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ty in anti-hapten antibodies of a phylogenetically primitive shark. Nature (Land.), 287, 639-340.

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Reyniud, C.-A., Mackay, CR, Miller, R.G. & Weill, J.-C. (I 99 1). Somatic generation of diversity in a mam- malian primary lymphoid organ: the sheep illeal Peyers patches. Cell, 64, 995.

Schlomchik, M., Mascelli, M., Shan, H., Radik, M., Pisetsky, D., Marshak-Rothstein, A. & Weigert, M. (I 9901, Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic muta- tion. J. Exp. Med., 171, 265-292.

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Reply to Kaufman and Salomonsen (K&S) by L&C :

At the outset we need to point out that K&S will have the last word in this exchange.

We were pleased to discover that despite a rocky beginning with the overuse of humour to make their points, K&S have now focused direct- ly on four issues of theory. While we would have preferred that they remain close to the chicken and rabbit data, K&S hammer away at already de- veloped issues that are still dear to our hearts. They insist that we play by their rules “That means clearing their [our] minds and using the in- formation that we [K&S] give them [us] . . . an exercise to help us [K&S1 distinguish between as- sumption and conclusions [i.e., ours].”

We tackle their four questions sticking reluc- tantly to their ground rules; in any case, our al- ready published fuller explanations proved too tiresome for us to restate here.

Question 1 : Why is antibody redundancy so bad ?

The K&S beast weighs 10’9, has 10’ total B cells, and is assumed to have IO’ “different B-cell clones each expressing a different anti- body”. This, we assume, means that there are 10’ B cells per gram, and this is the same as B cells per ml (within a factor of 2 or so). Thus, when K&S ask us to accept that “in each 1 ml of the animal there are a random assortment of lo5 cells”, and - IO5 different antibodies, we

have a problem in basic numerology. Our best guess at this point is that 10’ sequence-different B-cell clones represent only lo5 specificity- different antibodies. However, K&S continue fur- ther with the proposition that “one out of a thou- sand of these B cells [the IO5 in 1 ml1 produces an antibody that reacts with a given antigen”. In this case it seems as if there are only lo3 specificity-different antibodies; indeed, K&S con- clude that their ml of beast has “IO2 B cells per ml that produces specific antibody after immuni- zation”. But, from here we cannot seem to recon- struct the original assumption of IO’ different B-cell clones and 10’ total B cells. Then, when K&S tell us that another randomly chosen beast, is identical in all respects except that it has 10’ different (nonoverlapping) clones, we can only think that there are > > > IO” sequence differ- ent clones (where x =population size x IO’) and any sample of 10’ will contain lo5 specificity-different clones. If this beast of K&S is “diversity and redundancy together”, we can find no rational basis for a discussion, or, even the existence of such an animal.

A repertoire of IO5 functionally different specificities per ml that is present in every milliliter in a functionally equivalent though not identical form is, as we calculated, sufficient for protec- tion. That each milliliter of an elephant is NOT identical we agree; we also must agree that each milliliter of the elephant is equivalent in protection. A repertoire of IO’ (their guess, if this is what “different clones” means, or is it IO3 based on protection per ml ?), not 1 O5 (our calculation), is a confusion that needs straightening out. The lo2 B cells per mlger antigen they have (i.e., 1 in IO3 among 10 total B cells) applies to the BEGINNING, not “after” immunization, and is more than adequate (if we assume they agree with our estimates of antibody molecules secret- ed per second in plasma cells). We calculated that a copy number of IO2 was needed for the smallish germline repertoire of the mouse, and a copy number of 10’ applies to the mutants derived from this germline repertoire. In the chick- en, the germline repertoire is tiny (10’) and both the high and the low copy number repertoires must be extracted from the pseudogenes by gene conversion. In the rabbit the germline repertoire is very small (1 02) and while the V, contribution to the high copy number repertoire must be ex- tracted by gene conversion, the V, contribution is present as IO2 germline V segments. Paren- thetically, the notion of spreading the degenera- cy around to include CDRI and CDR2 might be more democratic, but not obviously necessary in any framework.

It was not a casual assumption that led us to

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propose 100 rig/ml as a typical threshold concen- tration for the elimination of common bacterial an- tigens. This number and the minimum threshold of 10 rig/ml were carefully extracted from the ex- perimental literature. However, if K&S can find better data leading to a quite different conclusion, we would be delighted to analyse it further. In any case, our Protecton calculations showed that these numbers fit well with the other available parameters to produce an internally consistent picture.

To try and settle the meaning of specificity without having to refer to a particular antigen, we reiterate that S antigens are defined with preci- sion and experience, whereas F antigens are only properly described as non-S antigens. Thus, we made the probability of an amino acid replacement changing any anti-F to any anti-S equal to a cons- tant K, and this, we argued was an objective es- timate of antibody resolving power (specificity) without having to resort to any particular refer- ence to an S or F antigen. Moreover, the S anti- gens of one individual are the F antigens of another, making the distinction between S and F truly circumstantial, and certainly not chemical. The ability of an average antibody molecule to dis- tinguish any pair of selected epitopes is of gener- al interest, but mostly when, how (or why) the two particular epitopes were selected. The abili- ty of a given antibody to distinguish members of a panel of known epitopes tells us very little about the functional specificity (resolving power) of the population of antibodies that are the target of evolutionary selection; these antibodies must track random mutations in viruses and bacteria. The concept of extra diversity is perhaps interest- ing to K&S, but again evolution selects for a suffi- cient degree of diversity and cannot select for more that this. Thus, if there were extra diversity as an unselected bonus, any loss of this would re- main evolutionarily invisible until the extra diver- sity shrunk to zero such that any further loss of a specificity would be lethal. A mechanism of diversification can be selected, but this must have a substrate to act on; hence our being driven to a two-stage diversification, one selected for a set of particular specificities (a small repertoire) and the other selected for a breadth of specificities (a largish repertoire).

The use of all lg loci in polyploid frogs, or even diploid mice, is expected because, as we have shown, the V regions on one haplotype cannot be very different from one individual to the next, otherwise the F, would have roughly four times the specificities of a homozygote, and in any case there is no evolutionary selection pressure that could maintain different V genes in every individu- al (Cohn and Langman, 1990, pp. 48-49).

2. What’s wrong with the D region?

Their sequence of “Consider.. . Assume.. . Pre- tend... ” is a rather complicated inquiry about the probability (frequency is the wrong concept here) that an amino acid substitution will change speci- ficity at one of three locations in the lg molecule. The CDR (complementarity determining region) is, as we took pains to explain, defined by function, not geography (Langman and Cohn, 1991). However, we will consider for the sake of their argument that there are three hypervariable regions called “CDRl “, “CDR2”, and “CDR3”. K&S want us to predict if an amino acid substitu- tion is more or less likely to change specificity at each CDR. The frequency with which an amino acid replacement is made has no bearing on the probability that the replacement will change speci- ficity. K&S state that the average frequency (prob- ability we assume) should be the same for each CDR although there might be some increase in fre- quency (probability) if one the of CDR’s is “more important”.

The oddities that can be envisaged by choos- ing particular antigens and experimentally ob- served events do not contribute significantly to the general case. A good index of this phenome- non under consideration by K&S is the silent (S) to replacement (R) ratio of amino acid inter- changes resulting from nucleotide interchanges. In framework the ration is > 10, in neutral regions it is - 1 (2/3) and in the hypervariable regions is closer to 0.1 ; in D we do not have sufficient data, but a good estimate of the S/R ratio would put the role of D unequivocally into framework or CD. Thus, to repeat in plain English - whereas CDR are hypervariable with respect to framework, hypervariability does not require that a region be complementarity-determining.

The possibility of D acting to regulate the ex- pression of V segments that need to associate with C is certainly not necessary in the case of L chains.

Question 3. What important function for the D region ?

The question is first turned into a question of what introns are doing if they are strongly con- served, because, by analogy, K&S argue that D is really just an evolutionary relic like the intron (even that is questionable). Relics, pseudogenes as they might also be called, are under no current selection for function, and therefore, cannot be stopped from mutational drift. So, let us take their D-dumb hypothesis (i.e., D is functionless and un- der no selection) and see how it helps us under-

CHALLENGES OF CHICKENS AND RABBITS TO MM!JNOLOGY 515

stand the next point about D; namely the preferred reading frame problem.

The preferred reading frame is a structural problem of one kind or another according to K&S. Given this, it would not be expected that B cells are “born” with lg molecules expressing D in all three reading frames, while secreted lg has D in one preferred frame. K&S want to have a random reading frame early and preferred reading frame late in ontogeny by analogy with waves of T-cell differentiation. While waves in T-cell ontogeny may not be well understood (i.e., related to func- tion), that does not justify lumping by analogy dis- comforting (or poorly understood) data on the D, LRF into a bag of waves and calling it an evolu- tionary relic. We have never denied the existence of relics and even suggested that D might be a relic in the chicken, but opted for a more intellec- tually challenging possibility that would enhance understanding of function. In any case, it is still not obvious how a once in a lifetime ontogenic process explains a step in B-cell differentiation that occurs continuously in the mouse, even if, in chicken, these two processes overlap during a limited period. Such guesses lack universality.

A preferred D, reading frame is found in lg, but in TAr-j3, Dp functions in all three reading frames. In reality, there are only two reading frames for D, because the third is largely filled with termination codons, which throws out l/3 of the diversity and leads to a lowering of the gene fusion efficiency for a reason that must be ob- scure to K&S. That a “specific” signal be gener- ated via D, is a misreading of our comments which clearly attempt to make the signal permis- sive or non-permissive as distinct from specific and all-or-none.

Question 4: Why allelic exclusion?

No doubt, the two’s, three’s, four’s, five’s... even sixteen’s can emerge as Ig-positive B cells that have no anti-S, and would be just fine as long as we ignore somatic mutation and the mechan- ism of the self-nonself discrimination. We assume that K&S accept somatic mutation and that some mutations will generate anti-S. Unless B cells with mutations to anti-S are distinguished from those with higher affinity for the immunizing antigen, anti-S must be inducible, and hence a problem. In unispecific (haplotype-excluded) B cells, the in- duction of anti-S depends on S-F complexes to drive the “help” via F to the B cell that is anti-S. In multispecific (non-haplotype-excluded “dou- bles”) the mutant anti-S is inducible via the other anti-F expressed in the cell. Which particular as- sumptions do K&S find unacceptable in this

scenario? We would like to see them tackle the assumption that some anti-S is tolerable in serum, but what is the limit threshold, and can this level of anti-S be calculated ? In any case the transgen- its with a complete H chain, L chain, or LH pair, all have B cells expressing the transgene with en- dogenous H and/or L chains, and this illustrates the essential leakiness of haplotype exclusion.

In retrospect

K&S end on a rather unhappy note, claiming all kinds of mistreatment by us. Their initial response was in a quite different style from all others in this Forum, and we tried to rise to the occasion and reply in kind. They were, and still are trying to reduce problems in evolution to non- issues by joking on the square and by claiming that any one proposed solution is trivial given that there are so many, and no way to determine which (if any) is correct. They constantly claim that our arguments are unconvincing, and that we failed in the past to answer the criticisms of others to their satisfaction. However, the existence of alternative interpretations is not a disproof of any one such interpretation. We were hoping for a rea- soned set of disproofs of our position. Instead we have four questions that K&S wanted answered and this we did.

Putting aside the polemics and hurt feelings of K&S, we were pleased to come one step closer to understanding what it would take to have them change their minds - their four questions and, hopefully, our answers. If this is not correct, then they might close by telling us precisely what would it take to have them change their minds?

The chickens come home to roost. Our response to the second rebuttal by Langman and Cohn (by J. Kaufman and J. Salomonsen)

We are grateful to L&C for taking the time to explain their concepts for us using our ground rules. We agree with them that a restatement of their “already published fuller explanations” would have proved too “tiresome”; in any case, we already had gotten lost reading those hundreds of pages. We take their points in turn.

Question 1. Why is antibody redundancy so bad?

We consider this issue very important, because L&C use the idea that there cannot be any “extra diversity” in the antibody repertoire as the basis for postulating an important function for the D region in signaling.

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We are quite sorry that the numerology seemed confusing, especially since we simply meant to duplicate L&C’s “back of the envelope calculations” from the section entitled “A mini- mum number of antigen-specific B cells per ml is required” in “Theory of the ontogeny of the chick- en humoral immune system”. We gave a medi- um sized animal the 1 O7 B-lineage cells per ml (or gram within a factor of two) suggested by L&C. We allowed 10’ B cells per ml to be non- functional to give lo5 functional B cells per ml (L&C say of IO7 per ml, only IO6 are iB, so we took another order of magnitude to be on their safe side, not that we necessarily agree with either number). We assumed one in a thousand of these cells were specific for a certain antigen (a number based on limiting dilution assays) to give IO’ specific antigen reactive functional B cells per ml. L&C are absolutely right that we meant cells before immunization, since their whole argument rests on the number of cells be- fore immunization that will divide to give the necessary number of plasma cells to achieve 10 rig/ml of specific antibody after immunization.

The actual calculations were not in themselves important; all we were trying to do was to create two L&C-compatible animals that had complete- ly different repertoires and yet were completely protected. We think that L&C must also believe this is possible, since they say (as they have said all along) “that each milliliter of an elephant is not identical we agree; we must also agree that each milliliter of the elephant is equivalent in protec- tion”. (Parenthetically, we are not sure that each sample of the antibody repertoire is equivalent - holes in the B-cell repertoire are harder to detect than holes in the T-cell repertoire; but that is for another discussion.)

We think that the crux of our difficulties with the L&C view are illustrated in two sentences. We agree with L&C’s statement that “evolution selects for a sufficient degree of diversity and can- not select for more than that”, but we have repeatedly argued that unless “extra diversity” is harmful in some way, evolution cannot select against it either. Of course, evolution is composed of accident as well as selection; L&C turn to ac- cident by saying that “if there were extra diversi- ty as an unselected bonus, any loss of this would remain evolutionarily invisible until the extra diver- sity shrunk to zero”.

We make two arguments against this superfi- cially reasonable statement. First, they are certain- ly right that the loss of “extra diversity” would be invisible, but they don’t mention that gain of “extra diversity” would also be invisible. There is no reason to assume (as their statement implies) that the process is unidirectional; for instance,

there is abundant evidence for both expansion and contraction in multigene families. Second, some biological features and processes are difficult to eliminate precisely or without wholesale disrup- tion. Antibody loci have complicated features that could be simplified only by very precise changes (e.g., germline joining of V to D and J); antibody gene rearrangement is a complicated process that shares enzymatic machinery with other important processes (e.g., the T-cell receptor rearrange- ment). It is not easy for accident to whittle away at the “extra diversity” until there is none, as im- plied by L&C’s statement.

We mentioned the example of polyploid frogs that use all of their antibody loci, thus having more diversity than the diploid species from which they arose. L&C counter that this is expected since they “have shown the V regions of one haplotype cannot be very different from one individual to the next, otherwise the Fl would have roughly four times the specificities of a homozygote, and in any case there is no evolutionary selection pressure that could maintain different V genes in every in- dividual”. As L&C suggested, we reread the ap- propriate pages of Cohn and Langman’s “The Protecton” (1990) and found that having 2N V,- segments “would leave the individual with... more V,-segments than necessary” as if that were a bad thing. We would argue that it doesn’t matter whether there are more V, segments than the absolute minimum required for survival (evolution doesn’t care about the extra as long as they are not harmful). Certainly, it would be interesting to know what experimental data show that a heter- ozygote has the same number of specificities for a particular epitope as a homozygote. Indeed the experimental evidence for more IgY spectrotypes against DNP in the polyploid frogs compared to the diploid frogs contradicts L&C’s expectation. As for the last phrase, it need not be selection that leads to different sets of V genes in two individu- als - accident would do as well (e.g., random divergence particularly after some time of ge- ographical isolation). Parenthetically, it is possi- ble to design an antibody system in which different individuals have different sets of speci- ficities - this is one possible result of our pro- posed selection of the chicken antibody repertoire on polymorphic B-G molecules (see our first set of comments).

Two other points that we should acknowledge. We did see where L&C stated that 10 ng specific antibody/ml was a minimum from the literature (section II.A.l of Langman and Cohn’s “The elephant-tadpole paradox” (I 98711, but failed to notice where 100 ng specific antibody/ml was a typical experimentally derived number (only that it was a “much more realistic antibody concen-

CHALLENGES OF CHICKENS AND RABBITS TO IMMUNOLOGY 517

tration” in section ll.A.4(3)). We agree that the functional specificity (or “resolving power”) of an antibody population is a difficult concept for us; the K parameter is very interesting if hard to quan- tify and we will think about it more in the future.

Question 2. What‘s wrong with the D region?

As we mentioned in the last set of comments, we are confused as to whether L&C think that CDR3 has no role in antigen binding, has no meaningful functional diversity and is only frame- work, or think that CDR3 has some role in anti- gen binding, has a higher level of structural but a similar level of functional diversity as CDR 1 and CDR2, and is therefore not really framework. For the second time, we asked as clearly as we could whether CDRl, CDR2 or CDR3 were similar ex- cept for all of the (potentially meaningless) “ex- tra structural diversity ” in CDR3. We were less interested in what is true in the animal than in try- ing to understand what L&C think they are telling us.

For some reason, L&C seem unable to answer this question directly in a manner that we under- stand. Whether frequency or probability is the cor- rect term to use was hardly the point (and in fact, we took the word “frequency” directly from their own statements on this issue in “The theory of the ontogeny of the chicken humoral immune sys- tem”.) We are certainly not convinced by L&C’s argument about the estimated silent to replace- ment ratio in the D region; we do not know of a way in which differently sized regions with low levels of identity can be compared to generate meaningful numbers.

As a conclusion, L&C “repeat in plain English - whereas complementaritydetermining regions are hypervariable with respect to framework, hypervariability does not require that a region be complementarity-determining”, but does this mean that they think that CDR3 does not bind an- tigen? Perhaps their sentence “the oddities that can be envisaged by choosing particular antigens and experimentally observed events do not con- tribute significantly to the general case” is meant to dismiss all of the experimental evidence that amino acids in the CDR3 region make contact with antigen. If so, then they have answered us, and we just don’t agree with them. If not, we go back to their statement that “it is not a question of whether amino acid replacements in the D-N-J region can change specificity, but with what fre- quency do they change specificity in a meaning- ful way”. We still do not see why it is a different frequency (or probability if they now think that is more correct) for CDRl, CDR2 or CDR3; there-

fore we do not see why CDR3 is characterized as “framework”.

(Parenthetically, we suggested another alter- native to both the “D-disaster” hypothesis and the “relic of the T-cell receptor” hypothesis - that split genes evolved to regulate the transcrip- tion of tandem V genes in order to avoid cells with multiple expressed antibody molecules. L&C’s response was that D acting to regulate expression of V segments is certainly not necessary in the case of L chains; we of course only mentioned split V genes, so either D or J could do the job. Since we have not dealt with the difference be- tween D and J, let us note here that Louis Du Pasquier suggested an interesting rationale for a second split in V genes (e.g., the emergence of D regions). A locus containing multiple V genes and multiple J regions but no D regions can se- quentially make several rearrangements, whereas the same locus with D regions becomes frozen (except for potential V gene replacements using cryptic recombination signal sequences) once a VDJ rearrangement has occurred.)

Question 3. What important function for the D region ?

The point of this question was to see whether L&C believed that every biological feature must have a current important function, by picking a simple structural feature (an intron) as an exam- ple. To our regret, L&C failed to answer the ques- tion about the intron in a direct way. Unfortunately, they equated an evolutionary rel- ic with a pseudogene, and asserted that a relic is under no current selection for function and thus cannot be stopped from mutational drift. It is clear from the example that an intron has conserved features under constant selection (otherwise a functional protein is unlikely to result) and yet it is a relic of bygone times with no important func- tion for the protein.

To our delight, L&C state that they “have never denied the existence of relics and even sug- gested that D might be a relic in the chicken, but opted for a more intellectually challenging possi- bility that would enhance understanding of func- tion”. Wow. Where did they make this suggestion? We never found it.

Intermission: why is there a preferred reading frame for the D region?

In our first set of comments, we remarked in passing that we thought that the Dreading frame problem was probably a structural problem, for in-

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stance “a bias in the recombinase specificity, a preference for small amino acids in the structure of CDR3 bend, and/or a requirement of the sur- face ‘germline Ig’ to bind to a bursal ligand, at least in the chicken”. We were not pleased by the way in which L&C responded to our presentation of these ideas (although they seemed to under- stand them when other participants made some of same suggestions!), so we expanded on these ideas in our second set of comments.

L&C again fail to respond to the arguments for these different possibilities and we will not bother to repeat them again, except to clarify the one point of the single model that they chose to con- test. We presented the example of T-cell waves emigrating from the thymus during ontogeny as an example of a process with obvious import in one taxum (frogs) and possible persistence as a relic in another taxum (birds). This example was meant to lend credibility to the idea that a change of recombinase specificity during ontogeny could be important in an ancestral species (e.g., an am- phibian with several lifestyles), but be retained in the chicken as a relic. We simply do not under- stand what L&C are trying to imply by their sen- tence “while waves in T-cell ontogeny may not be well understood, that does not justify lumping by analogy discomforting data on the D, LRF into a bag of waves”; perhaps they think that we were not being serious.

(Parenthetically, in all innocence we failed to catch on to the fact that there was an important difference between permissive/non-permissive and specific/all-or-none signaling in the L&C model. We will continue to ponder this, but we still have not been told how this putative signal is supposed to deal with the enormous size varia- tion in the D regions of recombined antibody genes - some rearranged antibodies have no rem- nant of the D region at all !I

Question 4. Why is there allelic exclusion?

We presented a simple-minded view of B-cell ontogeny in order to find out why L&C consider allelic exclusion to be necessary for self-nonself discrimination ; we explicitly left out peripheral tolerance (and mentioned that we would) because it made things one step more complicated. Natur- ally L&C respond with an argument that requires peripheral tolerance in our example : a B cell with only one specificity (that has become anti-self by somatic mutation in the periphery) can not mount an anti-self response because there is no T-cell help for that self antigen, whereas a B cell with multiple specificities (one of which has become anti-self by somatic mutation in the periphery) can

get help by presentation of nonself antigen- derived peptides after uptake of a foreign antigen via anti-nonself antibodies on the cell surface.

In answer to L&C’s question, we do not find these assumptions unacceptable and we think that they are making an entirely reasonable argu- ment. We would make four comments about it. First, L&C point out that antibody gene transgen- ic mice all have B cells expressing the transgene proteins with endogenous antibody chains and that “this illustrates the essential leakiness of haplotype exclusion”. We would take this as evi- dence that allelic exclusion is not essential for prevention of autoimmunity, since such transgen- ic mice are no more autoimmune than normal mice. Second, immune responses to pathogens often occur in tissues along with substantial in- flammation; under such conditions, bystander help may override the need for specific helper T cells leading to activation of anti-self B cells (a condition often invoked for the initiation of au- toimmune diseases) whether each B cell would have one or many different antibody specificities. Third, it seems to be generally accepted that var- ious mechanisms of peripheral B tolerance exist (see the reviews cited in our first set of com- ments), perhaps to deal with exactly the autoim- munity mentioned in the last sentence. If such peripheral B-tolerance mechanisms generally can take care of autoimmunity problems due to somat- ic mutation in B cells with one antibody specifici- ty, then they might work for B cells with many antibody specificities. More knowledge about the detailed mechanisms are needed before we could make that claim securely. Finally, even though L&C failed again to respond to the shark question, we would mention that there is no evidence for T-cell help for shark antibody responses, so one possibility is that the shark might not need allelic exclusion precisely because it doesn’t use T cells to control the B cells.

No one here but us chickens

We read L&C’s initial manuscript “Theory of the ontogeny of the chicken humoral immune sys- tem” with great interest, but long before we reached the parts that dealt with chickens and rabbits, we got stuck with fundamental assump- tions and arguments of the general Protecton theory. So we decided to deal with first things first (except for repertoire selection in the bursa and the origin of the chicken antibody loci, where we had already published specific proposals).

In our first set of comments, we were trying to be gentle, humble and humorous, but L&C evi- dently thought that we were not being serious and

CHALLENGES OF CHICKENS AND RABBITS TO IIvlMUNOLOGY 519

responded with ridicule, rhetoric or exasperated perplexity in those cases where they did not sim- ply ignore our arguments and examples entirely. L&C are quite right that we were unhappy with their first set of responses.

In our second set of comments, we tried to set up specific questions that would result in answers that we could understand. We are somewhat hap- pier with the tone of this second set of L&C responses, but what did we learn?

L&C’s response to question 1 suggests that they agree that redundancy in antibody specifici- ty can exist, but that “extra diversity” cannot be maintained over evolutionary time without selec- tion, a position that we argue is untenable for several reasons.

The most perplexing issue for us is illustrated by question 2 - we cannot figure out from their response whether L&C really believe that CDR3 has no role in antigen binding, has no meaningful functional diversity and is thus only “framework”. I f they believe that CDR3 does in fact have a role in antigen binding and some of the diversity in CDR3 is meaningful (and that it is therefore not entirely “framework”), then we see all three CDR as very similar except for the “extra diversity” in CDR3 due to the D region; we argue from ques- tion 1 that this “extra diversity” is not bad or good but just the typical redundancy possible but not necessary in a biological system.

In their response to question 3, L&C finally agree that the proposal of D as a relic (from the time that split V genes were T-cell receptors) is a reasonable alternative to the proposed impor- tant function of D for signaling in their Protecton theory; indeed they claim that the relic idea origi- nally was one of their ideas, but that they discard-

ed it as intellectually unchallenging. We of course would argue that is simple, definite, sufficient and reasonable.

And in their response to question 4, they sug- gest that B cells with more than one antibody specificity would not be properly controlled by T cells after somatic mutation. We think that there is more to learn about the mechanisms of peripheral tolerance before choosing between the idea that allelic exclusion is essential for self- nonself discrimination and the idea that it is a rel- ic from the time of multiple VDDJC loci. (L&C ig- nored nearly all the structural explanations for the preferred reading frame of D in mice and chick- ens, the examples and arguments about repertoire selection in the bursa and the origin of the chick- en antibody loci, and a number of other points that we made to respond to their first set of comments. 1

Of course, L&C are correct that “the existence of alternative interpretations is not a disproof of one such interpretation”. Not only do we agree, but it was the starting point for all three sets of our comments - we had understood from their writings that L&C thought that their model was the only possible explanation for the data. We are relieved to learn that they harbour no such delu- sions. As we stressed many times, we never thought that all of our ideas would turn out to be correct. But coming up with ideas, even slightly silly ideas, is part of the fun of doing science. We anticipate the time when we look back and laugh at what ridiculous notions we held. May L&C be there with us.

The Base1 Institute for Immunology was founded and is supported by Hoffman-La Roche SA, Basel, Switzerland.