The Take Home LESSON 1971

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THE TAKE-HOME LESSON-197 1*
Melvin Cohn

t

The Salk Institute for Biological Studies
San Diego, Calif. 92112

I have more than once suggested that at
the molecular level all real biological systems are impossibly complex.
Macfarlane Burnet (1970)

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If antibody synthesis can be understood at
the molecular level, a profound breakthrough might be achieved, not only with

implications for medicine, but also for all
aspects of the biology of multicellular
organisms.
James D. Watson (1970)
You have thrown many brightly colored patches into the sewing box. Usually
the tailor sews them into a crazy quilt; I will try to sew the patches into a Joseph’s
coat. Since they were thrown into the box without regard for the tailor’s skill,
some of them will be left over. This will be the way of deciding whether the coat
is well made, for it must be judged by the patches which are used as well as by
those which are not.

THEEVENTSPRECEDING
ANTIGENIC
ENCOUNTERS

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What genes coding for the variable region ( V ) are carried in the germ-line?
I will postulate that the germ-line V genes coding for the variable regions
specify between 100 and 1000 immunoglobulins, i.e., of the order of 25-50 V,

and 10-20 V, genes per haploid genome.
The V, genes are divided into two groups, V, and VA,which are unlinked
to each other, The ratio of the numbers of V, to VA genes varies from species
to species. In mouse this ratio is of the order of 30 (there being only one
germ-line VAgene), whereas in man it is close t o one.
The VH genes are taking on a new look. There appear to be two groups of
them, unlinked to each other. One group, familiar to all of us, is expressed
upon induction of bone marrow antigen-sensitive cells (B cells) to produce
humoral antibody. The second group has been postulated by McDevitt (this
*This paper is an expanded version of the taped copy of the summarizing talk.
I have made no effort to be complete in my bibliography but rather have referred to
the speakers at this conference whose papers are comprehensive. Further, many of
the discontinuities easily presented as asides while talking were put into a series of
intercalated comments which I invite, in fact warn, the light-headed reader to skip.
iThe work from my laboratory has been supported by United States Public Health
Service grant R01 A105875 and United States Public Health Service Training Grant
TO1 AI00430.

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monograph) to be expressed in thymus-derived antigen-sensitive cells (T cells).
This group is coded for in the mouse by the H-2 histocompatibility linked Ir-l
locus which determines unresponsiveness to certain antigens. I will call the
VH genes expressed upon induction of B cells VE genes. VE genes will be
those postulated to be expressed in T cells. In summary, four unlinked gene
loci have been postulated, V,, Vh, VL and V,”. (See comment 27.)
I picture an individual to be immunologically mature when he expresses a
total of 2 1000 V,> and 2 1000 V, genes or 2 los antibodies. This gives an
idea bf the efficacy of the somatic diversification process which must be envisaged, particularly if spontaneous mutation and selection by antigen is the
source of variability.

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Comments

1. The total number of germ-line V genes in a random-bred population, e.g. man
or rabbit, could be an order of magnitude larger than the estimated 25-50 VL and
10-20 Vir germ-line genes, because of polymorphism. My estimate is based on an
interpretation of the V sequences and a hypothesis concerning the initiation of
somatic evolution.20.94
2. I was pleased to discover recently that Jerne 4 2 , 4 3 9 4 5 had arrived at this estimate of 106 antibodies many years ago based on an argument independent of
those I have made.20 Talmagesg was the first to formulate the important question
of whether a small number of antibodies could distinguish a large number of antigens. In the present context, his argument that an antiserum is more specific than
an antibody molecule because the antiserum recognizes permutations and combinations of determinants is misleading. If a given antiserum can be made (e.g. by
cross-absorption) to distinguish N antigens unambiguously, then this antiserum has
at least N different antibody molecules in it.
It is a loaded assumption that the immune system is mature when it is capable
of expressing only 106 antibodies. This figure, by present consensus considered to
be very low (by two orders of magnitude), is very reasonable to me as a consequence of the increasing number of cases of restricted antibody responses (Krause,
Haber and Singer, this monograph), of “monoclonal” immunoglobulins with identical combining specificity, sequence and idiotype (Capra, Williams and Potter, this
monograph), of numerous examples of specific genetic unresponsiveness (McDevitt
and Benacerraf, this monograph), and of the ease with which one can clone antibody-producing cells of known specificity.2

3. I have described the products of the germ-line gene combinations (VLVH)as
immunoglobulins and those somatically derived by antigenic selection in the mature
animal as antibodies. I have done this to leave open the question of whether
the germ-line V genes were selected during evolution in order that all of the
VLVH combinations code for given specificities of survival value. I will deal with
which antibody specificities some of the germ-line VLVHgene combinations do code
for as well as the other selection pressures on these V genes when I analyze the
Jerne hypothesis.45

How are germ-line V genes expressed? I will assume that germ-line V genes
(like all other genes) are turned o n in a sequential and systematic way during
the ontogeny of antigen-sensitive cells in order to ensure that all permissible
combinations are expressed. Unlike V$ and V: genes, V, and VA genes are
postulated to be expressed in both T and B cells. As a consequence, the germline VLV, immunoglobulins used as receptors by T and B cells will be distinct

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only in their V, regions. I will assume now, and develop later (see section on
Origin of Diversity), the argument that germ-line genes are varied somatically
by spontaneous mutation.1s, zo

4. Under the Gally-Edelman :i? recombination model, germ-line V genes are
rarely expressed. Under the Hood-Talmage 3% 3 7 germ-line model ( 1 0 , VL and
10' VH germ-line genes), the assumption that V genes are turned on by a systematic
and sequential process presents a to-be-reckoned-with imposing problem in regulation.
5 . I wish to distinguish between two views of T-cell expression. The first view
that T cells are genetically restricted in the range of their specificities compared to
B cells.'" The second view is that T and B cells use equally discriminating receptors and generate a similar range of specificities.'? However, they differ in the
initial germ-line immunoglobulin receptors they express. Consequently, somatic
selection starts with different choices. In order to prevent this distinction from

becoming a semantic one, whatever formulations one uses to describe the families of
antibody specificities known in the Vh and V, classes should be applied to antibodies
in the postulated VE and V,,c
B lasses.
I should point out that genetic unresponsiveness (on a somatic mutation model)
due to structural V genes is a reasonable hypothesis, as has been pointed out by
Milstein and Munro.59 I will deal with evidence for this in one case involving the
anti-dextran response of mice.

How is the constant region (C) expressed? This question has taken o n
many new aspects at this conference. The following appear to be the rules of
expression :
1. Each antigen-sensitive cell expresses only one light and one heavy chain,
each coded by a unique VC combination.56 This means that each cell must
decide which light or heavy chain class and which allele t o express (allelic
exclusion). In order to make a subunit, a V gene is only expressed with a C
gene which is both closely linked and cis on the chromosome to it (Todd, this
monograph).
2. The T and B cells are postulated to behave identically as far as light
chain expression is concerned. It is only with respect to the heavy chain that

different behaviors may emerge.
a. T cells have been postulated to express a unique C, class 11, 12 as well
as a unique set of VL genes (McDevitt, this monograph). I will call this
immunoglobulin class IgT, to conform with Taylor and 1verson's9l nomenclature. ( I would have preferred to call this class IgC or IgX, in order not to
prejudice the possible finding that in some species (other than mouse) or under
special experimental conditions (even in the mouse) the function of the thymusderived cell making IgT might be carried out also by cells from another organ.)
IgT is postulated t o be coded for by the VE and CL genes comprising the Ir-1
locus which, in mouse, is H-2 linked.
b. B cells eventually express all of the known C i classes ( C p , Cy, GY,etc.)
associated with a unique set of Vyl genes (Putnam, Fudenberg, Pink, Edelman,
Frangione, Franklin and Terry, this monograph). C p is expressed as an antigenindependent step during ontogeny. Whether other classes, e.g. Ce, C6, GY, are
also in the antigen-independent category is unknown. However, C y is only
expressed in B cells which have previously expressed Cp. This phenomenon of
the IgM to IgG switch, discovered by Nossal, has recently been reinvestigated

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both by his group 7 2 and by Pernis (this monograph). It is not known which
of the Cy classes are involved when the species have more than one (mouse,
rat, man). In the rabbit, which Pernis investigated, there might be only one Cy
class defined by the a,,, aI2, a,,* and aI5 genetic markers which are associated
with > 90% of the IgG. The finding that the IgM to IgG switch requires an
immunogenic encounter raises many questions. Why switch? How does an
IgM B cell decide whether t o go on making IgM or to switch? What is the
switch mechanism? These questions should invite provocative speculation.
The generation of antigen-sensitive cells expressing germ-line genes for both
the variable and constant regions goes on throughout the life of the animal
(ignoring considerations of aging). From this point on, strong somatic selective
pressures due to tolerogenic and immunogenic encounters with antigen will

operate upon the “germ-line’’ antigen-sensitive T and B cell population expressing initially between 100 and 1000 different V,,V, combinations. In the adult
the C, region will, in large measure, be represented in the population, depending upon which variable regions it is associated with and which special physiological properties it confers o n the antigen-sensitive cell (see comments 17
and 27).
6. The antigen-independent expression of

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and the postulated CT, appear
reasonable at the moment. The assumption which has been challenged at this conference is that all CH genes are expressed independent of antigenic encounter.
Pernis (this monograph) has presented us with an impressive experiment showing,
in immunized adult rabbits, the existence of cells (presumably antigen-sensitive B
cells) which have IgM on their surface (putative receptors) and IgG in their cytoplasm (putative, eventually secreted, antibody). This study confirms Nossal’s
original contention that a cell could contain both IgM and IgG classes of antibody.
It could be argued that during the switch from IgM to IgG the antigen-sensitive
cell goes through a transitory stage in which it uses IgM receptors that it no longer
synthesizes. Since Pernis found no cells with IgG on the surface and IgM in their
cytoplasm, neither the switching from one class to the other nor the class from
which the cell starts ontogeny is likely to be random. There is no doubt, on purely
theoretical grounds, that the variable regions of the receptor and the induced antibody must be the same. Therefore, I expect the IgM receptor and the IgG product
to have identical V regions.

Let us consider the observations that bear on the requirements of the IgM to
IgG switch, which at present is the only one experimentally shown to exist. There
is a critical period in the ontogeny of the immune system of chickens. If they
are hormonally bursectomized too early, they cannot respond to antigen in either
the IgM or IgG class. They are agammaglobulinemic. If bursectomized at a later
stage, they can respond to antigen but only in the IgM class. No switch occurs.
A late bursectomy has no effect on the response and the switch from IgM to
IgG (R. A. Good in Reference 92). Two interpretations are evident. Either the
switch is antigen-independent and, at the time when bursectomy results in an IgM
response only, IgG antigen-sensitive cells have not been generated, or the switch is
antigen-dependent but some factor (decreased in concentration by hormonal bursectomy) is necessary for the switch (induction of IgG B cells) but not for the induction of IgM antigen-sensitive cells.
Fudenberg and coworkers and Nisonoff and associates (this monograph) have
described a human “biclonal gamma globulin, TIL.” which has identical variable
regions associated with IgM and IgG. It is likely that the conversion to neoplastic
occurred in one IgM producing cell which was dividing as a consequence of stimulation by an immunogen. This cell must then have divided asymmetrically to yield an
IgG producing cell that retained the neoplastic character. The two daughter lines,
Cp,

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one producing IgM and the other IgG, constitute the two myelomas in the individual
TIL. This observation is in essence similar to that made by Oudin and Michel 7 3
who demonstrated that a rabbit immunized with Sulmonella t y p h i possessed the
identical idiotype in the IgM and IgG antibody to this immunogen. Both findings
weigh albeit like gossamer in favor of an antigen-dependent switch only if one
assumes ( 1 ) in Fudenberg's case, that were two myelomas to arise as independent
events in an individual, they would have a negligible chance of expressing identical
V regions, and (2) in the Oudin-Michel case, that two genetically identical rabbits
would have a negligible chance of producing anti-Suhonellu ryphi of identical
idiotype. However, if the immunoglobulins produced in each case were coded for
by germ-line V genes present in small numbers, then the chance is not negligible
and the switch could well be antigen-independent. Conversely, if the switch were
antigen-independent, then the variable regions of the above-described immunoglobulins would be expected to be the products of a small number of germ-line V genes.
Since Potter (this monograph) has pointed out that myelomas of the inbred BALB/c
mouse often produce immunoglobulins of identical specificity and idiotype, i.e. V
regions, associated with different Ce classes, the above-mentioned evidence cannot
be cited as a compelling argument for an antigen-dependent IgM to I g G switch.
The key observation we owe McDevitt and coworkers (this monograph). Responder and nonresponder mice behave identically by producing, in an evanescent
fashion, an IgM antibody upon primary stimulation with the test antigen (T,G)A-L. Upon secondary stimulation the responder mice make IgG antibody, whereas
the nonresponder mice are not inducible in any class. Since McDevitt (this monograph) has shown us that it is the IgT function which is limiting in nonresponder
mice, two arguments can be made: ( 1 ) The inability of the nonresponder to mount
another IgM response on secondary stimulation with (T,G)-A-L
is due to the eventual rendering of the animal unresponsive by the primary does of antigen. This
implies that the induction of IgM B cells (like IgG B cells) requires the contribution
made by the IgT interaction. (2) The IgM+IgG switch is immunogen-dependent,
meaning that it requires both antigen and thymus-derived component, IgT.
For a somatic model in which selection by antigen of mutants is stepwise and
sequential, the postulate of an antigen-independent switch implies that antigenic
selection in each class is independent. As Jerne 4 5 points out in another context,
this could mean that the animal cannot use its accumulated somatic variants, associated with one C H class, in another C H class. It loses the ability to switch its hardearned specificities to different functions. At first glance, this seems to be a provocative criticism of any postulated antigen-independent switch. Hood's germ-line model
and Edelman's somatic recombination model are of course neutral with respect t o
whether antigen-dependent or independent switches operate, since all of the variants
can be accumulated in one giant step in all classes prior to antigenic selection.
It is the prediction that diversity is generated by a n antigen-independent mechanism
which will provide the experimental distinction between the Hood-Edelman models
and the somatic mutation model which requires sequential selection by antigen.
However, Jerne's argument45 would lose its force (1) if some CH classes
are expressed antigen-independent and some antigen-dependent, and (2) if once
switched, the cell expressing a new class produces a clone containing mutants which
can be selected upon by further immunogenic encounters. Another reason for the
switch might have to be envisaged. Suppose that the B cell expressing gem-line V
genes were born uniquely in the Cp class and the switch were antigen-dependent.
The decision as to which class to express next following induction could take one
of two routes, either sequential, e.g. Cp+Cy+Ca+CS+CE
etc., or parallel, e.g.,

I +CY
_.

I+&, etc., the latter being more likely. In either case, once switched, further
somatic selection by antigen could go on for mutants in the clone which has switched

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class. Of course, in the initial stages of diversification (by somatic mutation and
selection), the kinetics of the flow of increase in diversity into each CH class would
be different depending on whether the switch were operating sequentially or in
parallel. At later stages above a certain threshold of diversity, each CH class would
tend to be selected upon independently. Consider the origin of an IgA B cell which
is found in the gut (Heremans, this monograph). If one postulates that a B cell is
born expressing germ-line V genes on an IgA receptor, then this cell would be
pictured to home to the gut by virtue of a recognition unit for IgA (secretory piece?)
present there. This IgA B cell would be selected upon independently from an
analogous IgG B cell in the spleen, for example. On the other hand, if one postulates
that an IgM precursor cell which ends up by chance in the gut, and there encounters
an immunogen, will switch t o IgA production (signaled by a gut hormone), then this
cell must now remain there undergoing further and independent diversification as an
IgA cell. Therefore, except for the initial stages, whether or not the switch is
antigen-dependent its effect on the spreading of the diversity can only be very limited.
The picture which I expect to emerge is that B cells are born in the Cp class
uniquely, they can switch only once to any other C i class. Which class they switch

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to might be determined by factors acting locally. For example, the spleen might
signal a switch to Cy whereas the gut might signal a switch to Cn. The actual switching requires an immunogenic encounter i.e. antigen and IgT.
7. The mechanism of linking of V and C is an open quesion; in fact there is
something about this closet that makes the skeleton very restless. We all agree that
V and C are carried as separate germ-line genes and that a joining mechanism must
operate. Some five years ago16 I christened this the Todd Phenomenon, after its
discoverer. Further, it is established that VH is closely linked to CH, V, to C ,
and, by extension, V hto CA.The I ( , and two heavy chain gene loci are unlinked to
each other. Three kinds of arguments have been made to support the close linkage
of V to C: ( 1 ) Todd and Mage (this monograph) have shown that the VH and CH
genetic markers are linked in the rabbit; (2) Hood (this monograph) has identified
amino acid replacements in the V, region corresponding to the rabbit K-chain allotype (immunological marker) in addition to the ones already known in the C
region;’ ( 3 ) Terry, Franklin and Frangione (this monograph) have analyzed heavy
chain deletions which cover both VH and CH and must have occurred by the
excision of a segment of DNA between closely linked genes. This latter observation is compatible with but not proof that the linking of V to C occurs by translocation at the DNA level (see Reference 80 for discussion).
I am going to limit my comments to DNA-level models for joining and switching
because popularity as well as an illustrative (albeit weak) theoretical argument favors
them. The self-nonself distinction is the evolutionary selective pressure for “one cellone antibody” and it is only on V not C genes. Since it is found that both V and
C genes are expressed on a one cell-one antibody basis, V must determine the
expression of C. If allelic exclusion were to operate as an “on-off switch at the
DNA level on V only, then a peptide-joining mechanism cannot account for the
expression of only one of the two C alleles.
All DNA-joining models require recognition sequences either in, or contiguous
with, the V and C genes in order t o signal where to join or break. The models
differ in whether these sequences are translated.
A. Models in which the recognition sequences are translated. The usual proto62
type for this class of models is X-bacteri~phage.~~~
Three types of recognition systems have been envisaged: homology base pairing
between a sequence ending V and an identical one beginning C, an integration
enzyme recognizing the unrelated base sequences of the joining regions ending V and
beginning C, and a three-enzyme system (one for integration and two for excision)
recognizing the tertiary structure of the DNA-joining regi0n.2~ Formally, recombination and deletion joining of V to C are equivalent. It is not known precisely where
V ends and C begins, although it is generally (and erroneously) assumed that the

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amino acid no longer found to vary (-1 10) begins the region coded by the C gene.
This missing fact makes model building and testing against sequence difficult.
Before much was known about the integration of &bacteriophage, it seemed
simplest to assume that there was a pairing region (some 50 bases long) and
recombination integrated the A-DNA into the E. coli chromosome, a model which
Lennox and 1 5 2 simply transposed to VC joining. However, the later sequences
showed that if such a region exists, it would have t o be different in K and A. This
seemed unattractive enough at the time to make other models worth considering.*s
Gally and Edelman,32 employing the more up-to-date understanding of the integration and excision of lysogenic phages, introduced a specific integration enzyme,
which meant that the length of the nucleotide sequences to which the enzyme binds
could be short but which specificity dictates must be of the order of ten bases long
unless one postulates special (methylated) bases. Also aware of the difficulty that
long pairing or recognition regions were not revealed in the sequence of K and A,
they pointed out that "it is possible that the specific nucleotide sequences t o which
the integrating enzyme binds are neither transcribed nor translated "and, in fact,
illustrate V gene translocation to conform with this possibility (FIGURE7 in Reference 32; FIGURE7 of Edelman, this monograph). I wish t o make two points
concerning their model:
I . If no part of the recognition sequences postulated by Gally and Edelman 32
are transcribed or translated, then the V or C gene can no longer be excised (as
proposed by them) by the reversal of their integration mechanism and used for
switching of CH classes. Even if integration and excision were t o involve different
systems, as is now known for A-bacteriophage,Y' there must be a way to signal the
end of V or the start of C. The recognition nucleotide sequences or V end-C start
signals postulated by Gally and Edelman 3 z may be so short that they have thus
far gone unnoticed in studying amino acid sequence date but they must be there.
Putnam (this monograph) draws attention to the. . .Val.Ser.Ser. . .start of the
constant region of both human C, and C7,. This sequence, if it is to be a recognition region, must be present in every Cir class and absent from every CL class. For
light chains, the data are whispering paradoxically. As I pointed out, the exact end
of the region coded by the V gene and the exact beginning of the region coded by
the C gene are not known. However, from the middle of the light chain beginning
where replacements have not been found, the sequences are as follows:
Human

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. . . .Arg.Thr.Va1.A la.Ala.Pro.Scr.Val.Phe.1le.Phe.Pro.Pro.Ser.. . .
h . . . . G1n.Pro.Lys.A la.A/a.Pro.Ser.Va/.Thr.Leu.Phe.Pro.Pro.Ser
.. . .
K

Mouse

. . . . Arg.AIa.Asp.Ala.Ala.Pro.Thr.Val.Ser.1le.Phe.Pro.Pro.Ser.
. ..
. ...
In the case of human light chains, and A, . . . .Ala.Ala.Pro.Ser.Val.. . .has

K

h . . . .Gln.Pro.Lys.Ser.Ser.Pro.Ser.Va1
.Thr.Leu.Phe.Pro.Pro.Ser

K
been
a favorite recognition sequence for some time'" and is one candidate for the beginning of the C region. If a common sequence is used by the two C genes K and A,
which do not exchange V regions, I would guess this implies that one integration
system operates on both and regulatory mechanisms for expression prevent mixups.
Unfortunately, mouse K and h do not have a common sequence in these positions.
The common sequence. . . .Phe.Pro.Pro.Ser.. . . .seen further along turns up also
in an identical position in human K and A chains. This may be the starting sequence
coded by the C gene. If it is, we have one suspect for the recognition sequence.
If it is not, then either a peptide joining model operates80 or a return to our
original model 5'' (slightly more elaborate) might be considered. For example, a
r e c o m b i n e common to K and A could recognize the sequence coding for . . .Phe.

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Pro.Pro.Ser. . . . All V, genes could end with a sequence coding for the segment. . .
Arg.Ala. . . . . . . . . . .Ser.1le . . .and all Vh genes could end. . .Gln.Pro . . . . . . .Thr.Leu
. . . . .The C , and C h genes could start respectively with the identical sequences in
which the V, and Vh genes end. Thus, catalyzed recombination in this region would
leave it invariant. There are many objections to such a model related to the
problems of evolving such long, repeated invariant sequences (30 base pairs) attached
to the varying ones of each V subgroup.52. However, I am illustrating this extreme
case to encourage the development of precise translocation models which are more
compatible with known sequences than with A-bacteriophage, Of course, the assumption that K and h use different enzymatic systems for integration and excision might
be the trivial but perhaps correct solution.
It is expected that the light and heavy chains would use. different recognition
sequences and integration enzymes and, as expected, the above underlined light
chain sequences are not found in the heavy chains (Putnam, this monograph).
2. Jerne45 has stressed that “it is an advantage of the recombination model,
proposed by Gally and Edelman, that it so elegantly incorporates translocation
among its main features.” Gally and Edelman 32 argue that since “some kind of
somatic recombination is required to carry out translocation, the simplest theory
would assume that a similar mechanism underlies both translocation and variation,”
and they consider their model as an example of such a “single mechanism.”
First of all, elegance and parsimony notwithstanding. I am not convinced that the
mechanisms of diversification and VC joining need be “similar”. Secondly, even if
this ground rule were accepted, let us see in what sense the Gally-Edelman model
obeys it. According to their hypothesis, the diversity is generated by recombination
within a family (subgroup) of 10-20 V genes which are largely identical in sequence,
i.e., the V genes in a subgroup tend t o pair frequently with each other with the result
that the recombination rate is naturally and unavoidably high. No unique enzymes
recognizing these V gene sequences are needed for diversification as in the case of
VC joining in which a special integrating mechanism recognizing two nonhomologous specific V and C nucleotide sequences is postulated. The generation of diversity
by recombination due to homology base pairing does not intuitively or logically lead
to VC joining by a translocation mechanism which requires a special integration
enzyme (not involving homology pairing). The de facto separation of diversification by high frequency recombination and of VC joining by translocation is illustrated by Gally and Edelman’s 32 unwritten assumption that when switching of CH
classes occurs the diversification mechanism is inoperative. Not only is any model
of diversification equally compatible with the Gally-Edelman 32 proposal for joining
of V to C, but their model for diversification is completely compatible with any
mechanism of VC linkage, mRNA or even peptide-joining. For example, without
adding any ad hoc assumptions to prevent it,3* their postulated diversifying
recombining “V-episome” might be transcribed.
It was in an effort to sharpen this issue that Schubert and I S 0 proposed a VC
joining mechanism which operates at the peptide level and is compatible with all
data. This model ascribes the switching of the CH classes to regulatory events. The
key experiment is that of Lennox and colleagues 5 3 who showed that if joining were to
occur at the peptide level, V would have to be linked in peptide bond to C before
C was half completed as a nascent chain or the ribosome. This condition is so restrictive that DNA-level joining has been understandably the only popular model.
B. Models in which the recognition sequences are not translated. This class of
model was first conceived by Dreyer and Bennett.25 In view of their complexity,
these models seem less likely to me at the moment. They all involve variations on
a mechanism loosely called “copy-choice,’’ which is simple enough in that a replicating or transcribing enzyme reads along V then “skips” intervening DNA and continues along C. The result is a daughter cell in which V and C are joined because
of a precise deletion either at the DNA or mRNA level. The complexity comes
in arranging a reasonable and precise skipping mechanism and in switching V to

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another C. Looping out the intervening DNA by homology base pairing seems
unlikely because two identical sequences running in opposite directions are needed,
one contiguous with each V and one with each C gene. A special pairing or “looping
out” protein can be envisaged to recognize nonidentical sequences, or the DNA itself
can be rearranged by fancy enzymes in any one of several ways. The reason for introducing this class of models is to remind us of the sad possibility that an examination of sequences may yield no information about VC joining.
It should be stressed that V and C genes have a peculiar arrangement on the
chromosome and that the evolutionary selective pressure for this must be considered.
For example, there are two adjacent tandem sets, one for VH and one for CH on the
chromosome. Although only close linkage of VH t o C, genes has been demonstrated
(Mage, this monograph), I am prepared to extrapolate and say that all of the VH
genes are in a locus close to all of the CH genes which are also clustered in a locus.
(The f gene in rabbits has been assumed to code for C..3? Since f is unlinked to the a
locus, I am assuming this interpretation to be wrong and that j codes for the J
piece or for the secretory piece.) Given this optimized picture, it is still obvious
that any given V or C episome hunting its mate must stumble over a lot of genes.
It is not clear at the moment that genetic linkage of V to C is predicated with any
more reasonableness by considerations of how V and C are joined than by consideration of how V and C are regulated, i.e., the V and C genes are expressed only
in the cis configuration (Todd, this monograph) and show allelic exclusion.
A translocation mechanism in a normally regulated cell could not permit the
transcription of two CH genes associated with one VH gene. Finegold and coworkers 30 have isolated in tissue culture cloned, diploid human lymphoid cell lines
which synthesize both C, and C,. If these CH regions were to share an identical
Vtr region, either the VH sequence is germ-line (assuming identical alleles and that
allelic exclusion does not operate) or translocation models at the DNA level are
ruled out (assuming allelic exclusion does operate). I am not considering the possibility of specific V gene amplification only because there is no other problem posed
by the immune system at the moment that this assumption solves. However, disproof of a peptide-joining model and an extension of the Finegold 30 findings might
force us to such assumptions.
8 . On a priori grounds, an antigen-sensitive cell might differentiate in the absence
of antigen to the stage where allelic exclusion operates or it might stop short of
this step but, following induction, pass first through allelic exclusion before going
on the secrete antibody. These two situations are functionally equivalent because, in
the latter case, the immunogenic encounter signals the tetrareceptor cell expressing
both alleles to undergo allelic exclusion before further steps of induction select out
the resultant unireceptor cell.
At the present moment, it seems unlikely that the whole chromosome carrying the
light or heavy chain locus is inactivated as is the case for the X chromsome. Two
unlinked light chain loci, K and A, and two unlinked heavy chain loci, one, C,”,
known, and one, C$ postulated, are involved. Two regulatory decisions must be
made by the cell: (1) whether the K or X and C: or CE locus will be expressed,
and ( 2 ) which allele of the expressed locus will be activated. Possibly these two
decisions involve different mechanisms, therefore I will concentrate on allelic exclusion per se relegating to what is known as “ontogeny” the first decision as to
whether to express K or X and CE or C,’;.
A few attempts have been made to analyze this process.*G. 1 7 Although in 1968
I could discuss three possible classes of models, progress is such that two remain.
The first class e.g. the Ohno or Gally model, is that unireceptor antigen-sensitive
or antibody-secreting cells are in fact homozygous at the L and H loci and both
alleles are “on”. In other words, the progeny of a doubly heterozygous cell possessing
alleles LlL? and H1H2 at two unlinked loci, will be homozygous expressing all four
combinations L*L*H’Hl or L’LlHZH2 or LZHZHlH1 or L*LZH?H*. This is accomplished by random segregation of allelic chromosomes following, ar every divsion.

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tetrapolar spindle formation (Ohno model) or interchromosomal recombination
between the centromers and the L or H locus (Gally model). A dividing starting
population of doubly heterozygous cells would in five generations be over 95%
homozygous at both loci because heterozygotes segregate at each division homozygotes which then multiply to yield only homozygotes. If immunogenic selection
is imposed for a given LH pair, e.g. L1H2, which is complementary to that antigen,
then in five generations the doubly homozygous cell LlLlHZH2 would comprise over
95% of the population. The Ohno model predicts that the plasmacyte would be
homozygous at all loci while the Gally model creates homozygosity only for the
genes between the centromere and the L and H genes showing allelic exclusion. The
Ohno model is ruled out because plasmacytes express both alleles at the H-2 locus
for example. The Gally model would be ruled out if it would be demonstrated that
the Ir-1 locus, postulated to code for VE and C z undergoes allelic exclusion. Since the
Ir-1 locus is between the K and D ends of the H-2 locus Gally’s model would require
that one or the other end be rendered homozygous. While I believe both models to
be unlikely, the elegance of the idea that heterozygotes can be rendered homozygotes
will lead to many informative experiments. Suppose that a cloned plasrnacytoma
expressing two identical alleles L’L1 undergoes a mutation in one of them to create
the heterozygote expressing two different light chains, L1 and L2. This heterozygous
mutant cell undergoing the above processes of segregation would give rise to a clone
which would be a 1:1 mixture of homozygous L’LI and L2L2.
The second class are regulatory models which admit that only one of the two
alleles is transcribed. The building of a regulatory model is more than the simple
and frequent statement that translocation can occur in only one of the two chromosomes. A model of allelic exclusion must first account for the initial asymmetry
between the two allelic genes; then it must contain a mechanism which results in
one allele remaining on while the other remains off.
The asymmetry might arise because the allele first to complete the activation of
its subunit cistron (VC) produces a repressor which blocks the activation of its
mate. In other words, the process of turning off an allele is rapid compared to the
rate of activation of a VC gene. This is how I envisaged the process before experience increased my wisdom without reducing my folly.17 The competing model is
that a programmed operator constitutive Oc-event turns on one chromosome while
the other remains silent.10 The two models can be distinguished by the kinds of
experiments being carried out by Scharff (this monograph) and by ourselves
(Horibata, unpublished data), in which cloned cell lines are being treated with
mutagens and colchicine in order to isolate cells deregulated in this control mechanism. For example, suppose that the “on” chromosome were eliminated and a monosomy at one of the subunit loci (for L or H ) was isolated. One model 1 7 predicts
the automatic activation of the inactive allele which would be a germ-line gene
unselected upon somatically by antigen. The alternate model,IG that turning on
one allele is an Oc-event, predicts that the monosomy which has lost the “on”
chromosome will remain silent and not express its “off’ allele. This cell can now
be activated to express its germ-line genes by further mutation. These same kinds
of experiments should distinguish the two classes of models.

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TOLERANCE,
INDUCTION
AND ELICITATION
Now let us consider the antigen-dependent steps which operate in the soma.

1 will close by dealing with the relationship between the selective pressures o n
the germ-line V genes and the somatic derivatives of them, a problem to which
Jerne 45 so courageously addressed himself. Tw o conclusions based on
Gedanken experiments should be recalIed:l2
( 1) T h e self-nonself discrimination is the selective pressure driving antibody

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molecules to be specific. The counter selective pressure is that the greater the
specificity the more genes one needs to code for all of the antibodies required
to interact with all determinants. This tells us why antibody is neither a universal glue nor “infinitely” specific.
( 2 ) The self-nonself discrimination cannot be coded in the germ-line V
genes but must be learned or acquired. A most striking (nicht Gedunken)
experimental example of this is provided by Lee Herzenberg and associates
(this monograph). If antigen-sensitive cells expressing as receptors a given
immunoglobulin class are suppressed in early life, then this class never appears
in adult life. Since the animal generates cells of the suppressed class throughout life, some mechanism must exist to eliminate them. Herzenberg and coworkers (this monograph) postulate an autoimmune phenomenon in which
cells expressing as receptors immunoglobulin determinants of the suppressed
class are rejected by the immune system as foreign: hence, chronic suppression.
The animal had been tricked into treating self as nonself.
At this point, I will have to consider a precise model for tolerance and
induction. Bretscher and 1 1 2 have discussed a minimal model for the selfnonself discrimination, pointing out why any model must account for this in
terms of the past history of the animal. This is the meaning of the postulate
that the discrimination is learned. I am going to push on, supplying detail
beyond our original formulation stressing that the incorrectness of my present
guesses does not attack the original minimal formulation. I feel justified in
doing this because ( 1 ) there is no competing model that accounts for the
self-nonself distinction, and (2) I can relate most of the work we have discussed to one theoretical structure.
The two postulates of the minimal model are: ( 1 ) induction of antibody
formation normally requires the associated recognition of two determinants on
an antigen, one by the antibody receptor of the antigen-sensitive cell and the
other by a second antibody, in all likelihood thymus-derived IgT in the case
of the mouse, and ( 2 ) tolerance requires the recognition of only one determinant by the receptor on the antigen-sensitive cell.
Len Herzenberg and associates (this monograph) have shown us an important experiment in which the inductive responsiveness to an immunogen
is eliminated by specifically destroying the T cell population with anti-0 and
complement. This argues that thymus-derived antibody is required for induction, as we originally proposed.*Z One cannot conclude solely from the existence
of so-called thymus-independent antigens that T cells are “not a sine qua nod’
for induction of B c ~ I I s . ~ ~ ~ ~ ~
The precise model which I wish to consider is illustrated in FIGURE
1. The
tolerogenic interaction is a consequence of the interaction between a receptor on
the unipotential T or B antigen-sensitive cell and a determinant on the antigen.
The antibody-receptor is postulated to undergo a conformational change on
interacting via its combining site with an antigenic determinant. This change
is read by the cell as signal @ which leads to the death of that cell.
The inductive interaction normally requires associated recognition of antigen
by two receptors. The specific model (not minimal) proposed here
12 involves the participation of a third party effector cell (macrophage, reticulum
cell, dendritic cell, thymus cell, etc.) which derives its receptors passively as
cytophilic antibody (IgT) secreted by the induced T cell. This effector cell
carrying receptors of many different specificities can now interact with a uni-

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TOLERWENIC
INTERACTION

INDUCTIVE

INTERACTION

CYTOP

LIC

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HUYORAL
ANTI - a

FIGURE
1. A precise (not minimal) model of tolerance and induction.

potential antigen-sensitive cell of either T or B origin. T h e inductive interaction
is made up of two events: signal @ identical t o that of the tolerogenic interaction, and signal @ involving a communication between the effector cell and
the antigen-sensitive T or B cell. T h e tolerogenic signal @ is included in the
inductive interaction (signals '@ and @) t o assure that any cell which is
inducible can be tolerogenized, a consequence which is of key importance to
an understanding of the evolution of the immune system.

9. Sela (this monograph) has shown us that nonspecific charge effects can play
a major role in the response to an antigen. Positively charged antibody is elicited
by negatively charged antigens and vice versa. This should create no surprise, since
charge effects are the only long-range forces we know of in biology. If a determinant cannot interact with a combining site o n a receptor because of charge repulsion, then that antigen-sensitive cell can neither be tolerogenized or induced by that
antigen. There is no other possible result.
10. I would guess that the tolerogenic signal 1
0 involves the activation of
adenyl cyclase and is therefore mediated via CAMP. I make this guess ( 1 ) by
analogy, since. all known hormones which interact with a membrane-bound receptor,
mediate their action via CAMP, and (2) because of our findings (Epstein, unpublished data) that cloned cell lines of thymus-derived cells (carrying the e marker)
are killed by cAMP specifically. This proposal is to be compared with that of
Braun and colleagues 0 who consider cAMP as the mediator of an undefined inductive signal. Their very probing experiments do not yet permit dissection of the
cAMP effect in terms of signal @ or 0, but the direction these studies are taking
should do so.
11. I have guessed 2 1 that communication between the effector cells and the
antigen-sensitive T or B cell involves a chemical transmitter similar to that used
The effector
in chemical synaptic transmission in the nervous system (signal 0).
cell is signaled to release its transmitter by (1) a conformational change in any one
of its receptors possibly in an identical way to signal @, and (2) its contacting

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the membrane of the antigen-sensitive cell carrying bound receptors. Normally this
transmitter must act over a very short range and be short-lived, as in the nervous
system, in order to avoid nonspecific inductive events.
12. Unspecified in our theory at the moment (only because of the many arbitrary
solutions) are the number and ratios of doses of signal @ and signal @ required
to drive the T or B cell to tolerance or induction. Further, the number and ratios
might be different for T cells as against B cells. It should be clear, however, that
these two processes, tolerance and induction, compete at the cellular level (see
comment 18).
13. An asymmetry in the inductive interaction arises because thymus-derived
antibody (IgT) is required for both T and B cell induction. The reason that the
interaction of a B cell and B cell-derived antibody is not inductive comes from
a consideration of the functional roles of humoral and thymus-derived antibody
(IgT). The humoral antibody which protects against pathogens (viruses, bacteria,
neoplastic cells) must be made in large amounts and maintained at a high level.
The thymus-derived antibody (IgT) which regulates the self-nonself discrimination
must be maintained at a low level and turned over with appropriate rapidity. The
reason is that high levels of IgT would put the animal in danger of an autoimmune
reaction were it to encounter an antigen cross-reacting with a self-component. The
excess cytophilic IgT which is not fixed to an effector cells is postulated to be
rapidly eliminated and that which is fixed has an appropriate half-life.’? The
mechanism of regulation of the level of IgT must involve more than feedback
regulation by humoral antibody made by B cells.
Sela (this monograph) has given us an informative example of a specific response which is limited by the IgT concentration. A cell suspension from the spleen
of a rabbit primed with DNP-BSA responded in vitro to make anti-DNP when
exposed to BSA alone. Since some DNP-BSA must still have been present in the
in vitro system, exposure to the carrier protein (BSA) alone must have induced
the anti-carrier protein (IgT) to a level that made inductive encounters with the
DNP-carrier protein probable.
The switch from low zone tolerance to induction depends upon the threshold of
responsiveness of a cell to the numbers and ratios of signals @ and @ and upon
the level of IgT specific for the test antigen. High zone tolerance as a first approximation is independent of these factors. However, many subtleties are at play
here.I2 MollerGR argues that “tolerance in two zones of doses can be explained in
two basically different ways: ( 1 ) Each cell can distinguish signals for low zone
tolerance, immunity and high zone tolerance or (2) tolerance exists in only one
dose range and the two zones of tolerance depend upon induction of tolerance in
different cell populations.” Moller credits us with the first explanation, which I
would like to clarify by pointing out that the signal @ for low and high zone
tolerance under our model is the same. The transition from low zone tolerance to
induction depends upon kinetic factors.’? The point I wish to make now involves
the apparent contrast between the two explanations. Clearly, unresponsiveness
(tolerance is a misleading word) in the animal is due either to a lack of IgT or to
paralysis of the B cell itself, or both. The experiments cited by Moller involve
measurements of B cell response and are open to the two explanations above, except
that they are not mutually exclusive. Consider the T cell population only. Moller ex
argues that “T lymphocytes may have a lower threshold of immune triggering
[paralysis as well as induction] than B lymphocytes.” Therefore, these T cells can be
paralyzed at “low zone” or, to use Moller’s lang