The Take Home LESSON 1971

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THE TAKE-HOME

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

LESSON-197

1

*

Melvin Cohn

t

The Salk Institute

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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)

If antibody synthesis can be understood at the molecular level, a profound break- through might be achieved, not only with implications for medicine, but also for all aspects of the biology of multicellular organisms.

James D. Watson

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

(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.

THE EVENTS PRECEDING ANTIGENIC ENCOUNTERS

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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

V,

and 10-20 V, genes per haploid genome.

T h e V, genes are divided into two groups, V, and VA, which

are

unlinked to each other, T h e ratio of the numbers of V, to VA genes varies f r o m species t o species. In mouse this ratio is of the order of 30 (there being only

one

germ-line VA gene), whereas in man it is close t o one.

T h e VH genes are taking on a new look. There appear t o be two groups of them, unlinked t o each other. One group, familiar t o all of us, is expressed upon induction of bone marrow antigen-sensitive cells (B cells) t o produce humoral antibody. T h e 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.

i The work from my laboratory has been supported by United States Public Health Service grant R01 A105875 and United States Public Health Service Training Grant

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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 t o be immunologically mature when he expresses a

total of

2

1000 V,> and

2

1000 V, genes

or

2

los antibodies. This gives a n

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.

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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 esti-

mate of 106 antibodies many years ago based on a n 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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 VLVH gene combinations do code for as well as the other selection pressures on these V genes when I analyze the Jerne hypothesis.45

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97 1

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53

1

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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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. T h e following appear t o 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

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cis o n 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, c

lass 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 nomencla- ture. ( I would have preferred t o 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 thymus- derived 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 antigen-

independent step during ontogeny. Whether other classes, e.g.

Ce, C6,

GY,

are

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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, m a n ) . In the rabbit, which Pernis investigated, there might be only one Cy class defined by the a,,, aI2, a,,* and a I 5 genetic markers which are associated

with

>

90% of the IgG.

The

finding that the IgM to IgG switch requires a n immunogenic encounter raises many questions. Why switch? H o w 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.

T h e 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). F r o m 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 physio- logical properties it confers o n the antigen-sensitive cell (see comments 17 and 27).

6. The antigen-independent expression of

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

C p , and the postulated

CT,

appear reasonable at the moment. The assumption which has been challenged at this confer-

ence 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 a t 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

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533

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

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( 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). Re- sponder 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:

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( 1 ) The inability of the nonresponder to mount

another IgM response on secondary stimulation with (T,G)-A-L is due to the even- tual 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 hard- earned 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

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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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

C p class

uniquely, they can switch only once to any other C i class. Which class they switch 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

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Phenomenon, after its

discoverer. Further, it is established that VH is closely linked to CH, V, to C ,

and, by extension, V h to CA. The I ( , and two heavy chain gene loci are unlinked t o

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 allo- type (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 cell- one 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 proto- type for this class of models is X-bacteri~phage.~~~ 6 2

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

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The Take-Home

Lesson-1971

535

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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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

(FIGURE

7 in Refer-

ence 32; FIGURE 7 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 3 2

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

K

.

. . . Arg.Thr.Va1.A

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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

. . .

.

Mouse

K

. .

. . Arg.AIa.Asp.Ala.Ala.Pro.Thr.Val.Ser.1le.Phe.Pro.Pro.Ser. . .

.

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

. . .

.

In the case of human light chains, K and A,

. .

. .Ala.Ala.Pro.Ser.Val..

.

.has 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

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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 assump-

tion that

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 3 2 argue that since “some kind of

somatic recombination is required to carry out translocation, the simplest theory

would assume that a

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 nonhomolo- gous 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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 3 2 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 3 2 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 re- strictive that DNA-level joining has been understandably the only popular model.

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537

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 in- troducing this class of models is to remind us of the sad possibility that an examina- tion 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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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, dis- proof 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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 accom-

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538

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Sciences

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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 chromo- somes. 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 experi- ence 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.

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. T w o conclusions based

on

Gedanken experiments should be recalIed:l2

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Cohn:

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

The Take-Home Lesson-1971

539

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 uni- versal glue nor “infinitely” specific.

( 2 ) The self-nonself discrimination cannot be coded in the germ-line

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 self-

nonself 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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

( 1 ) there is

no

competing model that accounts for the self-nonself distinction, and (2)

I

can relate most of the work we have dis-

cussed 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 deter-

minant 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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

@ 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 in-

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540

TOLERWENIC

INTERACTION

Annals

New

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Academy of Sciences

INDUCTIVE INTERACTION

CYTOP

LIC THYYUS-

n n I n n

HUYORAL

[image:12.414.60.353.58.262.2]

ANTI - a

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 inter-

action, 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 repul- sion, 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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

who consider cAMP as the mediator of an undefined induc-

tive 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

in chemical synaptic transmission in the nervous system (signal

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

0).

The effector

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Cohn: The Take-Home Lesson-1971

54 1

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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

@ 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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

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 ap- proximation 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

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

B

lymphocytes.” Therefore, these T cells can be

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542

Annals New

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level. It is possible to wipe out low zone tolerance by making the number of doses

of the tolerogenic signal

@ needed t o silence a cell so large that low zone tolerance

merges into the intermediate zone of induction and is not seen. This is what Moller fis, 69 wants to do with B cells by setting their threshold for responsiveness

to signal @ (tolerance) high. However, for the T cell, setting the threshold low so

that “low zone” tolerance can occur before induction only emphasizes the three-zone phenomenon at the level of T cells and obscures the distinction between Moller’s 87

two explanations of the zones of tolerance.

It is argued, in order to explain self-tolerance, that “the very first cells to differentiate from stem cells to immunocompetent cells with immunoglobulin surface receptors have such a low threshold for triggering that any reaction with the relevant antigen results in tolerance.” ex This hypothesis, first stated in its general form by Lederberg,51 cannot account for the fact that the mature, inducible antigen-sensitive cells which emerge from the above postulated differentiation are also paralyzable (see Reference 12, footnote 7).

14. We have always favored l 1 , 12 the model of a multipotential effector cell-

unipotential antigen-sensitive T or B cell interaction over the unipotential T cell-B cell model because of three considerations: (1) The need to solve the problem of two rare antigen-sensitive cells having to find each other; (2) The paradox that a T

cell-B cell interaction (under our model) would lead to the tolerogenizing of the T cell and the induction of the B cell, since signal @ can only pass from T to B. Of course, a B cell-B cell “interaction” cannot be sensed by either cell and would lead to the tolerogenizing of both cells. ( 3 ) The desire to provide a way to prime responsiveness to immunogens during ontogeny.207 2 1

15. Associated recognition is the normal inductive interaction. I stress the word normal because it should be obvious that, given our theory, one could experimentally manipulate the system by providing signals @ and @ in various forms. For example, proper treatment with ‘‘CAMP’ alone (signal @) might nonspecifically wipe out the inductive response-“tolerance.” The combined administration of “CAMP” and the postulated chemical transmitter in high concentrations (signal @) might induce all antigen-sensitive cells. Since the responding antigen-sensitive T or B cell

can only know that associated recognition of antigen has occurred via signal

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

0,

any way of delivering this signal @ should convert the tolerogenic signal @ into an inductive interaction. This would be seen as an apparent bypassing of the

requirement for associated recognition; two examples 479

5 5 of this were pointed out

to me by Bretscher. Katz and coworkers47 use the fact that an animal primed with DNP-ovalbumin is not induced to make antibody to DNP when challenged with DNP-bovine gamma globulin. However, if signal @ is delivered via thymus- derived antibody (IgT) specific for the histocompatibility determinants on the anti- DNP B cells of the primed animal, then these cells can be induced to make anti- DNP. Signal @ is delivered via the B cell receptor-DNP-ovalbumin interaction, and signal @ via the effector cell carrying IgT receptors specific for histocompatibility determinants, thus leading to an inductive encounter. McCullagh55 uses T cells directed against the histocompatibility determinants of an animal unresponsive to SRBC in order to break the unresponsiveness. The animal is unresponsive because the level of IgT with specificity for SRBC is too low to induce the B cell. Here again, the anti-SRBC B and T cells are induced via IgT bound to the B and

T cells’ histocompatibility determinant (signal I@) and SRBC interacting with its receptor (signal

0).

Katz and coworkers 47 and McCullagh 55 attribute these

phenomena to products of the graft vs. host or cell-mediated killing reaction. This will likely prove wrong for cell cooperation via IgT and cell-mediated killing probably involve different thymus dependent systems. In any case, their experiments will be the first steps in proving that cell cooperation is not a mechanism primarily for concentrating or presenting antigen.

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Cohn:

The

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

Take-Home

Lesson-1971

543

It might be preferable to be formal and state that our theory contains the postulate that the inductive event consists obligatorily of two associated signals delivered simultaneously or sequentially, one via the receptor on the antigen-sensitive cell and the other via IgT. I am trying hard to prevent misinterpretations of our term “associated recognition” so that confusion reigns when we use this idea to account for the self-nonself discrimination (for an example, see discussion by R.

W.

Dutton

in Reference 92).

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

16. Following Mitchison,o*v 6 3 most immunologists treat the phenomenon of as-

sociated recognition of antigen as an enhancing or concentrating device and insist that it is not obligatory to induction. Rather, they treat cell cooperation as a fine- tuning regulatory device superimposed on the unknown mechanism of the self- nonself discrimination.

Taylor concludes that “the specific participation of thymus-derived cells in antibody responses is not mandatory, and probably consists in an antigen-handling function.” Somewhat later, Taylor and Iverson 91 repeat their position that “facilita-

tion is most simply interpreted as the action of IgT in

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

trapping antigen so as t o

form a local concentration at some critical site.”

Miller 5 7 sums up his position by stating that “the thymus is not essential for humoral antibody production in most cases” and that IgT acts on “weak determinants” which have “to be concentrated, as it were, on the antigen-sensitive cell by means of some mechanism which involves recognition of other determinants on the carrier molecule.”

Edelman 29 argues that

“. . .

the antigen-frapping step is not simply a n encounter

between an ‘antigen-gas’ and a ‘cell-gas’ but more likely depends on special mechanisms that have evolved to ensure efficiency of encouter.”

Moller 1;: thinks that “it is necessary to postulate either a very efficient focussing

mechanism of both antigen and cells, or a magnifying mechanism which is specific.”

A year later Mollerc‘ concludes “that cellular cooperation is not fundamental for antibody production in bone marrow lymphocytes. More likely, cooperation repre- sents a helper function in the induction of immunity.”

Mitchison“5 has always stressed the importance of low affinity as against high affinity antibody. “From the molecular point of view” cell cooperation or associated recognition “may be trivial, but not necessarily from a biological one. Cooperation

apparently concentrates antigen so that doses which would otherwise be too small

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

to

provoke an immune response do so.”

These formulations ignore the problem of how the distinction between tolerance and induction is made, as well as why there is an induction peak bounded by two tolerance zones (see comment 13). They d o not make “cell cooperation trivial.” They make it a side issue. Further, they predict that the concentrating device would be best seen for low zone tolerance where in fact several workers have postulated carrier effects, still without attempting t o use even this postulate to provide a way to determine whether the response will be that of tolerance or induction.

Miller and Mitchell 5 8 state their hypothesis bluntly:

“. . .

the induction of toler-

ance, like the induction of antibody formation” may require “the collaboration of thymus-derived cells.”

Mitchison‘” creates a dilemma by stating that “carrier effects occur in the induction of the secondary response, in the induction of immunological tolerance and in eliciting delayed hypersensitivity” (see comment 20). Having suggested two roles of cell cooperation (tolerance and induction GS), Mitchison must now tell us how

the cell cooperation mechanism, upon encountering a low dose of antigen, decides whether to mediate tolerance 64 or induction.65

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quences of an understanding of the mechanism of tolerance are enormous. Examples

of this position are as follows:

“.

. .

we assume that acquired immunological toler-

ance may be considered as the result of the process of terminal exhaustive differentiation

. . .

into short-lived antibody-producing cells.

. .

.”a‘

“It is conceivable that absence of repeated (antigenic) stimuli after an initial triggering (i.e., induction of antibody response) can lead to an abortive response and

to ‘low zone’ tolerance.”

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

14

“. . .

a small number of antigen molecules might stimulate the production of larger numbers of high-affinity antibodies which could be brought as antigen-antibody complexes to those cells committed t o some antigenic determinant on the antigen.

Such a complex might then inactivate those cells” [J. A. Gally, cited in Reference

271.

“. . .

you can produce tolerance specific for the hapten

. .

.

but it only can be

induced by substances that are immunogenic.”

5

Feldmann (discussion of his work with Diener in Reference 92) has shown that antibody to monomeric or polymeric flagellin greatly enhances the establishment of

the unresponsive state of an

zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA

in vitro culture system to specific immunogenic chal-

lenge by flagellin. He believes that his experiments bear on the general mechanism of tolerance (see comment 21) which he postulates to be humoral antibody- mediated. However, I am wary of any in vifro system for studying tolerance in which it is found that only the best in vivo immunogens such as polymerized flagellin can mediate the unresponsive state. I think it reasonable to point out that one must give reasons for believing that the mechanism for establishing unresponsiveness in vitro is the same as the normal mechanism in vivo, particularly in view of the above discrepancy.

Azar and Good,3 by proposing that tolerance is complement-mediated, imply that induction of antibody precedes tolerance (see comment 19). I view the Feldmann- Diener and Azar-Good phenomena as examples of ways of inactivating specifically antigen-sensitive cells by means of antigen-antibody complexes. In the Feldmann- Diener case, the complexes trap the cell in a cage and block an immunogenic encounter or gather together surface receptors into one island which is endocytosed, thereby leaving the antigen-sensitive cell temporarily without receptors, whereas in the Azar-Good case, complement activated by antigen-antibody complexes could be brought to the antigen-sensitive cell by fixation of the complex on the cell via the receptor with resultant killing of that cell. These phenomena may be very important examples of how antigen-antibody complexes inactivate antigen-sensitive and effector cells. This process, critical to problems of immune surveillance, is a side reaction as far as tolerance and