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Kaufman, J., Skjgdt, K. & Salomonsen, J. (1990), The MHC molecules of nonmammalian vertebrates. Immunol. Rev., 113, 83.

Kaufman, J., Flajnik, M. & Du Pasquier, L. (1991a), The MHC molecules of ectothermic vertebrates, in “Phylogenesis of immune functions” (Warr, G.W. & Cohen, N.) (pp. 125150). CRC Press, Boca Raton, FL.

Kaufman, J., Skjodt, K. & Salomonsen, J. (199lb), The B-G multigene family of the chicken MHC. Cril. Rev. Immunol., 11, 113-143.

Kaufman, J., Salomonsen, J., Riegert, P. & Skjoedt, K. (1991c), Using chicken class 1 sequences to unders- tand how xenoantibodies crossreact with MHC-like molecules in nonmammalian vertebrates, in “Procee- dings of the American Society of Zoology Centen- nial meeting”. Am. Zool., 31, 570-519.

Kaufman, J., Salomonsen, J. & Skjodt, K. (1991d), Evo- lution of MHC molecules in nonmammalian verte- brates, in “Molecular evolution of the major histocompatibility complex”, NATO conference on Evolution of the MHC April 1991 (Klein, J. & Klein, D.) (pp. 329-341). Springer-Verlag, Heidelberg, Berlin.

Kaufman, J. & Salomonsen, J. (1992) B-G : we know what it is, but what does it do? Immunol. Today, 13, l-3.

Knight, K. & Becker, R. (1990), Molecular basis of the alle- lit diversity of rabbit immunoglobuhn VH allotypes : implications for the generation of antibody diversity. Cell, 60, 963.

Litman, G., Varner, J. & Harding, F. (1991), Evblutio- nary origins of immunoglobuIin genes, in “Phyloge- nesis of immune functions” (Warr, G. & Cohen, N.) (p. 9). CRC Press, Boca Raton, FL.

Malissen, M., Trucy, J., Jouvin-Marche, E., Cazenave, P.- A., Scollay, R. & Malissen, B. (1992), Regulation of TCR a and p gene allelic exclusion during T-cell deve- lopment. Immunol. Today, 13, 315-322.

Mansikka, A., Sandberg, M., Lassila, 0. & Toivanen, P. (1990), Rearrangement of immunoglobulin light chain genes in the chicken occurs prior to colonization of the embryonic bursa of Fabricius. Proc. Nail. Acad. Sci. (Wash.), 81, 9416-9420.

McCormack, W., Tjoelker, L., Carbon, L., Petryniak, B., Barth, C., Humphries, E. &Thompson, C. (1989a), Chicken IgL gene rearrangement involves deletion of a circular episome and addition of single nonrandom nucleotides to both coding segments. Cell, 56, 785-791.

McCormack, W., Tjoelker, L., Barth, C., Carlson, L., Petryniak, B., Humphries, E. & Thompson, C. (1989b), Selection for B-cells with productive IgL rear- rangements occurs in the bursa of Fabricius during chicken embryonic development. Genes Develop., 3, 838-847.

McCormack, W., Carlson, L., Tjoelker, L. &Thompson, C. (1989c), Evolutionary comparison of the avian IgL locus : combinatorial diversity plays a role in the gene- ration of the antibody repertoire in some avian spe- cies. Int. Immunol., 1, 332-341.

McCumber, L., Sigel, M., Trauger, R. & Cuchens, M. (1982), RES structure and function of the fishes, in “The Reticuloendothelial System, vol. 3, Phylogeny and Ontogeny” (Cohen, N. & Sigel, M.) (p. 393). Ple- num Press, New York.

Nemazee, D., Russell, D., Arnold, B., Hammerling, G., Allison, J., Miller, J., Morahan, G. & Biirki, H. (1991), Clonal deletion of autospecific B lymphocy- tes. Immunol. Rev., 122, 117.

Parvari, R., Ziv, E., Lantner, F., Heller, D. & Schechter, I. (1990), Somatic diversification of chicken immu- noglobulin light chains by point mutations. Proc. Nall. Acad. Sci. (Wash.), 87, 3072-3076.

Reynaud, C.-A., Anquez, V., Dahan, A. & Weill, J.-C. (1985), A single rearrangement event generates most of the chicken immunoglobulin light chain diversity. Cell, 40, 283-291.

Reynaud, C.-A., Dahan, A., Anquez, V. & Weill, J.-C. (1986), The chicken immune system : a minimal gene model, in “Avian Immunology: basis and practice” (Toivanen, A. & Toivanen, P.) (pp. 101-I 11). CRC Press, Boca Raton, FL.

Reynaud, C.-A., Anquez, V., Grimal, H. & Weill, J.-C. (1987), A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell, 48, 379-388.

Reynaud, C.-A., Dahan, A., Anquez, V. & Weill, J.-C. (1989), Somatic hyperconversion diversifies the sin- gle V, gene of the chicken with a high incidence in the D region. Cell, 59, 171-183.

Reynaud, C.-A., Anquez, V. & Weill, J.-C. (1991a), The chicken D locus and its contribution to the immuno- globulin heavy chain repertoire. Eur. J. Immunol., 21, 2661-2670.

Reynaud, C.-A., Mackay, C.R., Miller, R.G. & Weill, J.-C. (1991b), Somatic generation of diversity in a mam- malian primary lymphoid organ: the sheep Illeal Peyers Patches. Cell, 64, 995.

Salomonsen, J., Dunon, D., Skjodt, K., Thorpe, D., Vai- nio, 0. & Kaufman, J. (1991a), Chicken major his- tocompatibility complex-encoded B-G antigens are found on many cells that are important for the immune system. Proc. Natl. Acad. Sci. (Wash.), 88, 1359.

Salomonsen, J., Eriksson, H., Skjodt, K., Lundgreen, L., Simonsen, M. & Kaufman, J. (1991b), The “adju- vant effect” of the polymorphic B-G antigens of the chicken MHC analyzed using purified molecules incorporated in liposomes. Eur. J. Immunol., 21,649.

Tegelstrom, H., Ebenhard, T. & Ryttman, H. (1983), Rate of karyotype evolution and speciation in birds. Here- ditas, 98, 235-239.

Thompson, C. & Neiman, P. (1987) Somatic diversifica- tion of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment. Cell, 48, 369-378.

Wysocki, L. & Gefter, M. (1989), Gene conversion and the generation of antibody diversity. Ann. Rev. Bio- them., 58, 509-531.

Response to Kaufman and Salomonsen by L&C:

“What in the dickens is with these chickens? An only slightly silly response to the first draft of Langman and Cohn”

Great expectations ! (or)

Fowl play?

CHALLENGES OF CHICKENS AND RABBITS TO IMMUNOLOGY 503

With due humility K&S make a merry muddle that is highly entertaining. They have much trou- ble separating the pure assumptions that we de- rive directly from the data from those assumptions that are our mere beliefs. This is worrying to us when they have such a broad command of the data. And, even we had trouble separating data used by the model (we therefore invented Sec- tion C) from data predicted by the model (and in- vented Sections D and El; except of course the unpublished (unknown) data of K&S, which is strictly predictable.

“We are all stupid, just about different things”

O.K. ! What were we so stupid about?

1. We have a bee in our bonnets about the D region

There must be swarms of bees as even K&S’s bonnets cannot reconcile a preferred LRF in D with D-diversity without resorting to some “structur- al problem - a bias in the recombinase specifici- ty, a preference for small amino acids in the structure of the CDR3 bend and/or, a requirement of the surface ‘germline lg’ to bind a bursal ligand, at least in the chicken” that thus selects against LRF 2 (i.e., D-disaster). We are about to rename this the D-depressing region.

The numbers racket continues to attract its fair share of suckers. K&S ask “what is wrong with the idea that there are potentially 1013 distinct antibody structures.. . but one out of IO4 anti- bodies will have a significant binding affinity with any particular antigen and therefore a random sample of 1 O5 is statistically enough for protec- tion?” True, such redundancy may not be a bad thing, and evolution may, as K&S like to point out, be a bit messy and has missed the cleanest and simplest solution to a problem. But how is “messiness” “ cleanliness” and “simplicity” be- ing assayed ?

What we have said is that there is a limit of around 1 O5 functionally different antibodies that constitutes a Protecton, and that not every Pro- tecton is identical while remaining functionally equivalent. So, what is wrong with the idea ? The idea needs to be thought through by K&S; our question “Has immunoglobulin come to a sticky end ?” needs their answer (see Stand. J., 33, 99, 1991 as a start). When K&S reach this next stage on the pathway to Nirvana, we can continue this discussion. Meanwhile our general comment and our reply to Maizels might be helpful.

Alas! someone had to fall into that trap. The model of the T-cell antigen complex put forward

by Davis and Bjorkman with CDR3 (i.e., D) giving specificity for peptides docked in the MHC groove is based on D being read in all three frames, not the one frame so stringently insisted on by lg. The question why D? must seem more obscure than ever. K&S ponder the possibility that the D region “is an evolutionary relic that evolved to fulfill a function in some long-dead ancestor”.

2. We invoke allelic exclusion often but K&S find it hardly necessary to the basic Protecton theory

So, why do we bother with allelic exclusion. First, it is not allelic but HAPLOTYPE exclusion. Second, if this is not too essential for Protecton theory in the minds of K&S, we shudder to think what, in their bonnets, Protecton theory is all about. Now, since it obviously does occur, what can be said about it? The one possibility that enters K&S’s minds is that it is another evolution- ary relic, one that was too complicated and difficult to eliminate without disrupting all the other related processes; in short, haplotype ex- clusion is another example like the D region of Ig to be added to the dust bin. They counter the ar- gument that doubles (non-haplotype-excluded B cells) of the type [anti-S + anti-f] are suscepti- ble to induction via F by noting in all seriousness that, in fact, tolerance works; OK! in passing, we note that autoimmunity also occurs, and how are we to accommodate this? Haplotype exclusion might have been important in the past according to K&S, but today it is uninteresting, as is the strikingly different mechanisms in use by various vertebrates to accomplish it.

Haplotype exclusion is a consequence of the necessity to make a S/NS discrimination. This is what justifies different mechanisms in different immune systems. In man and mouse, which use the cassette exchange mechanism for extracting the repertoire, haplotype exclusion is achieved at a cost of inactivating 90 % of B cells in order to keep the doubles below a threshold of destruction. In the chicken, which uses the copy cassette method to extract the repertoire, haplotype exclu- sion is achieved at a cost of generating a B cell system once in the lifetime of the animal because of limitations of continuous gene conversion in the bursal follicle, which, in turn, makes it necessary to select singly rearranged H and L chain B cells (H+“L+“).

3. “Who said that who said that who said... ?”

For the (broken) record, what did we plagiarize ?

K&S enjoy reducing our FF-anti-FF system as a theory for haplotype exclusion to a mere bind-

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ing of the germline lg to any old bursal ligand for any old reason, and thus lumping our model with any old similar Ig-binding-to-bursa proposal. However, they add the accusation of plagiarism. “This very attractive notion has, to put it mildly, occurred to everyone (Jerne, 1971 ; Reynaud et a/., 1986, 1989, 1991a; McCormack et al., 1989b; Mansikka et al., 1990; Salomonsen et al., 1991 a,b; Kaufman et a/., 1991 b).” Not quite so fast. First, to get the facts straight, Jerne never discussed the chicken and his anti-self germline repertoire was anti-MHC or anti-Id depending on the era under discussion; Reynaud and Weill have been concerned with getting 8 cells into the bur- sa (the homing device assumption), as were Man- sikka and Toivanen; McCormack and Thompson who were concerned with getting follicular 8 cells to proliferate and diversify, suggested a bursal ligand for B-cell lg that is initiating of this process; and K&S have their own “horn to toot” and we will plug that noise next. Our proposal deals with the mechanism for haplotype exclusion dispelling, we hope, the K&S confusion.

K&S continue to insist that all this diversity of lg at the VD and DJ junction, as well as the VJ junction of L chains, is a significant part of the generator of diversity. Pardon the hoarseness in our voices, but we obviously need to repeat the point that amino acid sequence diversity cannot be equated with an identical degree of combining site diversity at the level of different combining site specificities. The so called “CDR3 diversity” is useless for the mature lg repertoire, as it in- troduces vast levels of degeneracy, and it must be equally useless for the proposed nascent B-cell repertoire. There is little point to merely repeat- ing our arguments when no detailed criticism and rebuttal of them have been made (for example, Langman and Cohn, Mol. Immunology, 24, 675, 1987, see Section III pp. 681-686). It is not good enough to keep on repeating a mantra (D- diversity) and hope that it is true, unless of course we are trying to levitate the immune system in- stead of conducting a scientific discourse.

4. The toot of K&S’s horn

The “chicken B-G multigene family” encodes a set of polymorphic cell surface antigens present on most cells of most tissues. It is this set of tar- get antigens that K&S postulate is what the ini- tial repertoire of chicken lg recognizes (all due to “CDR3 diversity”). The B-G antigens determine when gene conversion can stop because loss of recognition of B-G is grounds for the B cell to exit the bursa. Details of positive and/or negative selection are not essential to the argument; they

are entirely Jernerian. There is a cross-section of data that the model accounts for, but one gets the feeling that it is the model’s “pleasing resonance with the selection of T cells into the thymus by polymorphic MHC class I and class II molecules” that K&S find so compelling. This resonance lacks timber given the absence of any data linking B-G antigens to B-cell ontogeny or function. They con- clude with the hope that they have made “clear during the description of the model, other reasona- ble simple alternatives exist to explain each fact”. Let us briefly review the facts they claim to explain.

I) “The disappearance of B cells with surface ‘germline lg’ molecules and massive cell death in the bursa.”

2) “It slips around the problem of allelic and joining diversity of ‘germline lg’ molecule by al- lowing the recognition of a multigene family of related but B-G molecules.”

3) “The preferential recognition of allogeneic B-G molecules as exemplified by the ‘natural an- tibodies’, the ‘preferential response’ and the ‘ad- juvant effect’ in terms of cross reaction.”

4) “Why B-G molecules are polymorphic.”

Hmm! after relegating the preferred LRF of D, D itself, and haplotype exclusion to the dustbin of evolutionary relics, it seems that there are only B-G molecules left to explain ; K&S certainly have a “simple” and an “alternative” to somebody’s model, but it’s reasonableness we will leave to the expert participants of this Forum. We do not wish to be accused of lack of impartiality by simply say- ing that what they claim to have explained is irrele- vant to understanding the origin of the B-cell repertoire of their fine feathered imaginary animals.

Salomonsen et al. (Proc. Nat. Acad. Sci. (Wash.), 88, 1359, 1991) used dead-reckoning to guess “that certain B-G molecules are involved in selection of B cells and the resulting antibody repertoire in the chicken”. In 1992, they specifi- cally refer to this as involving a bursal molecule (Kaufman and Salomonsen, Immune/. Today, 13, 1, 1992). As there is no experimental reason, one way or the other, to evaluate this guess as to the function of B-G molecules, we can only compare what is known about them with our proposed existence of a “bursal follicle-forming compo- nent” (FF). We would not expect the FF ligand to be present on many different cell types that are unrelated to the bursal elements, and to be a high- ly polymorphic multigene family of molecules, par- ticularly since it is recognized by a unique VLV, pair. Furthermore, in the end, humoral effector function is mediated by secreted lg, not by an ef- fector cell-target cell interaction of the T-cell type

CHALLENGES OF CHICKENS AND RABBITS TO IMMUNOLOGY 505

hat is dependent on restrictive recognition. If our postulated follicle-forming component, FF, hap- pened to be encoded in the MHC region (like com- plement, tumour necrosis factor, cytochrome P450 or steroid hydroxylase) its properties would remain unrelated to those characterizing class I and class II restriction elements. In essence, us- ing an analogy with the restriction elements used by T cells to describe B-G gene products in their assumed relationship with humoral antibody is simply misleading.

5. “Where did those poor cluckers go wrong?”

Being wrong is not for the birds!

There is a difference between the notion that, being GOD, I would have made the immune sys- tem thusly, and knowing GOD, could the immune system have evolved thusly. Blaming the oddities of the chicken immune system on its chromo- somes, lacks political correctness these days. At best, these “evolutionarily challenged” species are an opportunity to discover the selection pres- sures that would allow the chicken, the rabbit, the tadpole, the elephant, the mouse and man to all benefit equivalently from differently constructed immune systems. There is no point arguing, as do K&R, that we do not need to study barnyard biol- ogy in order to cure haemorrhoids and ingrown toe nails, let alone hayfever or other serious ills. It is sufficient to learn what can, and cannot, be tinkered with and hope to know the difference. The blind self-centered narcissim of Homo Sapiens, in general, is something above which to rise, and does not need feeding. Glick and Olah put it rather well : “the chicken happens to be an excellent experimental bird, and contributes to the economic and nutritional well-being of the world”.

We were a little hesitant to have God bless us; we would have preferred heaven help us.

Who laid the egg? Our response to the rebuttal by Langman and Cohn (J. Kaufman and J. Salomonsen) :

We thought that some sober messages in a light-hearted envelope might be fun, but apparent- ly it made the whole thing too obscure for L&G to take seriously. We thought that we were be- ing gentle with L&C; indeed, we used ourselves (rather than them) as bad examples, hoping that they would get the point. We made suggestions about the meaning of redundant antibody sys- tems, the origin of the D region, the reason for the preferred reading frame in D regions, the origin of allelic exclusion, the role of “germline lg” in bur-

sal B cells, and the origin of the compact chicken antibody loci. For some suggestions, we only laid out the possibilities, while for others we made our assumptions and predictions explicit. We did not claim to demonstrate that our ideas are correct and that the L&C model is wrong, but that the L&C model is not the unique explanation for the phenomena that they try to explain. We were, to put it mildly, taken aback by the tone of their responses.

We are attracted to what we naively assumed was the basic Protecton idea (that there is a minimal concentration of antibodies or cells or whatever needed to afford protection, Coleclough’s “immune reality” as described in the rebuttal, Cohn and Langman, 19901, but we have had difficulties following L&C past. that point. We did not understand what the fuss is about the de- generacy of antibody binding sites, and even if we gave L&C that point, we did not follow why they heap the responsibility on the D region, and even if we allowed them that as well, we were not driven therefore to assume that the D region car- ries out any mysterious signaling functions based on conformational changes. We also found their argument for the basis of allelic exclusion uncon- vincing, given our understanding of B-cell tolerance.

In our comments, we stressed that our lack of understanding might be a problem with our own intellects. As we were admonished to do, we have just struggled through the many hundreds of pages on the Elephant/Tadpole paradox (Langman and Cohn, 19871, the Protecton theory (Cohn and Langman, 1990) including the responses and rebuttals, “immunoglobulin sticky ends” (Lang- man and Cohn, 19911, the first draft of the L&C model for this Forum, and the general and speci- fic responses to the participants (including Maizels). We still do not understand L&C on these four points. What is more, we find that many of the participants in the Immunological Reviews dis- cussion also did not understand them (in particu- lar, Coleclough made some of the same points that we did, but much more elegantly; Coleclough, 1990); the rebuttals from L&C did not convince us. We were also unconvinced by the “sticky ends” paper, because most of the ar- guments hinge on the difference between mono- meric and aggregated antigen interacting with cell surface and secreted antibodies. Nearly all patho- gens (and many self antigens) exist as multimer- ic structures (either repeating proteins or carbohydrates as an efficient coat, or as molecules on a membrane) ; furthermore, antigens exist in a sea of “non-specific” or “low affinity” antibody (maternally derived early in ontogeny and self-generated later in life), so even monomeric an-

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tigens may have the chance to be polymeric. The use of “experimental evidence” was also hard for us to follow (among many examples : “Since the D segment is expressed in functional BAr in es- sentially one reading frame, then it must function by regulating the transmission of a conformational signal” (emphasis added)).

Yet, all in all, these papers represent a mag- nificent attempt by L&C to tie up everything that they know in one enormous neat package. We un- derstand more of it now, we admire the large amount of thought and work that went into it, but we don’t believe it. There was hardly an assump- tion or argument that we can accept whole- heartedly; accepting them all at the same time is too much for us.

So we will turn it around; we ask L&C to an- swer each of the following four questions, in clear language, without recourse to their secondary theoretical constructions (e.g., stage I and stage II repertoires, evolutionary selected germline genes, anti-P and anti-S and anti-N, invisible CD residues, and conformational changes). That means clear- ing their minds and using the information that we give them; it might be easiest for L&C to think of it as as an exercise to help us distinguish between assumptions and conclusions. We follow each set of questions with our viewpoint and some ap- proaches to test the questions using current tech- nology. But it should be remembered that we cannot prove that our evolutionary scenarios ac- tually occurred (as we pointed out in our original comments); the best we can do is to ask whether the models are consistent with everything that we know from nature and also to ask whether we can construct such systems in the laboratory that work as expected. We look forward to their clear and unencumbered explanations.

Why is antibody redundancy bad?

Question 1. Consider an imaginary 100 g animal containing 10’ B cells with, let us say, IO7 different B-cell clones each producing a different antibody. In each 1 ml of the animal, there are a random assortment of IO5 clones producing (on the average) some number close to IO5 different antibodies. If one out of a thousand of these B cells produces an antibody that reacts with any given antigen, then there are 10’ B cells per ml that produce specific antibody after immu- nization. In what way is this animal unprotected ? Now consider another imaginary 100 g animal identical to the first in every way, except that the IO7 B-cell clones all produce antibodies that are different from the antibodies produced in the first

animal. I f one out of a thousand of these B cells still produces an antibody that reacts with any given antigen, then this animal is still protected, and yet uses a completely different repertoire than the first. Why is this not diversity (dependent on the D region or not) and redundancy together?

Our answer to question 1. Both animals are protected (based on the initial calculations from the Protecton hypothesis, not the one used to justify stage I and stage II repertoires, involving a casual assumption that sufficient protection re- quires ten times the minimal amount of antibody already assumed to be protective), and the two antibody systems are both diverse and redundant. The point is that the antibody gene system (like many biological systems) may be degenerate without harm to the animal but that it need not be degenerate. We feel that the evidence of differ- ent but equivalent antibody spectrotypes found in isogenic frog clones (which we cited in our com- ments and which was not considered by L&C in their reply) is reasonable evidence that the anti- body repertoire may be both degenerate for a par- ticular antigen and yet may be sufficient for protection.

Maybe we got confused by L&C’s harsh descriptions of the D region diversity as “useless” and “framework” ; it sounds like they think that no D region diversity is important. Upon careful reading, we think that what they are saying is that some D-region diversity is important, but “extra diversity” is not helpful or even harmful. But we still get confused. If, as they apparently believe, only 1 O5 antigen-binding specificities are possible from all the different wals of generating diversi- ty, then whether the IO antigen-binding speci- ficities are due to 1 O5 or 10” structurally different antibodies is a matter of indifference to the antigen, as long as there is some antibody to bind it. (We leave aside the problems that it is not clear what a specificity is without specifying the antigen and that it is not clear that one antibody can only bind one antigen.) We do not see why this “extra diversity” is harmful to the animal - if it is truly redundant then it is invisible to evolu- tion. Alternatively, there may be intermittent selection for particular CDR3 regions - a continu- al selection for diversity per se rather than a con- tinual selection for particular sequences. In either case, there is nothing to indicate that the pro- posed “extra diversity” that they claim is due to the D region is harmful; the antibody system can be both redundant and protective. It is messy, but so what? As we said in the previous comments, “we feel that this unnecessary diversity in CDR3 will indeed be selected intermittently, but that it is not necessary and may in some animals not be present”.

CHALLENGES OF CHICKENS AND RABBITS TO IMMUNOLOGY 501

On the other hand, we do not know that the lower limit is L&C’s 200 V regions purportedly selected by pathogens during evolution. One approach to explore this might be to use the exis- ting C, and C, knock-out mice (Kitamura et al., 1991 ; Zou et al., 1993) bred so that only the X light chain loci would be functional. Then trans- genie mice could be made with constructs con- taining various numbers of unrearranged V, genes along with a single D,, a single J, and a single C, region. One might be able to answer the question : how many V, regions are required for the mouse to survive? Is one enough, if res- cue by somatic hypermutation is possible? (This experiment may already have been done in the sheep, which have very few V, genes and diver- sification by hypermutation (Reynaud et a/., 1991) - parenthetically, do L&C think that the pattern of hypermutation could be evolutionarily selected ?I

Whether the upper limit is L&C’s 200 V regions might be approached in a similar way - use trans- genies to load up the genome with V, regions (already cloned from many different organisms so as to be non-identical) in tandem with D,, J, and C, regions and see whether the animal survives. (Again, this experiment may already have been done in nature - polyploid frogs use all of their antibody loci without apparent harm ; Du Pasquier and Blomberg, 1982.)

What’s wrong with the D region?

Question 2. Consider a rearranged antibody gene (in other words, the equivalent in the real world of a hypothetical animal with a germline V gene without separated D and J regions) that gives rise to a 2-chain antibody molecule. Assume the protein structures of CDRI, CDR2 and CDR3 have no great distinguishing features in and of themselves. Pretend that the frequency with which an amino acid substitution would lead to a change in antibody specificity can easily be determined (leaving aside the fact that the con- cept is meaningless without knowing what anti- gen is being considered). Would the frequency be different between CDRl, CDR2 and CDR3? If so, why? Again consider the same germline rearran- ged antibody gene which gives rise to a two chain antibody molecule, but this time consider the fact that location of CDR3 is, if anything, more cen- tral to the whole antibody binding site than CDRI and CDR2 (and therefore might be, if anything, more decisive in the structure of the binding site than the othertwo loops). In this case, would the

frequency be greater for CDRI and CDR2 com- pared to CDR3, or less?

Ours answers to question 2. The frequency with which an amino acid change in CDR3 would affect binding of a particular antigen depends on how important the CDR3 contact sites are for that antigen, so clearly on average the frequency for CDR3 should be at least as high as for CDRl and CDR2, and higher if it is more important for bin- ding the particular antigen. The point is that even if the antibody repertoire is degenerate in a parti- cular individual, this does not mean that the D region is responsible, and it certainly doesn’t imply that the D region is therefore framework. All dif- ferent ways of creating diversity are responsible for the sequence diversity in antibodies, not just the D region. L&C state that they are concerned “with what probability an amino acid replacement in D-N-J will change specificity in a functional way”. We pointed out that it should be at least as high a frequency as with CDRl and CDR2, but L&C didn’t try to defend the position that CDRI and CDR2 are largely framework; they simply ignore the argument in their rebuttal.

They also ridiculed Davis and Bjorkman’s ele- gant model for the origin of split genes (Davis and Bjorkman, 1988) because it doesn’t explain the preferred reading frames of antibody D regions in mouse and chicken. Unfortunately, we didn’t sug- gest it as an explanation for the preferred reading frame problem (which we think has a relatively simple structural explanation), but as an explana- tion for why the split genes originated only in CDR3. Contrary to what L&C claim in their rebut- tal, the Davis and Bjorkman model does not make the origin of the D region “more obscure than ever” ; it is a clear alternative to the assertion by L&C that the D must have a function in antibo- dies. In any case, the Davis and Bjorkman hypo- thesis can be falsified: if the three-dimensional structure of a T-cell receptor/MHC molecule/ peptide complex shows that CDRI and CDR2 do not primarily contact the helices of the MHC mole- cule while CDR3 does not primarily contact pep- tide, then the model is probably wrong.

Another possibility is that split genes evolved to regulate the transcription of numerous tandem V genes; only one V region would be expressed in each antibody locus by requiring V genes to rearrange to D and/or J, close to the C region enhancer. Since L&C assign allelic exclusion the task of dealing with the dreaded “doubles”, then they should find this second possibility for the ori- gin of split genes indispensable for dealing with the much more worrisome “multiples”. This may have been a problem for the shark, as described below.

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What important function for the D region?

Question 3. Consider an antibody V gene which has the signal sequence encoded mostly on one exon and the mature V region encoded on another exon. (This is in fact the case for every V gene known, which means that this feature is conserved in thousands of published examples that span animals separated in evolution by nearly half a billion years). Are we automatically requi- red to postulate that the intron between the two exons has an important function that has some- how escaped notice?

Our answer to question 3. lntrons between a signal sequence exon and an extracellular region exon are widespread and have strong structural requirements (such as the minimal 80 nt length, the invariant 5’ GT and 3’ AG embedded in their respective consensus sequences, the 3’ pyrimi- dine tract, the invariant A responsible for the lariat), but in general do not have a critical func- tion since constructs in which they are deleted are usually perfectly functional. The accepted expla- nation is that exons originally specified particular protein motifs (“super-families”) that were mixed by recombination in the introns to give new func- tional proteins - the introns had a function during evolution, but not in the finished gene; they are evolutionary relics.

The point is that even if the D region is consi- dered to be largely responsible for the degeneracy of the antibody repertoire and furthermore (not therefore !I a framework region that has strong structural requirements but does not contribute to diversity, this does not imply that the D region has some other currently important function. It could simply be an evolutionary relic. Examples of mor- phological structures with obvious functional importance in ancestral species but not in descen- dent species are cited in every book on evolution since Darwin’s Origin of the Species, In ontogeny, structures come into existence and then pass away based on history rather than function. On the biochemical level, our little example about introns is only one of many that could be imagi- ned. All of these examples are based on the notion that processes that are complicated and tied into other systems may be difficult to get rid of without disrupting the whole organism, and thus may persist without any implication that they are performing some important task that is currently selected by evolution.

In other words, the D region might have had some important function in the distant past, and be retained, not because it is presently important, but because it is hard to “splice out” the inter- vening DNA at the genomic level. L&C didn’t res- pond to this argument in their rebuttal, except to

suggest that we were unable to think of anything else. In a scientific discussion, how would they propose to rule this possibility out?

Why is there a preferred reading frame for the D region ?

When we wrote our first comments, it was not obvious to us why L&C settled on the D region as the culprit for degeneracy or why they decided that it therefore must have some important function. Having read these hundreds of pages, it seems that they do so for different reasons in different places, but mainly to assign an important reason to the strong (but certainly not absolute) bias in the use of one reading frame for D regions in mouse and chicken antibodies. We did not say very much about this in our comments because we didn’t find it an interesting evolutionary problem; the expla- nations based on structure seem clear and direct. L&C claimed that we “resorted” to the idea that the preferred reading frame for D in mice and chic- kens might be due to some structural problem, for instance “a bias in the recombinase specificity, a preference for small amino acids in the structure of CDR3 bend, and/or a requirement of the surface “germline lg” to bind to a bursal ligand, at least in the chicken” instead of accepting their obvious view that the preferred reading frame is selected during ontogeny because it gives some conforma- tional signal to early B cells about tolerance. We will not belabour the arguments that have already been made against their view (and in our opinion not adequetely answered in the Immunological Reviews volume or in the “sticky ends” paper). We will set aside for the moment the lack of evidence for a preferred reading frame in frogs and sharks (and the difficulties with human D regions mentio- ned by L&C) in case that is due to insufficient data. However, we will note in passing that CDR3 regions vary enormously in length (due to the dele- tions and additions at the joining sites on both ends; see Kabat et a/., 1987); in some antibodies, there is virtually no D region left and in others it is not even clear where the D begins or ends; the capacity of these sequences to generate a speci- fic signal is not obvious! And we will explain our three suggestions for a structural basis of the pre- ferred reading frame, including some experiments to test them.

The most likely possibility for the change from random reading frames early in ontogeny to bia- sed reading frames later in ontogeny is selection at the protein level, but no one has seriously tes- ted the idea that the specificity of the recombinase changes with time. We find this an interesting pos- sibility because vertebrate development is loaded

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with relics that are hard to understand except in the context of the animals in which they develo- ped. As an example from the ontogeny of the immune system, it is not obvious why chickens export three waves of UP and yS T cells from the thymus with precise timing (Le Douarin et a/., 1984). Frogs also export three waves of T cells from the thymus, but there the rationale is clear from the timing - one wave emerges in tadpole, one wave emerges during metamorphosis and the final wave emerges in the adult frog (Turpin and Smith, 1989). The three waves of T cells in the chicken are likeliest to be an evolutionary relic, based on the requirements of amphibian T cells to function in three different internal environ- ments! It will be feasible to test the idea that the reading frame usage of D regions is determined by changing recombinase specificity during onto- geny when the components of the recombinase systems are isolated and reconstituted in vitro.

The CDR3 regions are central to antibody bind- ing sites, situated between the V, and V, domains. The unpreferred D reading frame gene- rally has larger and more hydrophobic amino acids than the preferred D reading frame (although there are exceptions that have many basic charges and bind DNA very well; Eilat et a/., 1988; Schlom- chik et a/., 1990). It would be a simple result if the amino acids of the unpreferred reading frame led to a problem in the association of the V, with the V, domain. The stability of the interdomain interactions could be estimated by molecular modeling (based on the detailed understanding of the atoms involved in interdomain contacts in known antibody structures) and determined directly by examination of mutants produced in myeloma cells transfected with in vitro mutage- nized antibody sequences. Another possibility is that the more hydrophobic binding sites generally resulting from the unpreferred reading frame bind fewer antigens (because the surfaces of most pro- tein antigens have only a sprinkling of hydropho- bic residues and carbohydrates are highly hydrophilic). This possibility might best be approa- ched by the appropriate use of the single chain antibody system (Griffiths, 19931, in which V and J regions would be joined with a large number of different (even random) D regions produced by polymerase chain reaction, the resulting antibody Fv regions would be selected by binding to some common antigen (for instance, a whole bacterium or virus or some large protein) and then the D regions actually used (before and after selection) would be determined by sequencing.

If the chicken “germline lg” molecules bind to bursal ligand(s) using the antibody binding site, then clearly particular sequences in the binding site (including the preferred reading frame of

CDR3) are required. Isolation of the proposed ligand would allow this idea to be tested; the hypothesis could be falsified by demonstration that the “germline lg” protein does not bind a ligand (for instance, demonstration that cytoplas- mic antibody triggers the expression of a relevant adhesion molecule). We (and undoubtedly others) have suggested that some B cells in neonatal mice might also be selected by some ligand ; this could contribute to the bias in mice.

Parenthetically, we were recently told of ano- ther possibility for a structural basis for the pre- ferred reading frame in mice: the existence of DNA homologies between the end of the V region and the beginning of the D region, so-called “over- lap” (Gu et a/., 1990).

Why is there allelic exclusion?

Question 4. Consider an imaginary diploid ani- mal that has two heavy chain loci and two light chain loci, all of which are allowed to rearrange within a single B cell located in a primary lymphoid organ, limited only by a fixed span of time. Assume that after this time, the cell is subjected to positive selection based on whether there is functional antibody on the cell surface and nega- tive selection based on whether that antibody binds (presumably self) antigen in the primary lymphoid organ. Finally, assume that after the B cell leaves the environment of the primary lymphoid organ, encounter with antigen leads to activation (leaving aside the idea of peripheral tole- rance for the moment). The result will be as fol- lows. Those cells which properly rearrange no loci or only heavy chain loci or only light chain loci will not be functional because there will be no com- plete antibody molecule produced. Those cells that properly rearrange two (one heavy and one light), three or four loci may be able form one, two or four antibody molecules, respectively (if all of the particular heavy and light chains do in fact interact properly). Only those cells which have no antibody molecules on their surface that react with self antigen will escape being tolerized in the primary lymphoid organ. In the periphery, encoun- ter with antigen leads to the production of either one, two or four antibodies per cell, some of which are specific. Is this animal either autoim- mune or unprotected and if so, why?

Our answer to question 4. Since the cell with four different antibody molecules can still be toler- ized and activated in the same way that a cell with one antibody molecule is tolerized and activated, such an animal would be neither autoimmune nor unprotected (at least no more than in an animal with allelic exclusion). The fact that a “four anti-

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body” plasma cell might make less specific anti- body than a “one antibody” plasma cell is offset by the fact that more “four antibody” plasma cells would be making antibodies with this specificity. The fact that the response to an antigen would lead to some non-specific antibody seems unim- portant in face of the large amount of serum anti- body with no clear specificity already present.

The point is that such an animal could easily exist, and thus allelic exclusion need not exist. The fact that in amniotes, a single B cell expresses only one heavy and one light chain does not mean that this phenomenon of allelic exclusion has some important basis in these taxa. It could be an evolutionary relic that was important for some ancestral animal but not in the descendants, being retained because it is too involved with other systems to easily delete. We suggested that there is no important function for allelic exclusion in mammals and chicken, but that the machinery for it is grinding on long after the need for it passed.

L&C didn’t respond to our suggestion that alle- lit (haplotype) exclusion could be an evolutionary relic nor did they respond to the example of the shark antibody system (similar arguments were made for hexaploid frogs; see Du Pasquier and Hsu, 1983). They ridicule the arguments about the response of cells with one and four antibody molecules, but we don’t understand why - in the scheme we present above, their “anti-S + anti-F doubles” would be eliminated in the primary lymphoid organ. (Or do they claim that every last antibody molecule on the surface of a cell must be engaged in order for tolerance to occur?) Finally, they reiterate the assertion that “haplotype exclusion is a consequence of the necessity to make a S/NS discrimination”, a de- rived result based on many assumptions that we question.

In fact, it is reported that sharks have an enor- mous amount of serum antibody compared to amniotes; in terms of antibody specificity and affi- nity, there is little difference between individual sharks and little change upon immunization (McCumber et al., 1982; MBkela and Litman, 1980; Litman et a/., 1982). So perhaps they do indeed have no allelic exclusion ! (What would L&C make of that?) One possibility is that some close relative of the shark ancestor developed allelic (locus) exclusion in order to generate specific anti- body responses, and was the ancestor of the bony fish, amphibians and all amniotes. An alternative that we like less is that the ancestor of both the shark and bony fish practiced allelic (locus) exclu- sion, and the system was lost in the cartilaginous fish. Interestingly, it appears that bony fish have heavy chain loci organized like mammals and frogs, but light chain loci organized like cartilaginous fish.

How would one test whether allelic exclusion is in fact necessary? When the mechanisms of allelic exclusion are known in detail, then homo- logous recombination in mouse ES cells might allow one to change the portions of the genes res- ponsible (for instance, parts of the transmembrane region in the p heavy chain gene) so that allelic exclusion no longer occurs, and then see whether more than one antibody is expressed for each B cell, whether the B cells can be triggered to pro- duce specific antibody, and whether the animal is protected or autoimmune.

Why do we like the B-G selection idea 7

We pointed out that many people (including ourselves) have played with the idea that “germ- line lg” molecules on the surface of bursal B cells bind a bursal ligand for one reason or another, that any such proposal must deal with the fact that there are multiple “germline lg” molecules due to genetic polymorphism and to joining diversity, and that there are reasonable alternatives to signaling by surface “germline lg” molecules. We quite carefully stated our assumptions, our model and our predictions. We finished with a caveat against believing too strongly in cute ideas, including our own. The response by L&C was moral outrage (to be dealt with at the end of this paper) and two counterarguments: one dependent on the idea that junctional diversity is not important and the other a dismissal of B-G molecules because they don’t fit the L&C model of an FF and are there- fore irrelevant.

Now we are confused again - their state- ments that “‘CDR3 diversity’ is useless for the mature lg repertoire” and “the FF ligand . . . is recognized by a unique VLV, pair” suggest that L&C really believe that CDR3 makes absolutely no contribution to antigen binding. We thought that we had finally understood their point that some CDR3 diversity was reasonable but not the “ex- tra diversity” (as discussed after question 2 above). If no CDR3 diversity is meaningful, then we are back to asking question 2 and all the other questions that most people ask - for instance, just where do L&C think the antigen is binding? If some CDR3 diversity is allowed, then they really do have to explain how their “germline lg” molecules (definitely not “a unique V,V, pair”, no matter what they assert!) deal with the fact that there is junctional diversity (and in any case, how they deal with the allelic forms of CDRl and CDR2). Of course, one way out is to posit that the FF only interacts with invariant portions of the “germline lg” molecules; as we pointed out in our first comments, this is reasonable but equivalent

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to any other molecule being expressed on the sur- face after antibody gene rearrangement.

Since we have explained in tedious detail the assumptions, predictions and faults of our model, we will not belabour them here, except to make three points. First, a multigene family of ligands is one way out of the problem of multiple and al- lelic “germline lg” molecules, but we did not say (as L&C assert) that the B-G molecules bind only to the CDR3 region. We also do not suggest that the whole multigene family is expressed in the bursa; indeed, we made it clear that we viewed the “preferential recognition” by the immune sys- tem as cross-reaction with B-G molecules found on other tissues. Just as for MHC class I molecules, not all members of the B-G multigene family need have the same function. Second, polyploidy of the chicken MHC has a profound in- fluence on bursal B-cell number and development (Hemendinger et al., 1992; Delany et al., 19921, as might be expected from a selection model. Of course, some other gene in the MHC might be responsible for the effect. The pleasing resonance of T-cell receptor recognizing class I and class II MHC molecules with “germline lg” molecules recognizing B-G molecules may strike L&C as mis- leading, but our point is that they are both poly- morphic and may both be involved in receptor selection. Third, even though we have suggest- ed a speculative model of B-cell selection by B-G molecules in the bursa, we would not be surprised if “germline lg” molecules on the surface of bur- sal B cells in fact have no ligand. We don’t expect every biological phenomenon to have a deep func- tional meaning ! On the other hand, L&C seem to fully expect that “germline lg” molecules bind the hypothetical bursal FF molecule. Again they em- brace the dictum - if it exists, it must be im- portant.

Microchromosomes and macroevolution

The last question posed by L&C was “what was it that prompted the choice” between the B-cell systems of chickens and the B-cell systems of mouse and man. We proposed that the birds were driven to this extreme by catastrophic mac- roevolutionary events - the appearance of microchromosomes. Again, we very carefully stated our assumptions, our model and our predic- tions along with a caveat about plausibility ver- sus reality. L&C simply ignored the arguments in favour of easy ridicule. Again, since the details are spelled out in our first comments, we will not be- labor them except to ask L&C - what do they think happened? Naturally we are interested in al- ternative proposals.

Fowl play, no lie!

It was probably a mistake to present our seri- ous comments in a light-hearted wrapping; L&C jumped on the chance to make hay by forcing un- natural meanings on our remarks. We would just as soon laugh at this as well, except that it might appear to some readers as though L&C really un- derstood what we meant.

“OK, what were we so stupid about ?” As we mentioned in the first comments, “We are all stupid, just about different things” is a famous saying from the comedian Will Rogers to say that no matter how much one knows about some things, there are always areas that one doesn’t understand. We were referring to our problems with the L&C Protecton theory, but of course it applies to all of us.

“For the (broken) record, what did we plagia- rize?” ” Who say dat who say dat who say dat ?” (“Who is that who is saying: who is that who is saying : ‘who is that’?“) is an old joke about a group of people in the dark who don’t want to divulge their identities to each other. This seemed fitting, because most of us are in the dark about “germ-line lg”. Apparently L&C feel that they have already seen the light, but are afraid that we will offend someone with this language (we will take our chances, thank you very much!). However, L&C go way too far by stating that we accused them of plagiarism! Plagiarism is a seri- ous accusation that we would never make light- ly, and we in fact never used the word. Our sentence deals explicitly with the notion that “the ‘germline lg’ molecules on the surface of bursal B cells have specificity for some bursal ligand” and that has been considered by the many peo- ple we cited. Every such proposal has had unique elements and we are happy to acknowledge those unique portions of the L&C model.

In contrast to the L&C version of history, we found that Jerne (1971) proposed “that ver- tebrates have developed specialized organs for breeding lymphocyte mutants, such as the bone marrow, the thymus and the bursa of Fabricius” (emphasis added); he used the thymus simply as a convenient example. In this paper, he discuss- es many details of serum antibodies in the frame- work of his hypothesis, he cites Mel Cohn for the notion of selection after somatic mutation, and he concludes that the basic idea of his hypothesis is “that the germline V genes code very precisely for antibodies that fit to a certain set of histocom- patibility antigens” (emphasis added).

“Being wrong is not for the birds!” This para- graph amazed us, since L&C summarized some of the reasons that we study phylogeny (see the second paragraph of our first comments; the in-

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troduction to Kaufman et al., 1991). Despite their repeated use of the word “evolution”, their glar- ing assumption that each biological phenomenon must have an important functional meaning and that all these important meanings must be ex- plained in one tidy package is still a problem for us. As we tried gently to point out, one learns such a notion from studying engineering design, not by studying evolution. Of course reductionists would love to simplify everything to neat little packages where nothing is out of place - we did it ourselves with the antibody selection by B-G molecules hypothesis and again with the compact antibody gene loci by microchromosomes hypothesis, but we tried with humour and due hu- mility to make it clear what difficulties there are with such an approach!

“Blaming the oddities of the chicken immune system on its chromosomes lacks political correct- ness these days.” As must be clear from our dis- cussion in the first comments of antibody repertoire selection by B-G molecules, we do not consider the chicken an oddity, but as an exam- ple of a different strategy for diversity than that which is dominant in mammals. In fact, we point- ed out that we would like to believe that the differ- ent strategies are present in many taxa, but one or another predominates; thus the birds did not need to invent a new system, but merely increase the usage of one that was already present (a typi- cal evolutionary selection). L&C use the notion of “political correctness” several times in their rebut- tal of our comments. We hope that this is an at- tempt at jocularity, since we find the idea that a scientific theory can be “politically correct” real- ly obnoxious and, as far as we read it, the oppo- site of what L&C intend. Scientific theories should stand on scientific data, and not on social expec- tations! The fact that this is sometimes difficult and often not the case is all the more reason to be careful with such statements. One only has to remember Lysenko or McCarthy to realize why the notion of “politics “ in science is worrisome.

And our remarks about the hayfever and illness are quite tongue-in-cheek (both afflictions figured prominently in our lives this spring). In fact, we have an abiding interest in the diversity of immune mechanisms used in evolution, having worked on all vertebrate taxa at one time or another in the last 15 years. In addition to the chicken, we study unfashionable animal models (crocodiles, salamanders and lampreys) that make no econom- ic contribution to the well-being of humankind. All this despite the fact that we continue to suffer hayfever and influenza. Amen.

We thank Louis Du Pasquier for critical comments. The Base1 Institute for Immunology was founded and is support- ed by Hoffman-La Roche SA, Basel, Switzerland.

References

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Coleclough, C. (19901, A critique of the Cohn-Langman protecton theory. Immunol. Rev., 115, 173-l 81.

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Delany, M., Dietert, R. & Bloom, S. (19921, The effects of MHC chromosome dosage on bursal 8 cell sub- populations in embryonic and neonatal chickens. Develop. Comp. Immunol., 16, 313-327.

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Du Pasquier, L. & Hsu, E. (1983). lmmunoglobulin ex- pression in diploid and polyploid interspecies hybrids of Xenopus: evidence for allelic exclusion. Eur. J. Immunol., 13. 585-590.

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Gu, H., Farster, I. & Rajewsky, K. (19901, Sequence homologies, N sequence insertion and J, gene utilization in V,DJ, joining: implications for the joining mechanism and the ontogenetic timing of Lyl B cell and B-CLL progenitor generation. EMBO J., 9, 2133.

Griffiths, A. (19931, Production of human antibodies us- ing bacteriophage. Current Opinions Immunol., 5, 263-267.

Hemendinger, R., Putnam, J. & Bloom, S. (19921, MHC dosage effects on primary immune organ develop- ment in the chicken. Develop. Comp. Immunol., 16, 175-186.

Kabat, E.A., Wu, T.T., Reid-Miller, M., Perry, H.M. & Gottesman, K.S. (19871, Sequences of proteins of immunological interest, U.S. Department of Health and Human Development.

Kaufman, J., Flajnik, M. & Du Pasquier, L. (1991). The MHC molecules of ectothermic vertebrates, in “Phylogenesis of immune functions” (Warr, G.W. & Cohen, N.) (pp. 125-l 50). CRC Press, Boca Ra- ton, FL.

Kitamura, D., Roes, J., Ktihn, R. & Rajewsky, K. (19911, A B cell deficient mouse by targeted disruption of the membrane exon of the immunoglobulin p chain gene. Nature (Land.), 350, 423-426.

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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