TCR) plays its major role in haplotype exclusion as D-disaster, not in the generation of diversity, is being surprisingly ignored by the immunological community. The problem arises because amino acid diversity when translated directly into combining site diversity, is too much of a good thing.1, 2 Rather than argue that point here (see References 2, 6–8 for discussion), let’s consider how D-disaster might function in haplotype exclusion.
As a minimum we consider three steps: First, a branching ratio that describes the order in which the H- and L-loci become targets for gene fusion events. Typical values for mouse are H: kappa = 10, kappa:lambda1 = 100, lambda1:lambda2 = 1. Second, a fusion efficiency ( f ) describes the probability that a given fusion event will result in an arrangement of gene segments that allows in frame translation of an entire H or L chain. Reasonable values for fH = 0.17, fkappa = 0.2, flambda = 0.3 for mouse. Third, a STOP signal that results in the cessation of gene fusion events: there is an agreed upon LH-STOP. The branching ratio, H:kappa = 10, places the burden of reducing the doubles on the H-locus. As an example, given fH = 0.17 then: H +/− = 2(1 − 0.17)(0.17) = 0.2822 H +/+ = (0.17)2 = 0.0289.
and
First, D in the non-preferred frame might block a conformational change rendering the BCR nonsignaling. This implies that a large proportion of B cells would be non-functional although they might still bind antigen. This interpretation is testable by determining the distribution of D reading frames expressed in antigen-unselected populations of B cells. The presence of a high proportion of the non-preferred frame in B cells, and its absence in secreted Ig, would confirm this assumption.2 Second, D in the non-preferred frame might inactivate the H-chain so that it never appears in the BCR. This assumption would be confirmed by the above experiment if the non-preferred frame was simply absent from the normal antigen-unselected B cell population. This latter explanation has been ruled out in the case of the avian immune system (see next). In any case, no competing interpretation of the preferred D reading frame has surfaced. Third, if selection is operating at the level of triggering effector function, then so long as the DN-fit effects do not stop the formation of paratopes, then they would not change the proportion of molecules with mixed specificities and, we predict, would not contribute to the selection on haplotype exclusion. In other words, the DN-fit would apply only if selection operates at the level of B cell induction. As an aside we note that the finding in mice of promoter and an initiation codon 5 to the DH cluster can result in a DJC transcript when the DJ joint is in the
H+/+
The proportion of doubles is 0.0289/0.3111 = 9.3%. If this level is to be reduced, what mechanisms are there and how effective are they? Two have been proposed: (i) H-STOP-H: the haplotype that produces the first H-chain peptide turns off rearrangement at the sister locus. The efficacy of such a mechanism depends on the ratio of the time it takes to rearrange to the time it takes to STOP. If rearrangement is long compared to STOP, then this mechanism would be efficient. However, this seems unlikely because transcription, RNA processing, export from nucleus, translation on ribosomes, complementation with surrogate light chain, insertion in outer membrane and then signaling back to the nucleus to cease H-locus rearrangement can hardly take less time than joining alone. As an aside we should point out that arrests in B cell development caused by the lack of surrogate light chains is not sufficient to conclude the surrogate light chain combined with H chain forms a STOP signal. If the formation of a membrane Ig receptor complex is required in order for B cell differentiation to proceed, then the ‘STOP’ signal is not simply one of stopping further H locus rearrangements, but B cell development as a whole. In any case, H-STOP-H would be disproved if the proportion of H+/− in the 157
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non-preferred frame,9 is as far as we can tell unique to mice. In other words, the explanation in mice for the missing D reading frame is the production of a toxic DJC peptide, but this explanation does not apply to other species. In fact in the rabbit it has been found that the various D segments have a preferred reading frame, but it differs among different D segments. All of the other known examples of D reading frames and their usage points to all D’s in the cluster have the same preferred frame. One of the difficulties in this area is the arbitrary naming of reading frames. We suggested3 some time ago that a logical reading frame determined by the boundary of the conserved nonamer/heptamer region would help simplify the comparison of species.
haplotype exclusion. A strictly random R:S is 2.8:1.The observed R:S for framework regions of NAR is 1.6:1, and this implies that 57% of replacements are lost. Rounding this down to 50% gives us the probability that an otherwise functional gene will be inactivated. We calculated next the effects of various mechanisms that contribute to haplotype exclusion, starting with 1/3 for VJ fusions, assuming no effects due to D, we then added a 50% mutational inactivation factor. This set of assumptions was then compared with adding in the D-disaster effect (i.e. a 2/9 fusion efficiency) and again adding the 1/2 for mutational inactivation. We have assumed that after mutation, the probability of two functional V segments having the same specificity is vanishingly small. To illustrate the calculation we name the two binding sites a (the paratope under selection) and b (any paratope not under selection) and calculate the fraction of gene rearrangements that are a+b+ (i.e. hetero-specific doubles) versus the a+b− (i.e. homo-specific singles). In cells that express two functional V-segments there is a random pairing of the chains so that in an a+b+ cell, half of the paratopes are a, and half are b, and the probability of producing aa is 0.25, ab is 0.5 and bb, 0.25. Thus, the probability of any secreted dimer containing an a binding site is 0.75, but the fraction of a binding sites present in mixed ab molecules is half that (i.e. the probability that an a chain will end up in a mixed (heterodimer, monovalent) molecule is 0.375). Applying these probabilities to the NAR system we obtain the results shown in Table 1. These calculations show that under the best conditions, the combined effects of D-disaster plus mutational inactivation result in about 4% of the total anti-a produced being present as mixed ab hetero-specific heterodimers. The next step is to evaluate the effects of different concentrations of ab in a system of polymerization that requires aa. We calculated the theoretical effects of hetero-specific heterodimers that comprise between 3 and 15% of the total dimers containing an anti-a site. Assuming different lengths of polymerization starting with those that are at least 3 long, then 4, 5, and 10, we calculated the probability of obtaining a chain of the desired length before a chain-terminating hetero-specific ab molecule attaches. The calculation depends on the probability that there will not be a mixed molecule in 3–5, or 10 consecutive binding events in the presence of 3–15% of mixed molecules. The results are summarized in the Table 2.
A new lesson from the shark The simplest informative system we found to analyze was the antibody-like molecule termed NAR that is found in the Nurse shark.10–13 This antibody is a homodimer of two heavy-like chains and this allows us to put aside the effects of any STOP signals. A reasonable assumption is that NAR has two identical binding sites per molecule as would be required for it to drive aggregation reactions with antigen in solution. There are two apparently similar gene clusters that encode separate V, D, J, and C segments. A single V is present at each locus along with three D segments, a single J, and C. The D segments can be joined in all three frames. In cDNA libraries, which represent presumptive functional NAR, only two reading frames appear to be used. The C region is characterized by classical exons, including separate membrane and secretory C-terminal exons. Extensive sequencing of cDNA clones shows that the membrane-bound NAR predominantly uses V in the germline form whereas, the secreted form of NAR is extensively mutated with an average of five replacements per V segment, with an overall replacement to silent ratio of 1.6:1 in the framework regions. From these data some rough calculations have been made using gene fusion efficiencies of 2/3 for D (based on two reading frames) and 1/3 for V–J, making for an overall fusion efficiency of 2/9. Based on the assumption that the two NAR loci do not form mixed molecules, we were able to estimate the frequency of B cells that might produce two different specificities via the expression of two different genes at a given NAR locus. The replacement to silent ratio (R:S) allows us to calculate the contribution of mutational inactivation to 158
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Table 1. The proportion of a given specificity being present as a hetero-specific dimer 1/3 No D effect
1/6 Add mut (D−)
2/9 Add D effect
2/18 Add mut (D+)
a+b+
(a+ab) 0.75 (ab) 0.375
0.0833 (1/9) 0.0417
0.02083 (1/36) 0.01042
0.03704 (4/81) 0.01852
0.0926 (4/324) 0.00463
a+b−
(a+ab) 1.0 (ab) 0.0
0.2222 (2/9)
0.13889 (5/36)
0.1728 (14/81)
0.9876 (32/324)
Sum
(ab) (a+ab) Percentage of mixed ab
0.01042 0.015972 6.5%
0.01852 0.20984 8.8%
0.00463 0.10802 4.2%
0.0417 0.3055 13.7%
to somatically generate a repertoire.14 Gene conversion as a generator of diversity poses a problem for haplotype exclusion that is distinct from that posed by somatic mutation. Gene conversion can correct an out-of-frame joint restoring it to functional. This means that haplotype exclusion becomes dependent on cells that express the configuration L+/0 H+/0 , one haplotype functional, L+ H+ the other in the germline configuration, L0 H0 . In order to accomplish this, a mechanism to select and sequester the very first rearranging pre-B cells that express membrane bound Ig (mIg) is required. The first B cells to express mIg would be L+/0 H+/0 in which an LH-STOP operates. To optimize the proportion of L+/0 H+/0 that appear initially, simultaneous expression of L and H (the branching ratio must be 1:1) is required. Unlike mouse where the burden of reducing doubles is placed on the H-chain locus by having an order of expression H then L, in chicken the order of expression of the two loci is random. Under conditions where the fusion efficiency at the H locus is 0.22 and at the L-locus 0.33, about 20% of a population rearranged to exhaustion would be H+/0 L+/0 . Of course, the initial mIg+ population after two rearrangements would be all H+/0 L+/0 . Due to our postulated role of the D gene segment, only 1/2 of these are targets for follicular entry.
The results illustrate the sharp drop in the percentage of antigen that is in complexes as the proportion of mixed antibody molecules increases. Even when 3% of the NAR antibody is mixed, only 91% of the antigen (a potential pathogen) is in complexes of three or more antibodies. In order to have >99% of antigen in complexes of three or more the mixed molecules have to be less than 0.3% of the total (i.e. 0.9973 = 0.991). The shark NAR which produces under our calculations around 4% of doubles under the best conditions would be sufficient to have 90% of the antigen in polymers of three or more. These calculations are only illustrative because we simply have no way to determine from the available data how many antibody molecules need to be in a minimum-sized aggregate in order trigger ridding. An aggregate of three is a theoretical lower limit if three different antibodies have to bind, but this seems intuitively too small, and as the calculations in Table 2 show, as the minimum size of the polymer increases, the mixed molecules have to be reduced to very low levels. An old lesson from the chicken (gene conversion) The chicken (as well as several other species) use gene conversion from a donor pool of V gene segments
Table 2. The effect of chain-terminating hetero-specific heterodimers on polymerization by homo-specific homodimers Percent mixed
Percentage in 3 or more
Percentage in 4 or more
Percentage in 5 or more
Percentage in 10 or more
15 10 8 6 4 3
61 73 79 83 89 91
52 66 72 78 85 89
44 59 66 73 81 86
20 35 43 54 67 74
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Next a mechanism to capture the very first cells expressing membrane bound Ig (mIg) is required. This is accomplished by encapsulating a minimum number of these mIg+ cells into bursal follicles where they divide and undergo conversion. How might mIg+ be detected? It can be assumed that the germline BCR expressed by these cells recognizes a follicular component the interaction with which triggers encapsulation. Or, it can be assumed that the follicle recognizes mIg or something associated with its expression and this interaction triggers encapsulation of the initial L+/0 H+/0 cells. The two suggestions are not equally explicative because the first one explains what maintains the germline recipient genes functional, whereas the second explanation leaves this question open. In either case, haplotype exclusion in a system that gene converts requires a selectively sequestering mechanism for L+/0 H+/0 cells and this is the postulated role of the follicular structure of the bursa. The chicken immune system has highlighted the role of the D gene segment.15 In the absence of a bursa, the germline VL and VH genes rearrange to produce a population of B-cells that can be induced to secrete IgM, IgG and IgA. This serum Ig is essentially monoclonal by physical criteria presumably encoded by the germline recipient VL and VH genes. It does not react with a large variety of randomly chosen antigens, its ligand being unknown. The key here is that, in the absence of a bursa, a proportion of the mIg+ B cells resulting solely from gene rearrangement are functional antigen-responsive iB cells, even if their ligand is unknown. The cells are born without N-additions and expressing two of three D reading frames, the third being chain terminating. If their ligand is a follicular component, those iB cells with D in the preferred frame would be deleted as anti-self; only those in the non-preferred frame could initiate follicular formation. This non-preferred frame must then be corrected to the preferred frame by gene conversion from a set of donor VH DH ‘JH ’ gene segments with D in the preferred frame, thus making the converted B-cells functionally responsive to antigen. If it is argued that no signal via the mIg is required to seed follicles then it is an open question as to why the donor gene segments are not just VH rather than VH DH ‘JH ’. Further, if the recipient germline VL VH has not been selected by ligand for function as a paratope, what is the source of the serum Ig in the embryonically bursectomized animals? Lastly, the D-reading frame expressed in the secreted Ig would
predictably be in the preferred frame, a formal test of D-disaster. To this might be added that some B cells enter the follicle with two to three concatenated D segments, presumably a mixture of reading frames, but they leave with the concatenation of Ds in the preferred frame corrected by conversion. This, too, implies a role in BCR function for D (D-disaster), not one of D-diversity. Receptor editing or run-on kappa rearrangements In a murine B-cell expressing an H-chain, the expression of a kappa-chain results in an LH-STOP to further rearrangement. It has been argued that if such a cell expresses an anti-self specificity, rather than waste the cell by deletion, it overwrites its expressed kappa-chain to result in a new LH-pair that is now anti-nonself. For such a cell saving device to be selectable, it must be a high frequency event and this translates into the assumption that a large part (surmised to be 63%) of the somatically generated random repertoire must be anti-self. This assumption alone is not sufficient to justify this interpretation of receptor editing. The probability that the overwriting by one rearrangement will successfully replace the anti-self specificity, is only 0.037. The probability of rearranging the kappa+ haplotype is 0.5, the probability that the rearrangement will be in-frame is 0.2 and the surmised probability that it will be anti-nonself is 0.37 (0.5 × 0.2 × 0.37 = 0.037). Correcting only 4% of anti-self is hardly a waste-saving device when a small increase in total B cell output would have much the same effect. Further, the postulate that 63% of the unselected repertoire is anti-self is open to question12 not to mention whether or not an anti-self signal1 normally overrides the LH-STOP to any significant extent. If it did, in an animal expressing an anti-self transgenic BCR, the endogenous Ig-loci should rearrange to exhaustion, clearly contrary to fact. The cells are deleted.16, 17 Finally, if our estimate is correct that the probability of being anti-self is actually ∼0.01, receptor editing to correct anti-self-ness becomes unselectably rare. Repeated rearrangement of the kappa-locus that is sequential from J1 to J4 and the eventual exhaustion of Jkappa , has been proposed18 to explain findings of kappa:lambda ratios >10 in the apparent absence of antigenic selection (see below). The fundamental difference between ‘receptor editing’ and this ‘iterative rearrangement’ is that the former, initiated by signal,1 overrides an LH-STOP whereas the latter continues to rearrange over an out-of-frame joint in a kappa−/− 160
Haplotype exclusion: the solution to a problem in natural selection
Table 3. The virgin kappa:lambda ratios as a function of the apparent fusion efficiency at the kappa locus fkappa
kappa:lambda1
kappa:lambda2
kappa:lambda(1+2)
0.6 0.5 0.4 0.3 0.2
10:1 6:1 3:1 2:1 1:1
21:1 12:1 7:1 4:1 2:1
7:1 4:1 2:1 1:1 0.7:1
sion efficiency is by sequential repeat joining of Vkappa from Jkappa1 → Jkappa4 . Random repeat joining does not change the apparent fusion efficiency. The reason to introduce the unlikely assumption of sequential rearrangement is the dispute at the experimental level as to what is the virgin kappa:lambda ratio (reviewed in Reference 19). If the value is less than 2–3 as the Minimum Stochastic Model predicts, then sequential rearrangement to exhaustion at the kappa locus is ruled out (albeit independently testable). If the virgin kappa:lambda ratio is >10, then the apparent fusion efficiency, fkappa , must be at least 0.6 and this requires sequential rearrangement to exhaustion of the Jkappa -gene segments, the prediction being that Jkappa1 will be used at higher frequency than Jkappa4 in functional kappa chains. By comparison random repeat rearrangements results in Jkappa1 being present less frequently than Jkappa4 in the same population of functional kappa chains. Even were we to entertain the fustian of sequential rearrangement to exhaustion uniquely at the kappa locus in mice, we would be left with the question, ‘what would be the selection pressure to go to such lengths just to achieve an antigen-unselected kappa:lambda expression ratio >10 in the first place?’ Our argument has been that kappa:lambda ratios >10 are due to antigen-selection.20 The virgin ratio is <10, closer to 2–3, and with good reason! It allows evolution to encode in the minor locus, lambda in this case, a critical specificity that would be expressed in very high copy number, thus allowing an exceedingly rapid response to the target pathogen that appears soon after birth. As an illustration, a kappa:lambda of 1:1 implies the 1 Vlambda -gene segment is expressed antigen-independently in mice at a 50-fold higher frequency than any Vkappa -gene segment. Nothing seems to have changed since 1992 when we wrote,19 ‘there is an irreconcilable inconsistency in the data that must be faced. Either the kappa:lambda ratios in antigen-unselected B cells are <10:1 (more correctly ∼1:1), the proportions of doubly and singly rearranged kappa and lambda haplotypes are correct as measured, and kappa:lambda ratios of >10 are the result of antigenic selection, or the kappa:lambda ratio in antigen-unselected B cells is >10 and the kappa- and lambda-gene rearrangement data are faulty’.
cell until a STOP is produced or there are no more V’s or J’s left to rearrange. Any role for receptor editing has to be restricted to those loci which lack a D segment and have clusters of multiple V and J segments, thus ruling out editing at the H chain locus and the lambda loci. If multiple rearrangements occur at kappa because there is no way to stop them in the absence of an LH-STOP signal, then any examples of productive joints at kappa being lost in a second rearrangement must be the result of a leakiness in the LH-STOP signal. The kappa:lambda ratio To begin with, there are two or more light chain loci in most species. The oft advanced argument is weak that in the mouse for example the one or two functional Vlambda gene segments encode unique antibodies that could not have been encoded in kappa V segments. Making matters worse is the observation that in many species, one of the two loci carries the vast majority of V segments; why not, for example, spread them evenly as in the exceptional case found in humans? These and other considerations led us to analyze the consequences of isotype exclusion, that is embodied in the kappa:lambda ratio. The antigen-unselected or virgin kappa:lambda ratio is a fundamental test of the proposed pathways of expression of the light chain Ig loci. In the light of the Minimum Standard Stochastic Model, only the fusion efficiency contributes in a significant way to the virgin kappa:lambda ratio. In Table 3 the kappa:lambda ratios for mice as a function of the fusion efficiency at the kappa locus is calculated ( f lambda is 0.3). Most measurements involve the kappa:lambda1 ratio and, as is obvious, to attain a kappa:lambda1 ratio greater than 10 the apparent fusion efficiency at the kappa locus must be at least 0.6. The fusion efficiency at the kappa locus per joint is 0.2 due to nonfunctional Vkappa gene segments that have STOP codons, yet join. The only way to increase the apparent fu-
Hapex: a computer model We have put on the web http://www.cig.salk.edua program ‘Hapex’ under the link ‘Ig Haplotype Exclusion’ on the main page that allows one to deter161
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mine the output of any set of assumptions concerning the gene rearrangements that result in BCR (Ig) expression in cells.
References 1. Langman RE, Cohn M (1987) The E–T (elephant–tadpole) paradox necessitates the concept of a unit of B-cell function: the Protecton. Mol Immunol 24:675–697 2. Cohn M, Langman RE (1990) The Protecton: the evolutionarily selected unit of humoral immunity. Immunol Rev 115:1–131 3. Langman RE, Cohn M (1993) A theory of the ontogeny of the chicken humoral immune system: the consequences of diversification by gene hyperconversion and its extension to rabbit. Res Immunol 144:421–446 4. Claverie J-M, Langman R (1984) Models for the rearrangements of immunoglobulin genes: a computer view. TIBS 9:293–296 5. Ehlich A, Martia V, Muller W, Rajewsky K (1994) Analysis of the B-cell progenitor compartment at the level of single cells. Curr Biol 4:573–583 6. Langman RE, Cohn M (1992) What is the selective pressure that maintains the gene loci encoding the antigen-receptors of T- and B-cells? Immunol Cell Biol 70:397–404 7. Cohn M (1997) A new concept of immune specificity emerges from a consideration of the self-nonself discrimination. Cell Immunol 181:103–108 8. Langman RE (2001) The specificity of immunological reactions. Mol Immunol 37:555–561 9. Kitamura D, Rajewsky K (1992) Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion. Nature 356:154–156 10. Diaz M, Flajnik MF (1998) Evolution of somatic hypermutation and gene conversion in adaptive immunity. Immunol Rev 162:13–24 11. Diaz M, Greenberg AS, Flajnik MF (1998) Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: possible role in antigen-driven reactions in the absence of germinal centers. Proc Natl Acad Sci USA 95:14343–14348 12. Roux DH, Greenberg AS, Greene L, Strelets L, Avila D, McKinney EC, Flajnik MF (1998) Structural analysis of the nurse shark (new) antigen receptor (NAR): molecular convergence of NAR and unusual mammalian immunoglobulins. Proc Natl Acad Sci USA 95:11804–11809 13. Diaz M, Velez J, Singh M, Cerny J, Flajnik MF (1999) Mutational pattern of the nurse shark antigen receptor gene (NAR) is similar to that of mammalian Ig genes and to spontaneous mutations in evolution: the translesion synthesis model of somatic hypermutation. Int Immunol 11:825–833 14. Langman RE, Cohn M, eds (1993) The challenge of chickens and rabbits to immunology, in 52nd Forum in Immunology, vol. 144 pp. 421–520 15. Reynaud CA, Dahan A, Weill JC (1991) The chicken D locus and its contribution to the immunoglobulin heavy chain repertoire. Eur J Immunol 21:2661–2670 16. Goodnow CC, Adelstein S, Basten A (1990) The need for central and peripheral tolerance in the B cell repertoire. Science 248:1373–1379 17. Goodnow CC (1992) Transgenic mice and analysis of B cell tolerance. Annu Rev Immunol 10:489–518 18. Takeda S, Sonoda E, Arakawa H (1996) The k/l ratio of immature B cells. Immunol Today 17:200–201 19. Langman RE, Cohn M (1992) What determines k/l ratio? Res Immunol 143:803–811 20. Langman RE, Cohn M (1995) The proportion of B cell subsets expressing kappa and lambda light chains changes following antigenic selection. Immunol Today 16:141–144
Conclusions While details of mechanism are important, they are easily misinterpreted in the absence of a consistent framework based on selectable function. Our knowledge of gene fusion events, initiation factors, and STOP conditions far outstrips our ability to map them onto their roles in explaining physiological behavior. The goal of this paper was to formulate as consistent a position as possible in a way that highlights the data that do not fit. We have explored the assumption that haplotype exclusion is driven by selection on the requirements for coupling antibody binding to effector function. A Minimum Stochastic Model ties the mechanisms of initiation, fusion, and STOP together in an attempt to rationalize the role of D and of the kappa:lambda ratio. We have not resolved the paradox that, if haplotype exclusion is driven by the need to limit the level of hetero-specific Ig, then D must operate at the level of signaling. Yet we do not have a convincing model as to how D might act. A cell producing two paratopes, one with a functional D and one with a nonfunctional D must be selected against at the level of signaling, and this implies a role for D in the antigen-induced formation of dimers (D-dimerization?) and higher order polymers. While this is not ruled out by the data, it would have to be regarded as a totally surprising conclusion. The striking differences in mechanism used by sharks (inactivation by hypermutation), birds (selective sequestering of the first Ig+ L+/0 H+/0 cells in follicles), and human/mouse (ordered initiation, a low fusion efficiency, and STOP) illustrate the various tacks taken by evolution to achieve the same end of haplotype exclusion. In all cases the role of D remains to be settled. We do not have a consensus view on the most fundamental question, ‘why haplotype exclusion in B cells?’ Further, we have not successfully explained: 1. Why D, why a preferred reading frame in functional Ig, and why only at the H locus 2. Why multiple nearly homologous J’s? 3. Why have two homologous L chain loci with grossly unequal numbers of V gene segments? 162