IMMUNOLOGICAL COMMUNICATIONS, 5 ( 4 ) , 281-301 (1976)
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EVOLUTIONARY AND DEVELOPMENTAL ASPECTS O F T-CELL RECOGNITION Gregory W. W a r r , Janet M. Decker a n d John J. Marchalonis Laboratory of Molecular Immunology, The Walter and Eliza Hall Institute of Medical Research, P. O., Royal Melbourne Hospital, Victoria 3050, Australia
ABSTRACT Studies relating to the nature of the antigen-specific T-cell receptor a r e reviewed in the light of present knowledge of phylogenetic and ontogenetic development. It i s suggested that this evidence supports the concept that immunoglobulin (Ig) is the T-cell receptor, and that the following conclusions may be tentatively drawn. 1) T cell membrane Ig differs in its physical and functional properties from that on the B cell membrane. 2 ) The divergence between the T and B c e l l surface Ig molecules was apparent a t the time of emergence of the ancestors of modern fish. 3 ) Specific T cell recognition in the form of antigen binding appears e a r l y in ontogeny, and is blocked by antisera to Ig.
The nature of the antigen-specific surface receptor of T lymphocytes has generated much interest and debate.
The obvious can-
didate f o r this receptor is immunoglobulin (Ig) which possesses variable region combining s i t e s f o r antigen s i m i l a r to those of s e r u m Ig and B cell surface Igs (1, 2).
Controversy has surrounded attempts to estab-
lish this hypothesis because, in a formal sense at least, positive
-
attempts to demonstrate ( 1 5 ) o r isolate ( 6
- 10) surface Ig-like mole-
cules of T cells could be countered by negative reports of workers using "similar" (but not identical) experimental approaches ( 1 1 - 14).
Studies
281 Copyright 0 1976 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
282
WARR, DECKER, AND MARCHALONIS
from a number of laboratories now document the conclusion that murine T cells express a type of surface Ig termed IgM(T) o r IgT, which is distinct from the 7s IgM and IgD-like molecules isolated from the B cell surface ( 2 , 7
- 10,
15
- 20).
Evidence showing that this T-cell
Ig is synthesized by T cells and not by contaminating B cells o r plasma-
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cytes w a s obtained using monoclonal T lymphoma cells (15, 21, 2 2 ) characterized by s t r i c t T cell phenotypes.
Moreover, it has now been
established in some cases that T cells and B cells possess surface receptors for antigen which bear the same variable ( V ) region idiotypes a s do circulating antibodies directed against the s a m e antigen ( 2 3 - 2 5 ) . We believe that much of the difficulty reported in the isolation of T cell Ig by some workers ( 1 1, 1 2 ) s t e m s from the use of detergent extraction procedures shown by other workers to be unsuitable for isolation of T cell Ig (10, 2 6 ) .
This work has been reviewed elsewhere (2, 27, 28),
and w i l l not be reiterated h e r e except insomuch a s it impinges upon the phylogenetic and developmental results under consideration. The c r i t e r i a which a r e classically considered necessary for the demonstration of a true immune phenomenon a r e specificity of the evoked response, and an altered level of reactivity to subsequent antigenic exposure.
Although apparently all vertebrate c l a s s e s show
-
immune responsiveness of this type ( 2 9 31), the bulk of o u r knowledge of the immune system comes from intensive investigations of a few species, namely, man, the laboratory rodents, and to a l e s s e r extent chickens, and the comparative aspects of the generation and expression of immune competence have been unduly neglected.
This is, in o u r
opinion, unfortunate, since i t is through an investigation of the prototypical and universal features of immunity that we may eventually hope to achieve a comprehensive understanding of the subject, and be able to place in perspective the overwhelming amount of often contradictory data which abounds in current cellular immunology. The present article represents an attempt in this spirit to r e view the current state of our knowledge regarding the nature, evolution and ont ogenetic emergence of receptors on antigen-specific T - c e l l s .
T-CELL RECOGNITION
283
The salient conclusions arising from phylogenetic considerations a r e that ( a ) all vertebrates possess cells which exhibit specific T c e l l functions, (b) antigen-specific T cells bearing Ig receptors appear e a r l y in ontogeny of mammalian species, and ( c ) virtually all lymphocytes of lower species, including thymus lymphocytes, express and synthesize
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readily detectable surface Ig.
Furthermore, recent evidence supports
that hypothesis that surface Igs of T and B cells r e p r e s e n t distinct molecules which diverged from a common p r e c u r s o r e a r l y in vertebrate evolution.
Phylogenetic Origins of T Cell Functions Lymphocytes in certain birds, mice and other mammals have been subdivided, on the basis of origin, function and surface m a r k e r s , into thymus-derived T cells, and bursa (or bursa1 equivalent) derived
B cells (32, 33).
The B cells a r e the p r e c u r s o r s of antibody-secreting
cells and bear l a r g e amounts of readily detectable membrane immunoglobulin.
The T cells appear to be m o r e heterogeneous in function than
B cells, being involved in the phenomena of cell-mediated immunity
(CMI) such a s graft rejection, delayed-type hypersensitivity, graftversus-host reaction (GVHR). and the in vitro mixed lymphocyte r e a c tion (MLC), and also being implicated in collaborating with B cells to bring about efficient antibody production by the latter.
In considering
the nature of the T c e l l receptor, i t is a s well to b e a r in mind that the heterogeneity of c l a s s e s of T cells subserving various functions may be reflected in a heterogeneity of receptors.
Since the t r u e immunological
nature of GVHR and MLR phenomena has been questioned (31, 34), we will confine o u r attention to the recognition of classical foreign antigens and strong histocompatibility antigens. It is c l e a r (Table 1) that the thymus plays a central role in the generation of immune competence in teleost fish (35), amphibians (36, 37), birds and mammals (32, 33), and that lymphocytes of lower vertebrates show responses typical of T cells, e. g. graft rejection (38). MIF pro-
?
- ve + ve + ve + ve
+ ve
+ ve
+ ve
+ ve
REPTILIA
AVES
MAMMALIA ( o r d e r Rodentia
( o r d e r Artiodactyla)
?
-
ve
+ ve
+ ve
?
+ ve
+ ve
+ ve
?
?
Depression of Immunity by Thymec tomy*
+ ve
- ve
-
+ ve
+ ve + ve
+ ve ive
ve
- ve
-
?
- ve - ve ?
+ ve
MLR or GVH Reactivity
?
+ ve(larva1)
+ ve
+ ve
+ ve
?
Readily detectable Ig on Thymo cytes
All extant vertebrates show classical antibody production. This table w a s compiled from data in refs. 32, 35, 36, 38, 40 48, 53, 5 4 D 88 95. * Thymectomy a t an e a r l y developmental stage.
?
+ ve
+ ve
+ ve
+ ve
( o r d e r Anura)
+ ve
- ve
+ ve
?
?
~~
Carrier effect in AB production
+ ve + ve
-
+ ve
+ ve
OSTEICHTHYES ( s u p e r o r d e r Teleostii)
AMPHIBIA ( o r d e r Urodela)
-
+ ve
CHONDRICHTHYES ve
- ve
+ ve
AGNATHA
I
Graft Rejection
r
Class
Presence of strong H-Antigens
STATUS O F IMMUNE RESPONSIVENESS IN VERTEBRATE CLASSES
TABLE I
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205
T-CELL RECOGNITION
duction, MLR (in anuran amphibians, 35), responsiveness to c e r t a i n mitogens ( 3 9 ) . GVH reactions (40), and a c a r r i e r effect on secondary challenge ( 4 1 - 4 4 ) .
However, the inference that T and B c e l l s con-
stitute distinct populations of lymphocytes in all v e r t e b r a t e s p e c i e s can only be drawn indirectly from this evidence, a s appropriate c e l l m a r k e r s
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and cell separation techniques a r e not available,
However, t h e r e
e m e r g e s a consistent phylogenetic picture suggesting that all v e r t e brates show responses typical of those mediated by T and B c e l l s in the higher vertebrates (Table 1) even in those animals such a s the agnathan hagfish which do not p o s s e s s organized lymphoid t i s s u e ( 3 5 , 38).
T h e possibility that T and B c h a r a c t e r i s t i c s a r e s h a r e d by
a single c l a s s of lymphocytes is intriguing, but a number of observations suggest that t h e r e is a distinct and apparently universal division of function,
The possibility that T and B c e l l functions a r e e x p r e s s e d
by one c e l l type in lower species might be supported by observations which demonstrate that thymocytes in fish ( 4 5 - 4 7 ) and l a r v a l amphibians ( 4 8 ) b e a r readily detectable, endogenously synthesised m e m b r a n e Ig.
This property is generally considered a B c e l l c h a r a c t e r i s t i c in
m a m m a l s and birds; however, demonstration of binding of anti-Ig to thymus ( 4 , 18, 49) and T lymphoma c e l l s (22, 50) r a i s e s questions r e garding the stringency of this criterion.
Antibody production c a n occur
in the thymus of certain fish (51), although all this may suggest is that a s m a l l number of B c e l l s occur in f i s h thymus, as they do in mouse thymus.
The following evidence can be marshalled in support of the
idea that T and B cells a r e functionally distinct populations throughout v e r t e b r a t e phylogeny: (a) In a l a r v a l amphibian (37), thymus, but not spleen, is essential to the reconstitution of graft rejection,
(b)Lympho-
cyte differentiation into functional subgroups has o c c u r r e d in fish, because subpopulations of lymphocytes reactive to c e r t a i n mitogens a r e demonstrable (39).
( c ) In the goldfish, the membrane of thymocytes
d i f f e r s in s e v e r a l physical r e s p e c t s f r o m that of splenocytes (47).
In
particular, goldfish thymus lymphocytes r e s e m b l e those of m i c e i n that u s e of the nonionic detergent Nonidet P - 4 0 under conditions routinely
286
WARR, DECKER, AND MARCHALONIS
used for mouse B cells ( 1 1, 26), does not allow fully efficient extraction of Ig.
By contrast, "solubilization" of goldfish spleen lymphocytes
under conditions used f o r mouse B cells allows surface Ig to be precipitated in good yield.
Since surface iodination and various solubilization
conditions will be used increasingly in attempts to characterize mem-
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brane receptors and m a r k e r s of lymphocytes of lower species, we would caution the reader that techniques which w o r k satisfactorily for a subpopulation of mammalian lymphocytes cannot be expected to be directly applicable to cells of other subpopulations within a species or to cells of other species.
As an illustration, the Nonidet P - 4 0 approach w a s
not suitable f o r the isolation of surface Ig of chicken B cells ( 5 2 ) . Surface Immunoglobulins of Lymphocytes of Lower Species Data collated in Table 1 show that surface Ig is demonstrable by immunofluorescence on thymus lymphocytes of adult elasmobranchs (45), teleosts (46. 47), urodele amphibians ( 5 3 ) and larval anuran am-
phibians (48).
Moreover, studies involving shedding and reappearance
provided evidence 'that the thymus lymphocyte populations were capable of synthesizing this surface receptor.
This point might prove of
crucial importance because species such a s rays and urodeles a r e thought to lack strong histocompatibility antigens (381, but, nevertheless, c a r r y out antigen-specific T cell functions and bear surface Ig. The presence of readily detectable surface Ig on thymus lymphocytes of lower species provides a s h a r p contrast with the result usually obtained in studies of adult mice (1, 2, 54).
It is worthwhile
considering possible explanations for this discrepancy.
One obvious
possibility is that thymus lymphocytes of lower vertebrates and mammals differ in the number of Ig molecules expressed on the membrane. Another explanation might involve the u s e of antisera made in rabbits o r other mammals to investigate Igs of other mammals.
Since
mammals evolved relatively recently and their Igs show extensive crossreactions, it is possible that antibodies made in another vertebrate class, e. g. chickens, might possess stronger reactivities for minor
T-CELL
287
RECOGNITION
d e t e r m i n a n t s , p a r t i c u l a r l y t h o s e of V - r e g i o n s and t h e F d f r a g m e n t of the h e a v y chain.
Consequently, non- m a m m a l i a n a n t i b o d i e s m i g h t
s e r v e a s b e t t e r p r o b e s for t h e d e m o n s t r a t i o n of cell s u r f a c e I g s of m a m m a l i a n c e l l s than do antibodies m a d e in c l o s e l y - r e l a t e d s p e c i e s . C o n s i s t e n t with t h i s notion a r e the o b s e r v a t i o n s t h a t a n t i s e r a m a d e in
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c h i c k e n s to p u r i f i e d m o u s e o r human Igs will bind to m o u s e t h y m u s (18) and human t h y m u s lymphocytes (49), r e s p e c t i v e l y .
A third possible
f a c t o r i s that s u r f a c e Ig of m o u s e T c e l l s m i g h t b e "buried" with o n l y i t s VH-VL combining s i t e exposed (55, 56), w h e r e a s m o s t of t h e H-chain c o n s t a n t r e g i o n i s e x p o s e d on B c e l l s ( 5 7 ) .
T h e s e e x p l a n a t i o n s are not
m u t u a l l y e x c l u s i v e and d a t a to be c o n s i d e r e d below s u g g e s t t h a t s u r f a c e Igs of T and B c e l l s p o s s e s s d i f f e r e n t H-chain c o n s t a n t r e g i o n s ( 1 5 - 17,
47) and consequently m a n i f e s t a n u m b e r of functional ( 5 8 - 60) a n d p h y s i c a l d i f f e r e n c e s ( 1 6 , 47, 61). A meaningful r o l e f o r s u r f a c e Ig of l y m p h o c y t e s of l o w e r s p e c i e s is indicated by s t u d i e s which show that all s p e c i f i c antigenbinding lymphocytes of a n u r a n a m p h i b i a n s ( 6 2 ) including p u t a t i v e " c a r r i e r - s p e c i f i c ' ' h e l p e r c e l l s ( 6 2 ) a r e inhibited by a n t i s e r a d i r e c t e d a g a i n s t l i g h t c h a i n s and p c h a i n s .
Antigen s p e c i f i c functions of T cells
of c h i c k e n s ( 6 3 ) and m i c e (64, 65) h a v e a l s o been inhibited by a n t i s e r a to Igs, p a r t i c u l a r l y t h o s e p o s s e s s i n g a n t i b o d i e s t o light c h a i n s , A t t h i s time, s u r f a c e Ig h a s been i s o l a t e d f r o m l y m p h o c y t e s of only one l o w e r s p e c i e s , v i z . , t h e goldfish ( C a r a s s i u s a u r a t u s ) , a teleost.
T h i s s p e c i e s , l i k e o t h e r c y p r i n o i d s (66, 67), p o s s e s s e s o n l y
o n e s e r u m Ig, a t e t r a m e r i c IgM molecule.
Rabbit a n t i b o d i e s s p e c i f i c
f o r this s e r u m Ig p r e c i p i t a t e d s i g n i f i c a n t c o u n t s i n '251-labelled s u r f a c e p r o t e i n s f r o m both goldfish t h y m u s a n d s p l e e n l y m p h o c y t e s ( T a b l e 2). T a b l e 2 a l s o i l l u s t r a t e s t h a t Ig of goldfish t h y m u s l y m p h o c y t e s , l i k e that of m o u s e T l y m p h o m a c e l l s a n d t h y m u s l y m p h o c y t e s , is n o t a d e quately e x t r a c t e d by u s e of Nonidet P - 4 0 u n d e r m o u s e B cell conditions. A distinction between s u r f a c e I g s of goldfish t h y m u s a n d s p l e e n l y m p h o c y t e s is o b s e r v e d when polypeptide c h a i n s of t h e s e I g s are c o m -
WARR, DECKER, AND MARCWONIS
288
TABLE I1 EFFECT O F EXTRACTION CONDITIONS UPON ISOLATION O F SURFACE IG
I 2 Macromolecular ~~~~
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Lymphocyte Source
Extraction Procedure
~
' %-counts Precipitate(
L Specifically Precipitated
GOLDFISH Spleen
1yo N P 40
3.1
Thymus
1% NP40
0.5
Thymus
Metabolic release
4.5
1.5
3.0
CBA Thymus
0. 570 N P 4 0
0.3
0.2
0.1
nu/nu Spleen
0. 5% NP40
3.6
0.4
3.2
CBA Thymus
Acid Urea
1.7
0.3
1.4
nu/nu Spleen
Acid Urea
1.4
0.3
1.1
WEHI 22
1% NP40
0.1
0.1
0
WEHI 22
1% N P 401
1.2
0.1
1.1
MOUSE
T LYMPHOMA
6M Urea WEHI 22
Acid Urea
6. 1
1.7
4.4
WEHI 22
Metabolic release
2.9
0.8
2.1
In all experiments, lymphocytes were surface labelled with 1251in a lactoperoxidase-catalysed reaction. Details of experimental techniques employed w i l l be found in refs, 10, 26, 47, from which some of these data were taken. pared by polyacrylamide gel electrophoresis in SDS-containing buffers under conditions which provide high resolution of proteins in range of apparent mass, 40,000
- 90,000
daltons.
Most importantly, the Ig
heavy chain shows a distinctly f a s t e r mobility in gel electrophoresis than that of splenocyte Ig heavy chains.
This difference is illustrated
T-CELL RECOGNITION
289
in Fig. 1, and i t is significant that analogous differences in the mobility of splenocyte and thymocyte Ig heavy chains have been described f o r the mouse (15, 16).
Fig. 2 presents a composite diagram based upon
studies which have compared the mobilities on SDS- PAGE of s u r f a c e p and
& -like chains of
B cells with heavy chains of surface Igs of thymus
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( 16 ) and T lymphoma cells ( 1 7).
Surface Ig of in vitro grown murine
fetal thymus anlagen ( 6 8 ; Haustein, D. and Mandel, T. E.
in p r e p a r a -
tion) also possesses a heavy chain which is slightly, but significantly, f a s t e r than p chain.
i
FIGURE 1.
A COMPARISON O F THE SURFACE IMMUNOGLOBULINS O F SPLEEN AND THYMUS LYMPHOCYTES O F THE GOLDFISH ) and Polypeptide chains of Ig from i251-surface labelled spleen -( thymus ( - - - - - ) lymphocytes were analysed by SDS-polyacrylamide gel electrophoresis after reduction with mercaptoethanol. It can be seen that the Ig from both c e l l s dissociated into light (L)chains and heavy chains. Whereas the heavy chain of the splenocyte Ig migrated like a t r u e p chain, the thymocyte Ig heavy chain showed a significantly f a s t e r Positions of standard murine and human p, y and L chain mobility. m a r k e r s a r e indicated by a r r o w s ( & ). Both thymus and spleen lymphocytes show a polypeptide migrating f a s t e r than mammalian y chain, and which appears to be analogous to the molecule described in mammalian lymphocytes which has affinity f o r antigen-antibody complexes (See a l s o Fig. 2 ) .
W A R R , DECKER, AND M A R C W O N I S
290
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I
EL
6 1 j
c cr
I
DISTANCE MIGRATED
L
c
-
FIGURE 2. A COMPARISON O F THE SURFACE IMMUNOGLOBULINS O F MURINE T CELLS AND SPLENOCYTES Polypeptide chains of Ig from 1251-surface labelled spleen -( ) and T ( - - - - - ) lymphocytes analysed after reduction with mercaptoethanol, The positions of standard by SDS-polyacrylamide gel electrophoresis. murine and human p, y and light (L)chain m a r k e r s a r e indicated by a r r o w s ( & ). Whereas the splenocytes show a light chain and two heavy chains (one a true p, the other designated 6 '-like (9 6 ) and marked ( I ) as such in the figure, although i t does not migrate like a human 6 chain, ref. 971, the T cell Ig resolves into a light ( L ) chain and a heavy chain with a mobility distinctly f a s t e r than p. Both T and spleen c e l l patterns show a polypeptide chain migrating in a position intermediate between y and L chains, This molecule has the property of binding to antigen-antibody complexes. The data shown in this figure is a composite representation of that presented elsewhere (15, 16, 68).
Although mobility in SDS gels should not be taken as a s o l e criterion for heavy chain homology, these r e s u l t s suggest that the p-like heavy chain of T cells is physically distinct from that of B cells and that this distinction a r o s e (unless we a r e dealing with convergent evolution) in or p r i o r to the divergence of the a n c e s t o r s of modern teleosts and mammals.
Quite clearly, further comparative studies
w i l l be needed to confirm this hypothesis, and to determine whether or
T-CELL RECOGNITION
291
not the T cell Ig heavy chain differs from p chain i n some minor aspect (e. g. carbohydrate content) o r whether the differences lie in the p r i m a r y protein s t r u c t u r e of the molecule. Ontogeny of T Cell Recognition
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The mammalian immune system is relatively immunoincompetent at birth.
Stimulation with a foreign antigen will elicit a t best only
a weak antibody o r cellular immune response, while the induction of tolerance in neonatal life is relatively easy(69 - 71).
Nevertheless, the
ability to recognize antigen, as measured by specific antigen binding to lymphocytes, appears rather early in the development of the lymphoid system during ontogeny.
Antigen- binding lymphocytes have been
detected in the thymus at 14 days gestation in the mouse and chicken, 20 days in the rat, and 12 weeks in the human ( 7 2 - 74).
The frequency
of binding cells in the thymus is high when f i r s t detected, often close to one percent of lymphocytes, and declines during gestation and childhood,
This decrease in frequency might be explained by a change in
the cell populations comprising the thymus ( f o r example, a dilution of the binding population by a non-binding one), o r by a decrease in the amount of receptor Ig p e r binding cell.
In all c a s e s antigen binding
can be inhibited with antisera specific for immunoglobulin p and kappa .chains (72, 74, 75). Although the newborn mammal i s relatively immunoincompetent, there have been s e v e r a l reports of T c e l l function detectable a t birth o r even before.
In the mouse, helper cells a r e present in the thymus at
48 hours after birth (76), and GVH reactivity has been measured in yolk s a c cells a t 9 days gestation (77).
Silverstein has demonstrated helper
activity in the fetal lamb, which is able to make s o m e antibody responses in utero (78). In general, conditions which demonstrate the presence of s u r face Ig on B cells by autoradiography will not detect Ig on T cells, although a high percentage of thymus c e l l s from adult mice can be shown to have Ig when m o r e sensitive conditions are used ( 3 ) .
It has been
29 2
WARR, DECKER, AND M A R C W O N I S
shown, however, that approximately 20% of thymocytes from young children do have surface Ig (49).
The facts that only a subpopulation
of cells show anti-Ig binding and that not all anti-Ig s e r a bind to thymocytes argue against passive-acquisition of Ig by the cells.
Moreover,
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of in vitro grown murine fetal thymus anlagen ( 6 8 ; Haustein, D. and
Mandel, T. E., in preparation). Discussion On the basis of the evidence available a t present, we therefore incline to the view that lymphocytes exist, a s low in the phylogenetic o r d e r a s bony fish, which show distinct homologies, at both a functional and a molecular level, with the T cells of mammals.
If this view is
to be accepted, i t implies that the thymocyte Ig, readily detectable by immunofluorescence in fish ( 4 5 - 47), has become, by the phylogenetic level of the birds and mammals, less readily detectable.
The obser-
vations that larval amphibia have readily detectable Ig on their thymocytes, which becomes more difficult to detect during development (48), a r e readily compatible with this hypothesis.
Possibly a parallel situa-
tion might obtain for fetal and neonatal mammalian thymus lymphocytes. If this interpretation of the comparative and developmental studies of thymocyte Ig is correct, several questions remain to be answered. Firstly, the teleological question: why should the display of T cell membrane Ig change?
Secondly, do T cells possess receptors, other
than Ig. that may, for example, be expressed in a reciprocal relationship?
In answer to the f i r s t question, the most attractive hypothesis is that the great bulk of membrane Ig on, f o r example, fish thymocytes and all B cells, is functionally redundant.
Circulating human lympho-
cytes possess only about 2000 membrane bound receptors for insulin
(79), so why should mammalian B cells require 80,000 o r more membrane Ig receptor molecules (1, 8 0 ) ?
What distinguishes the Ig mole-
cule from the insulin receptor is the capacity of the lymphocyte to syn-
T-CELL RECOGNITION
293
thesize and s e c r e t e in response to stimulation l a r g e numbers of molecules closely related to i t s membrane Ig receptors.
This l a r g e quantity
of membrane Ig may merely represent a by-product of p r e s e c r e t o r y activity of the protein synthetic apparatus of the lymphocyte, with very few of the molecules being involved in the process of triggering.
As
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f a r a s we a r e aware, no one has yet investigated this important question,
It is c l e a r that the antibody s e c r e t o r y apparatus becomes m o r e complex and diversified with movement up the phylogenetic scale, with appearance of plasma cells (30). increasing complexity of lymphoid t i s s u e s ( 3 5 ) and diversity of Ig c l a s s e s ( 2 9 , 30).
With this increasing complexity of the
B cell Ig s e c r e t o r y system, i t is possible that T cells would not have
been subjected to selective p r e s s u r e to retain superfluous membrane Ig since the function of mammalian T cells is clearly not to s e c r e t e l a r g e amounts of Ig.
The notion that some thymocytes a r e capable of
antibody secretion is however, supported by observations on the fish thymus, and there appears to be an interesting intermediate s t a g e in the g r a s s frog ( 6 2 ) , where i t has been reported that the thymus can respond to immunization with an increasing number of antigen-binding cells, but that these c e l l s a r e non-secretors of antibody. The dispute over the existence of Ig on mammalian T c e l l s has been argued for many years, but a t the r i s k of boring the r e a d e r we feel i t is worth while summarizing what we consider to be the p r e s e n t state of the field.
The evidence relating to the detection of T c e l l Ig
has recently been reviewed in detail (21, and although many investigat o r s have reported positive results (4-10, 1 5 - 22), i t is c l e a r that s o m e workers have experienced difficulties.
In o u r opinion, these difficul-
ties s t e m f r o m the facts that ( a ) Ig in the membrane of T c e l l s is obviously not readily accessible to anti- Ig reagents, ( b ) moreover, the complication a r i s e s that readily detectable surface Ig of T cells is often of passive nature, ( c ) Membrane Ig in T cells is not satisfactorily ex-
tracted by all methods used f o r B c e l l s (10, 17, 26).
Some workers,
in addition, consider that Ig is present in the membrane of thymocytes in relatively s m a l l amounts ( 1 , 81), although no assumption-tree methods
WARR, DECKER, AND MARCHALONIS
294
presently exist for the accurate quantitation of membrane molecules which a r e neither completely accessible to binding reagents nor completely extractable under all "solubilization" conditions.
The recent
phylogenetic evidence establishes that a l l lymphocytes of lower species express surface Ig which is easily detected using antisera specific for
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s e r u m IgM. The second question posed by the comparative studies, i. e . , is it possible that phylogenetic development has seen the partial replace-
ment of Ig by another receptor molecule? is worthy of serious consideration.
It is c l e a r that the plasma membrane of lymphocytes b e a r s a
range of receptors ( f o r hormones, antigens, Fc, heterologous erythrocytes, lectins), but it is difficult to s e e why the T cells should bear two distinct types of antigen specific receptors both of which would bear the V region of the Ig molecule (23
- 25).
While a DNA translocation model
f o r the sharing of V regions by the various C regions, quite probably linked to each other (82). has been proposed (821, it is l e s s likely that translocation between the unlinked V region genes and major histocompatibility complex ('MHC) genes ( 8 2 ) is a feasible molecular mechanism. The majority of published reports on the role of MHC specified cell surface antigens suggest that they play a role in cell collaboration, rather than mediating specific recognition (83
, but
c. f. 8 4 ).
Many animals appear to have some powers of discriminating self from non-self, a capacity which manifests itself in such phenomena
as incompatibility reactions in non-vertebrate groups like c o r a l s (85) and tunicates (86).
Such recognition does not appear, from o u r present
knowledge, to possess the characteristics of a true immune response, and while such "quasi-immune" phenomena may have persisted in the vertebrates, it is unclear to what extent they contribute to cell-mediated immunity o r a r e related to reactions against strong histocompatibility antigens.
Certainly all fish s e e m to lack MLR and elasmobranchs do
not show rapid first-set graft rejection as is also the c a s e in urodele amphibians (38).
However, this apparent lack of major histocompati-
bility complex antigens does not prevent these animals f r o m developing
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T-CELL RECOGNITION
295
WARR, DECKER, AND MARCHALONIS
296
adequate immune responsiveness c h a r a c t e r i s t i c in o t h e r r e s p e c t s of T and B c e l l function.
While i t is unwise to draw conclusions f r o m nega-
tive experiments, we do not feel that phylogenetic studies support the view that antigen recognition by T c e l l s is due in any g r e a t m e a s u r e to c e l l s u r f a c e components specified by the m a j o r histocompatibility complex.
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The recent phylogenetic and ontogenetic data reviewed here, taken in conjunction with c u r r e n t investigations of the p r o p e r t i e s of s u r face Igs of murine T cells,indicate that (a) Ig of T c e l l s is distinct f r o m the surface IgM and IgD-like molecules of B cells, and ( b ) the genes specifying heavy chains of IgM(T) o r IgT probably diverged e a r l y in v e r tebrate evolution from those encoding the p and
d
chains.
T h e s e con-
cepts a r e depicted in Fig. 3 which gives a schematic representation of the emergence of genes specifying p chains and lymphocyte s u r f a c e H-chains.
This model proposes that the pT and pB genes diverged
p r i o r to the divergence of the a n c e s t o r s of m a m m a l s and teleosts, whereas the divergence of pB and in evolutionary time.
s
probably o c c u r r e d much l a t e r
T h e possibility is also included h e r e that sur-
face pB differs f r o m s e r u m p in physical properties (87) and that this might represent another gene duplication event.
This s c h e m e m u s t
be taken as tentative until sufficient p r i m a r y sequence data accumulate to allow a p r e c i s e formulation. ACKNOWLEDGMENTS The authors' work was supported by grants f r o m the U. S. P. H. S. (AI- 12565,
(AH 75-877).
AI-10886) and the American H e a r t Association
JMD is a Postdoctoral Fellow of the National Multiple
S c l e r o s i s Society, and GWW is in receipt of a Postdoctoral Fellowship f r o m the Science Research Council (U. K. 1.
W e thank Sir F. M. Burnet
f o r helpful discussions, T h i s is publication number 2205 f r o m T h e Walter and Eliza Hall Institute.
297
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EVOLUTIONARY AND DEVELOPMENTAL ASPECTS O F T-CELL RECOGNITION Gregory W. W a r r , Janet M. Decker a n d John J. Marchalonis Laboratory of Molecular Immunology, The Walter and Eliza Hall Institute of Medical Research, P. O., Royal Melbourne Hospital, Victoria 3050, Australia
ABSTRACT Studies relating to the nature of the antigen-specific T-cell receptor a r e reviewed in the light of present knowledge of phylogenetic and ontogenetic development. It i s suggested that this evidence supports the concept that immunoglobulin (Ig) is the T-cell receptor, and that the following conclusions may be tentatively drawn. 1) T cell membrane Ig differs in its physical and functional properties from that on the B cell membrane. 2 ) The divergence between the T and B c e l l surface Ig molecules was apparent a t the time of emergence of the ancestors of modern fish. 3 ) Specific T cell recognition in the form of antigen binding appears e a r l y in ontogeny, and is blocked by antisera to Ig.
The nature of the antigen-specific surface receptor of T lymphocytes has generated much interest and debate.
The obvious can-
didate f o r this receptor is immunoglobulin (Ig) which possesses variable region combining s i t e s f o r antigen s i m i l a r to those of s e r u m Ig and B cell surface Igs (1, 2).
Controversy has surrounded attempts to estab-
lish this hypothesis because, in a formal sense at least, positive
-
attempts to demonstrate ( 1 5 ) o r isolate ( 6
- 10) surface Ig-like mole-
cules of T cells could be countered by negative reports of workers using "similar" (but not identical) experimental approaches ( 1 1 - 14).
Studies
281 Copyright 0 1976 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
282
WARR, DECKER, AND MARCHALONIS
from a number of laboratories now document the conclusion that murine T cells express a type of surface Ig termed IgM(T) o r IgT, which is distinct from the 7s IgM and IgD-like molecules isolated from the B cell surface ( 2 , 7
- 10,
15
- 20).
Evidence showing that this T-cell
Ig is synthesized by T cells and not by contaminating B cells o r plasma-
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cytes w a s obtained using monoclonal T lymphoma cells (15, 21, 2 2 ) characterized by s t r i c t T cell phenotypes.
Moreover, it has now been
established in some cases that T cells and B cells possess surface receptors for antigen which bear the same variable ( V ) region idiotypes a s do circulating antibodies directed against the s a m e antigen ( 2 3 - 2 5 ) . We believe that much of the difficulty reported in the isolation of T cell Ig by some workers ( 1 1, 1 2 ) s t e m s from the use of detergent extraction procedures shown by other workers to be unsuitable for isolation of T cell Ig (10, 2 6 ) .
This work has been reviewed elsewhere (2, 27, 28),
and w i l l not be reiterated h e r e except insomuch a s it impinges upon the phylogenetic and developmental results under consideration. The c r i t e r i a which a r e classically considered necessary for the demonstration of a true immune phenomenon a r e specificity of the evoked response, and an altered level of reactivity to subsequent antigenic exposure.
Although apparently all vertebrate c l a s s e s show
-
immune responsiveness of this type ( 2 9 31), the bulk of o u r knowledge of the immune system comes from intensive investigations of a few species, namely, man, the laboratory rodents, and to a l e s s e r extent chickens, and the comparative aspects of the generation and expression of immune competence have been unduly neglected.
This is, in o u r
opinion, unfortunate, since i t is through an investigation of the prototypical and universal features of immunity that we may eventually hope to achieve a comprehensive understanding of the subject, and be able to place in perspective the overwhelming amount of often contradictory data which abounds in current cellular immunology. The present article represents an attempt in this spirit to r e view the current state of our knowledge regarding the nature, evolution and ont ogenetic emergence of receptors on antigen-specific T - c e l l s .
T-CELL RECOGNITION
283
The salient conclusions arising from phylogenetic considerations a r e that ( a ) all vertebrates possess cells which exhibit specific T c e l l functions, (b) antigen-specific T cells bearing Ig receptors appear e a r l y in ontogeny of mammalian species, and ( c ) virtually all lymphocytes of lower species, including thymus lymphocytes, express and synthesize
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readily detectable surface Ig.
Furthermore, recent evidence supports
that hypothesis that surface Igs of T and B cells r e p r e s e n t distinct molecules which diverged from a common p r e c u r s o r e a r l y in vertebrate evolution.
Phylogenetic Origins of T Cell Functions Lymphocytes in certain birds, mice and other mammals have been subdivided, on the basis of origin, function and surface m a r k e r s , into thymus-derived T cells, and bursa (or bursa1 equivalent) derived
B cells (32, 33).
The B cells a r e the p r e c u r s o r s of antibody-secreting
cells and bear l a r g e amounts of readily detectable membrane immunoglobulin.
The T cells appear to be m o r e heterogeneous in function than
B cells, being involved in the phenomena of cell-mediated immunity
(CMI) such a s graft rejection, delayed-type hypersensitivity, graftversus-host reaction (GVHR). and the in vitro mixed lymphocyte r e a c tion (MLC), and also being implicated in collaborating with B cells to bring about efficient antibody production by the latter.
In considering
the nature of the T c e l l receptor, i t is a s well to b e a r in mind that the heterogeneity of c l a s s e s of T cells subserving various functions may be reflected in a heterogeneity of receptors.
Since the t r u e immunological
nature of GVHR and MLR phenomena has been questioned (31, 34), we will confine o u r attention to the recognition of classical foreign antigens and strong histocompatibility antigens. It is c l e a r (Table 1) that the thymus plays a central role in the generation of immune competence in teleost fish (35), amphibians (36, 37), birds and mammals (32, 33), and that lymphocytes of lower vertebrates show responses typical of T cells, e. g. graft rejection (38). MIF pro-
?
- ve + ve + ve + ve
+ ve
+ ve
+ ve
+ ve
REPTILIA
AVES
MAMMALIA ( o r d e r Rodentia
( o r d e r Artiodactyla)
?
-
ve
+ ve
+ ve
?
+ ve
+ ve
+ ve
?
?
Depression of Immunity by Thymec tomy*
+ ve
- ve
-
+ ve
+ ve + ve
+ ve ive
ve
- ve
-
?
- ve - ve ?
+ ve
MLR or GVH Reactivity
?
+ ve(larva1)
+ ve
+ ve
+ ve
?
Readily detectable Ig on Thymo cytes
All extant vertebrates show classical antibody production. This table w a s compiled from data in refs. 32, 35, 36, 38, 40 48, 53, 5 4 D 88 95. * Thymectomy a t an e a r l y developmental stage.
?
+ ve
+ ve
+ ve
+ ve
( o r d e r Anura)
+ ve
- ve
+ ve
?
?
~~
Carrier effect in AB production
+ ve + ve
-
+ ve
+ ve
OSTEICHTHYES ( s u p e r o r d e r Teleostii)
AMPHIBIA ( o r d e r Urodela)
-
+ ve
CHONDRICHTHYES ve
- ve
+ ve
AGNATHA
I
Graft Rejection
r
Class
Presence of strong H-Antigens
STATUS O F IMMUNE RESPONSIVENESS IN VERTEBRATE CLASSES
TABLE I
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205
T-CELL RECOGNITION
duction, MLR (in anuran amphibians, 35), responsiveness to c e r t a i n mitogens ( 3 9 ) . GVH reactions (40), and a c a r r i e r effect on secondary challenge ( 4 1 - 4 4 ) .
However, the inference that T and B c e l l s con-
stitute distinct populations of lymphocytes in all v e r t e b r a t e s p e c i e s can only be drawn indirectly from this evidence, a s appropriate c e l l m a r k e r s
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and cell separation techniques a r e not available,
However, t h e r e
e m e r g e s a consistent phylogenetic picture suggesting that all v e r t e brates show responses typical of those mediated by T and B c e l l s in the higher vertebrates (Table 1) even in those animals such a s the agnathan hagfish which do not p o s s e s s organized lymphoid t i s s u e ( 3 5 , 38).
T h e possibility that T and B c h a r a c t e r i s t i c s a r e s h a r e d by
a single c l a s s of lymphocytes is intriguing, but a number of observations suggest that t h e r e is a distinct and apparently universal division of function,
The possibility that T and B c e l l functions a r e e x p r e s s e d
by one c e l l type in lower species might be supported by observations which demonstrate that thymocytes in fish ( 4 5 - 4 7 ) and l a r v a l amphibians ( 4 8 ) b e a r readily detectable, endogenously synthesised m e m b r a n e Ig.
This property is generally considered a B c e l l c h a r a c t e r i s t i c in
m a m m a l s and birds; however, demonstration of binding of anti-Ig to thymus ( 4 , 18, 49) and T lymphoma c e l l s (22, 50) r a i s e s questions r e garding the stringency of this criterion.
Antibody production c a n occur
in the thymus of certain fish (51), although all this may suggest is that a s m a l l number of B c e l l s occur in f i s h thymus, as they do in mouse thymus.
The following evidence can be marshalled in support of the
idea that T and B cells a r e functionally distinct populations throughout v e r t e b r a t e phylogeny: (a) In a l a r v a l amphibian (37), thymus, but not spleen, is essential to the reconstitution of graft rejection,
(b)Lympho-
cyte differentiation into functional subgroups has o c c u r r e d in fish, because subpopulations of lymphocytes reactive to c e r t a i n mitogens a r e demonstrable (39).
( c ) In the goldfish, the membrane of thymocytes
d i f f e r s in s e v e r a l physical r e s p e c t s f r o m that of splenocytes (47).
In
particular, goldfish thymus lymphocytes r e s e m b l e those of m i c e i n that u s e of the nonionic detergent Nonidet P - 4 0 under conditions routinely
286
WARR, DECKER, AND MARCHALONIS
used for mouse B cells ( 1 1, 26), does not allow fully efficient extraction of Ig.
By contrast, "solubilization" of goldfish spleen lymphocytes
under conditions used f o r mouse B cells allows surface Ig to be precipitated in good yield.
Since surface iodination and various solubilization
conditions will be used increasingly in attempts to characterize mem-
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brane receptors and m a r k e r s of lymphocytes of lower species, we would caution the reader that techniques which w o r k satisfactorily for a subpopulation of mammalian lymphocytes cannot be expected to be directly applicable to cells of other subpopulations within a species or to cells of other species.
As an illustration, the Nonidet P - 4 0 approach w a s
not suitable f o r the isolation of surface Ig of chicken B cells ( 5 2 ) . Surface Immunoglobulins of Lymphocytes of Lower Species Data collated in Table 1 show that surface Ig is demonstrable by immunofluorescence on thymus lymphocytes of adult elasmobranchs (45), teleosts (46. 47), urodele amphibians ( 5 3 ) and larval anuran am-
phibians (48).
Moreover, studies involving shedding and reappearance
provided evidence 'that the thymus lymphocyte populations were capable of synthesizing this surface receptor.
This point might prove of
crucial importance because species such a s rays and urodeles a r e thought to lack strong histocompatibility antigens (381, but, nevertheless, c a r r y out antigen-specific T cell functions and bear surface Ig. The presence of readily detectable surface Ig on thymus lymphocytes of lower species provides a s h a r p contrast with the result usually obtained in studies of adult mice (1, 2, 54).
It is worthwhile
considering possible explanations for this discrepancy.
One obvious
possibility is that thymus lymphocytes of lower vertebrates and mammals differ in the number of Ig molecules expressed on the membrane. Another explanation might involve the u s e of antisera made in rabbits o r other mammals to investigate Igs of other mammals.
Since
mammals evolved relatively recently and their Igs show extensive crossreactions, it is possible that antibodies made in another vertebrate class, e. g. chickens, might possess stronger reactivities for minor
T-CELL
287
RECOGNITION
d e t e r m i n a n t s , p a r t i c u l a r l y t h o s e of V - r e g i o n s and t h e F d f r a g m e n t of the h e a v y chain.
Consequently, non- m a m m a l i a n a n t i b o d i e s m i g h t
s e r v e a s b e t t e r p r o b e s for t h e d e m o n s t r a t i o n of cell s u r f a c e I g s of m a m m a l i a n c e l l s than do antibodies m a d e in c l o s e l y - r e l a t e d s p e c i e s . C o n s i s t e n t with t h i s notion a r e the o b s e r v a t i o n s t h a t a n t i s e r a m a d e in
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c h i c k e n s to p u r i f i e d m o u s e o r human Igs will bind to m o u s e t h y m u s (18) and human t h y m u s lymphocytes (49), r e s p e c t i v e l y .
A third possible
f a c t o r i s that s u r f a c e Ig of m o u s e T c e l l s m i g h t b e "buried" with o n l y i t s VH-VL combining s i t e exposed (55, 56), w h e r e a s m o s t of t h e H-chain c o n s t a n t r e g i o n i s e x p o s e d on B c e l l s ( 5 7 ) .
T h e s e e x p l a n a t i o n s are not
m u t u a l l y e x c l u s i v e and d a t a to be c o n s i d e r e d below s u g g e s t t h a t s u r f a c e Igs of T and B c e l l s p o s s e s s d i f f e r e n t H-chain c o n s t a n t r e g i o n s ( 1 5 - 17,
47) and consequently m a n i f e s t a n u m b e r of functional ( 5 8 - 60) a n d p h y s i c a l d i f f e r e n c e s ( 1 6 , 47, 61). A meaningful r o l e f o r s u r f a c e Ig of l y m p h o c y t e s of l o w e r s p e c i e s is indicated by s t u d i e s which show that all s p e c i f i c antigenbinding lymphocytes of a n u r a n a m p h i b i a n s ( 6 2 ) including p u t a t i v e " c a r r i e r - s p e c i f i c ' ' h e l p e r c e l l s ( 6 2 ) a r e inhibited by a n t i s e r a d i r e c t e d a g a i n s t l i g h t c h a i n s and p c h a i n s .
Antigen s p e c i f i c functions of T cells
of c h i c k e n s ( 6 3 ) and m i c e (64, 65) h a v e a l s o been inhibited by a n t i s e r a to Igs, p a r t i c u l a r l y t h o s e p o s s e s s i n g a n t i b o d i e s t o light c h a i n s , A t t h i s time, s u r f a c e Ig h a s been i s o l a t e d f r o m l y m p h o c y t e s of only one l o w e r s p e c i e s , v i z . , t h e goldfish ( C a r a s s i u s a u r a t u s ) , a teleost.
T h i s s p e c i e s , l i k e o t h e r c y p r i n o i d s (66, 67), p o s s e s s e s o n l y
o n e s e r u m Ig, a t e t r a m e r i c IgM molecule.
Rabbit a n t i b o d i e s s p e c i f i c
f o r this s e r u m Ig p r e c i p i t a t e d s i g n i f i c a n t c o u n t s i n '251-labelled s u r f a c e p r o t e i n s f r o m both goldfish t h y m u s a n d s p l e e n l y m p h o c y t e s ( T a b l e 2). T a b l e 2 a l s o i l l u s t r a t e s t h a t Ig of goldfish t h y m u s l y m p h o c y t e s , l i k e that of m o u s e T l y m p h o m a c e l l s a n d t h y m u s l y m p h o c y t e s , is n o t a d e quately e x t r a c t e d by u s e of Nonidet P - 4 0 u n d e r m o u s e B cell conditions. A distinction between s u r f a c e I g s of goldfish t h y m u s a n d s p l e e n l y m p h o c y t e s is o b s e r v e d when polypeptide c h a i n s of t h e s e I g s are c o m -
WARR, DECKER, AND MARCWONIS
288
TABLE I1 EFFECT O F EXTRACTION CONDITIONS UPON ISOLATION O F SURFACE IG
I 2 Macromolecular ~~~~
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Lymphocyte Source
Extraction Procedure
~
' %-counts Precipitate(
L Specifically Precipitated
GOLDFISH Spleen
1yo N P 40
3.1
Thymus
1% NP40
0.5
Thymus
Metabolic release
4.5
1.5
3.0
CBA Thymus
0. 570 N P 4 0
0.3
0.2
0.1
nu/nu Spleen
0. 5% NP40
3.6
0.4
3.2
CBA Thymus
Acid Urea
1.7
0.3
1.4
nu/nu Spleen
Acid Urea
1.4
0.3
1.1
WEHI 22
1% NP40
0.1
0.1
0
WEHI 22
1% N P 401
1.2
0.1
1.1
MOUSE
T LYMPHOMA
6M Urea WEHI 22
Acid Urea
6. 1
1.7
4.4
WEHI 22
Metabolic release
2.9
0.8
2.1
In all experiments, lymphocytes were surface labelled with 1251in a lactoperoxidase-catalysed reaction. Details of experimental techniques employed w i l l be found in refs, 10, 26, 47, from which some of these data were taken. pared by polyacrylamide gel electrophoresis in SDS-containing buffers under conditions which provide high resolution of proteins in range of apparent mass, 40,000
- 90,000
daltons.
Most importantly, the Ig
heavy chain shows a distinctly f a s t e r mobility in gel electrophoresis than that of splenocyte Ig heavy chains.
This difference is illustrated
T-CELL RECOGNITION
289
in Fig. 1, and i t is significant that analogous differences in the mobility of splenocyte and thymocyte Ig heavy chains have been described f o r the mouse (15, 16).
Fig. 2 presents a composite diagram based upon
studies which have compared the mobilities on SDS- PAGE of s u r f a c e p and
& -like chains of
B cells with heavy chains of surface Igs of thymus
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( 16 ) and T lymphoma cells ( 1 7).
Surface Ig of in vitro grown murine
fetal thymus anlagen ( 6 8 ; Haustein, D. and Mandel, T. E.
in p r e p a r a -
tion) also possesses a heavy chain which is slightly, but significantly, f a s t e r than p chain.
i
FIGURE 1.
A COMPARISON O F THE SURFACE IMMUNOGLOBULINS O F SPLEEN AND THYMUS LYMPHOCYTES O F THE GOLDFISH ) and Polypeptide chains of Ig from i251-surface labelled spleen -( thymus ( - - - - - ) lymphocytes were analysed by SDS-polyacrylamide gel electrophoresis after reduction with mercaptoethanol. It can be seen that the Ig from both c e l l s dissociated into light (L)chains and heavy chains. Whereas the heavy chain of the splenocyte Ig migrated like a t r u e p chain, the thymocyte Ig heavy chain showed a significantly f a s t e r Positions of standard murine and human p, y and L chain mobility. m a r k e r s a r e indicated by a r r o w s ( & ). Both thymus and spleen lymphocytes show a polypeptide migrating f a s t e r than mammalian y chain, and which appears to be analogous to the molecule described in mammalian lymphocytes which has affinity f o r antigen-antibody complexes (See a l s o Fig. 2 ) .
W A R R , DECKER, AND M A R C W O N I S
290
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I
EL
6 1 j
c cr
I
DISTANCE MIGRATED
L
c
-
FIGURE 2. A COMPARISON O F THE SURFACE IMMUNOGLOBULINS O F MURINE T CELLS AND SPLENOCYTES Polypeptide chains of Ig from 1251-surface labelled spleen -( ) and T ( - - - - - ) lymphocytes analysed after reduction with mercaptoethanol, The positions of standard by SDS-polyacrylamide gel electrophoresis. murine and human p, y and light (L)chain m a r k e r s a r e indicated by a r r o w s ( & ). Whereas the splenocytes show a light chain and two heavy chains (one a true p, the other designated 6 '-like (9 6 ) and marked ( I ) as such in the figure, although i t does not migrate like a human 6 chain, ref. 971, the T cell Ig resolves into a light ( L ) chain and a heavy chain with a mobility distinctly f a s t e r than p. Both T and spleen c e l l patterns show a polypeptide chain migrating in a position intermediate between y and L chains, This molecule has the property of binding to antigen-antibody complexes. The data shown in this figure is a composite representation of that presented elsewhere (15, 16, 68).
Although mobility in SDS gels should not be taken as a s o l e criterion for heavy chain homology, these r e s u l t s suggest that the p-like heavy chain of T cells is physically distinct from that of B cells and that this distinction a r o s e (unless we a r e dealing with convergent evolution) in or p r i o r to the divergence of the a n c e s t o r s of modern teleosts and mammals.
Quite clearly, further comparative studies
w i l l be needed to confirm this hypothesis, and to determine whether or
T-CELL RECOGNITION
291
not the T cell Ig heavy chain differs from p chain i n some minor aspect (e. g. carbohydrate content) o r whether the differences lie in the p r i m a r y protein s t r u c t u r e of the molecule. Ontogeny of T Cell Recognition
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The mammalian immune system is relatively immunoincompetent at birth.
Stimulation with a foreign antigen will elicit a t best only
a weak antibody o r cellular immune response, while the induction of tolerance in neonatal life is relatively easy(69 - 71).
Nevertheless, the
ability to recognize antigen, as measured by specific antigen binding to lymphocytes, appears rather early in the development of the lymphoid system during ontogeny.
Antigen- binding lymphocytes have been
detected in the thymus at 14 days gestation in the mouse and chicken, 20 days in the rat, and 12 weeks in the human ( 7 2 - 74).
The frequency
of binding cells in the thymus is high when f i r s t detected, often close to one percent of lymphocytes, and declines during gestation and childhood,
This decrease in frequency might be explained by a change in
the cell populations comprising the thymus ( f o r example, a dilution of the binding population by a non-binding one), o r by a decrease in the amount of receptor Ig p e r binding cell.
In all c a s e s antigen binding
can be inhibited with antisera specific for immunoglobulin p and kappa .chains (72, 74, 75). Although the newborn mammal i s relatively immunoincompetent, there have been s e v e r a l reports of T c e l l function detectable a t birth o r even before.
In the mouse, helper cells a r e present in the thymus at
48 hours after birth (76), and GVH reactivity has been measured in yolk s a c cells a t 9 days gestation (77).
Silverstein has demonstrated helper
activity in the fetal lamb, which is able to make s o m e antibody responses in utero (78). In general, conditions which demonstrate the presence of s u r face Ig on B cells by autoradiography will not detect Ig on T cells, although a high percentage of thymus c e l l s from adult mice can be shown to have Ig when m o r e sensitive conditions are used ( 3 ) .
It has been
29 2
WARR, DECKER, AND M A R C W O N I S
shown, however, that approximately 20% of thymocytes from young children do have surface Ig (49).
The facts that only a subpopulation
of cells show anti-Ig binding and that not all anti-Ig s e r a bind to thymocytes argue against passive-acquisition of Ig by the cells.
Moreover,
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of in vitro grown murine fetal thymus anlagen ( 6 8 ; Haustein, D. and
Mandel, T. E., in preparation). Discussion On the basis of the evidence available a t present, we therefore incline to the view that lymphocytes exist, a s low in the phylogenetic o r d e r a s bony fish, which show distinct homologies, at both a functional and a molecular level, with the T cells of mammals.
If this view is
to be accepted, i t implies that the thymocyte Ig, readily detectable by immunofluorescence in fish ( 4 5 - 47), has become, by the phylogenetic level of the birds and mammals, less readily detectable.
The obser-
vations that larval amphibia have readily detectable Ig on their thymocytes, which becomes more difficult to detect during development (48), a r e readily compatible with this hypothesis.
Possibly a parallel situa-
tion might obtain for fetal and neonatal mammalian thymus lymphocytes. If this interpretation of the comparative and developmental studies of thymocyte Ig is correct, several questions remain to be answered. Firstly, the teleological question: why should the display of T cell membrane Ig change?
Secondly, do T cells possess receptors, other
than Ig. that may, for example, be expressed in a reciprocal relationship?
In answer to the f i r s t question, the most attractive hypothesis is that the great bulk of membrane Ig on, f o r example, fish thymocytes and all B cells, is functionally redundant.
Circulating human lympho-
cytes possess only about 2000 membrane bound receptors for insulin
(79), so why should mammalian B cells require 80,000 o r more membrane Ig receptor molecules (1, 8 0 ) ?
What distinguishes the Ig mole-
cule from the insulin receptor is the capacity of the lymphocyte to syn-
T-CELL RECOGNITION
293
thesize and s e c r e t e in response to stimulation l a r g e numbers of molecules closely related to i t s membrane Ig receptors.
This l a r g e quantity
of membrane Ig may merely represent a by-product of p r e s e c r e t o r y activity of the protein synthetic apparatus of the lymphocyte, with very few of the molecules being involved in the process of triggering.
As
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f a r a s we a r e aware, no one has yet investigated this important question,
It is c l e a r that the antibody s e c r e t o r y apparatus becomes m o r e complex and diversified with movement up the phylogenetic scale, with appearance of plasma cells (30). increasing complexity of lymphoid t i s s u e s ( 3 5 ) and diversity of Ig c l a s s e s ( 2 9 , 30).
With this increasing complexity of the
B cell Ig s e c r e t o r y system, i t is possible that T cells would not have
been subjected to selective p r e s s u r e to retain superfluous membrane Ig since the function of mammalian T cells is clearly not to s e c r e t e l a r g e amounts of Ig.
The notion that some thymocytes a r e capable of
antibody secretion is however, supported by observations on the fish thymus, and there appears to be an interesting intermediate s t a g e in the g r a s s frog ( 6 2 ) , where i t has been reported that the thymus can respond to immunization with an increasing number of antigen-binding cells, but that these c e l l s a r e non-secretors of antibody. The dispute over the existence of Ig on mammalian T c e l l s has been argued for many years, but a t the r i s k of boring the r e a d e r we feel i t is worth while summarizing what we consider to be the p r e s e n t state of the field.
The evidence relating to the detection of T c e l l Ig
has recently been reviewed in detail (21, and although many investigat o r s have reported positive results (4-10, 1 5 - 22), i t is c l e a r that s o m e workers have experienced difficulties.
In o u r opinion, these difficul-
ties s t e m f r o m the facts that ( a ) Ig in the membrane of T c e l l s is obviously not readily accessible to anti- Ig reagents, ( b ) moreover, the complication a r i s e s that readily detectable surface Ig of T cells is often of passive nature, ( c ) Membrane Ig in T cells is not satisfactorily ex-
tracted by all methods used f o r B c e l l s (10, 17, 26).
Some workers,
in addition, consider that Ig is present in the membrane of thymocytes in relatively s m a l l amounts ( 1 , 81), although no assumption-tree methods
WARR, DECKER, AND MARCHALONIS
294
presently exist for the accurate quantitation of membrane molecules which a r e neither completely accessible to binding reagents nor completely extractable under all "solubilization" conditions.
The recent
phylogenetic evidence establishes that a l l lymphocytes of lower species express surface Ig which is easily detected using antisera specific for
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s e r u m IgM. The second question posed by the comparative studies, i. e . , is it possible that phylogenetic development has seen the partial replace-
ment of Ig by another receptor molecule? is worthy of serious consideration.
It is c l e a r that the plasma membrane of lymphocytes b e a r s a
range of receptors ( f o r hormones, antigens, Fc, heterologous erythrocytes, lectins), but it is difficult to s e e why the T cells should bear two distinct types of antigen specific receptors both of which would bear the V region of the Ig molecule (23
- 25).
While a DNA translocation model
f o r the sharing of V regions by the various C regions, quite probably linked to each other (82). has been proposed (821, it is l e s s likely that translocation between the unlinked V region genes and major histocompatibility complex ('MHC) genes ( 8 2 ) is a feasible molecular mechanism. The majority of published reports on the role of MHC specified cell surface antigens suggest that they play a role in cell collaboration, rather than mediating specific recognition (83
, but
c. f. 8 4 ).
Many animals appear to have some powers of discriminating self from non-self, a capacity which manifests itself in such phenomena
as incompatibility reactions in non-vertebrate groups like c o r a l s (85) and tunicates (86).
Such recognition does not appear, from o u r present
knowledge, to possess the characteristics of a true immune response, and while such "quasi-immune" phenomena may have persisted in the vertebrates, it is unclear to what extent they contribute to cell-mediated immunity o r a r e related to reactions against strong histocompatibility antigens.
Certainly all fish s e e m to lack MLR and elasmobranchs do
not show rapid first-set graft rejection as is also the c a s e in urodele amphibians (38).
However, this apparent lack of major histocompati-
bility complex antigens does not prevent these animals f r o m developing
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T-CELL RECOGNITION
295
WARR, DECKER, AND MARCHALONIS
296
adequate immune responsiveness c h a r a c t e r i s t i c in o t h e r r e s p e c t s of T and B c e l l function.
While i t is unwise to draw conclusions f r o m nega-
tive experiments, we do not feel that phylogenetic studies support the view that antigen recognition by T c e l l s is due in any g r e a t m e a s u r e to c e l l s u r f a c e components specified by the m a j o r histocompatibility complex.
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The recent phylogenetic and ontogenetic data reviewed here, taken in conjunction with c u r r e n t investigations of the p r o p e r t i e s of s u r face Igs of murine T cells,indicate that (a) Ig of T c e l l s is distinct f r o m the surface IgM and IgD-like molecules of B cells, and ( b ) the genes specifying heavy chains of IgM(T) o r IgT probably diverged e a r l y in v e r tebrate evolution from those encoding the p and
d
chains.
T h e s e con-
cepts a r e depicted in Fig. 3 which gives a schematic representation of the emergence of genes specifying p chains and lymphocyte s u r f a c e H-chains.
This model proposes that the pT and pB genes diverged
p r i o r to the divergence of the a n c e s t o r s of m a m m a l s and teleosts, whereas the divergence of pB and in evolutionary time.
s
probably o c c u r r e d much l a t e r
T h e possibility is also included h e r e that sur-
face pB differs f r o m s e r u m p in physical properties (87) and that this might represent another gene duplication event.
This s c h e m e m u s t
be taken as tentative until sufficient p r i m a r y sequence data accumulate to allow a p r e c i s e formulation. ACKNOWLEDGMENTS The authors' work was supported by grants f r o m the U. S. P. H. S. (AI- 12565,
(AH 75-877).
AI-10886) and the American H e a r t Association
JMD is a Postdoctoral Fellow of the National Multiple
S c l e r o s i s Society, and GWW is in receipt of a Postdoctoral Fellowship f r o m the Science Research Council (U. K. 1.
W e thank Sir F. M. Burnet
f o r helpful discussions, T h i s is publication number 2205 f r o m T h e Walter and Eliza Hall Institute.
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