Table Of ContentADVANCES IN
PROTEIN CHEMISTRY
EDITED BY
FREDERIC M. RICHARDS DAVID E. EISENBERG
Department of Molecular Biophysics Department of Chemistry and Biochemistry
and Biochemistry University of California, Los Angeles
Yale University Los Angeles, California
New Haven, Connecticut
PETER S. KIM
Department of Biology
Massachusetts Institute of Technology
Whitehead lnstitute for Biomedical Research
Howard Hughes Medical lnstitute Research Laboratories
Cambridge, Massachusetts
VOLUME 49
Antigen Binding Molecules:
Antibodies and T-cell Receptors
EDITED BY
EDGARHABER
Department of Biological Sciences
Harvard School of Public Health
Harvard Medical School
Boston, Massachusetts
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PREFACE
Immunology began at the end of the nineteenth century when Paul
Ehrlich became fascinated by precipitation reactions in the serum of
immunized animals. His work was followed by a systematic analysis by Karl
Landsteiner of the exquisite specificity of what we now know to be the
antigen-antibody interaction. Yet the precipitin reaction remained in the
realm of phenomenology until the 1950s, during which Rodney Porter
and Gerald Edelman defined the modular structure of the antibody
molecule and demonstrated that antigen binding was the property of only
one domain of this multifunctional molecule. Renaturation experiments
from my laboratory and those of Michael Sela and Charles Tanford soon
implied that there had to be a great many different antibody molecules to
account for the wide range of antigen binding specificities, since the amino
acid sequence of an antibody determined its specificity. After the earliest
amino acid sequences of myeloma proteins (homogeneous surrogates for
the very heterogeneous serum antibodies) became available in 1965 from
Norbert Hilschmann and Lyman Craig, it was apparent that a hypervari-
able region accounted for the antigen binding domain. The ability to clone
examples of real antibodies by the hybridoma method of George Kohler
and Cksar Milstein then led to a surge of activity in the 1970s that defined
many aspects of the structure and function of the antigen binding site.
Soon it was clear that the same principles of binding applied not only to
humoral antibodies but to T-cell receptors and many members of what we
now call the immunoglobulin superfamily.
Interest in antigen binding waned for a decade while the detailed
mechanisms of the cellular immune response were explored and defined.
Now in the 1990s we are witnessing a revival of interest in antigen bindings
as its broad relevance to many immunologic processes has become appar-
ent and a variety of new analytic tools have allowed for deeper insights into
the nature of antigen binding. Advances in molecular engineering permit
the recapitulation of clonal selection and affinity maturation of antibodies
in vitro. It is possible to produce antibodies to selected antigens without the
mediation of an animal host, and it is even possible to turn an antigen
binding site into a highly selective enzyme.
This volume brings the reader up to date on the covalent and three-
dimensional structures of the antibody molecule’s antigen binding domain
and the synthesis and use of this domain as a separate small molecule. The
reader will find a full account of antibody three-dimensional structure (as
xi
xii PREFACE
revealed by x-ray crystallography and computational biochemistry) as well
as an analysis of how antigens bind. The nature and the structure of an
antigen are defined, and the affinity maturation of antibodies is examined
in relation to gene structure and diversification. The exciting field of cata-
lytic antibodies has advanced to include a range of enzymatic functions not
even contemplated when the first examples were described a few years ago.
The T-cell receptor, which has some elements in common with the anti-
body molecule, is far more complex in its antigen recognition function.
The T-cell receptor is analyzed here in the context of its binding to anti-
gen and to the essential major histocompatibility complex. The role of the
T-cell receptor’s accessory proteins in binding and activation is also defined.
Not only immunologists but also biologists and chemists should profit
from reading this volume: it reveals a mature yet evolving field of broad
interest to other areas of science.
EDGAR HABER
ANTIGEN-SPECIFIC T-CELL RECEPTORS AND THEIR REACTIONS
WITH COMPLEXES FORMED BY PEPTIDES WITH MAJOR
HISTOCOMPATIBILITY COMPLEX PROTEINS
By HERMAN N. EISEN, YURl SYKULEV, and THEODORE J. TSOMIDES
Center for Cancer Research and Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts0 2139
I. Overview ............................................... 1
11. History a ound .......... 3
111. T-cell Receptor Genes ............................................ 8
IV. T-cell Receptor Proteins 11
V. T-cell Receptor Ligands: Peptide-MHC Complexes. ................... 15
VI. T-cell Responses to PepMHC 22
A. Affinity: Intrinsic Equilibri 23
B. Kinetics ................. 26
C. Time Required to Approach .......................... 29
D. Epitope (PepMHC) Density 30
E. TCR-PepMHC Engagement: .................... 32
F. Specificity, Degenerac 35
VII. T-cell Receptor Accessory Proteins. ................................. 39
A. CD3andc.. ......... 39
B. CD4andCD8.. ...... 40
VIII. Altered Peptide Ligands: Partial Agonists and Antagonist 41
IX. MHC Restriction by Self and Nonself MHC: The Paradox 43
X. Concluding Remarks. ............. 47
References ...................................................... 48
I. OVERVIEW
The most distinctive feature of the vertebrate immune system is its
ability to recognize an enormous number of organic molecules and molec-
ular complexes, termed antigens, distinguishing broadly between those
that are foreign to the responding animal (nonself) and those that are
indigenous (self). This property is due to antigen-specific receptors on
lymphocytes, small cells that comprise -5% of all cells in the body (esti-
mated at 101'-lO1z out of about loL3ce lls in an adult human). The recep-
tors on the two major classes of lymphocytes, B and T cells, are similar
structurally but profoundly different functionally. On B cells the receptors
1
ADVANCES IN Copyright 63 1996 by Academic Press, Inc.
PROTEIN CHEMISTRY W1. 49 All rights of reproduction in any form reselved.
2 HERMAN N. ElSEN ET AL.
are immunoglobulins (Ig) embedded in the cell surface as integral mem-
brane proteins; in response to recognition of antigens, B cells produce
large amounts of the receptors and secrete them as soluble antibody mole-
cules. The antigen-specific receptors on T cells (T-cell receptors or TCR)
are also Ig-like cell surface integral membrane proteins; their recognition
of antigens triggers T cells to exercise a great variety of functions but not
to secrete the receptors.
Both Ig and TCR molecules are heterodimers, each subunit consisting
of two or more domains, with each domain having a characteristic three-
dimensional shape called the Ig fold. The N-terminal domains (termed
variable or V domains) differ in amino acid sequence from one lymphocyte
clone to another. The variable domains of each heterodimer pair to form
a single antigen binding site that determines the unique ability of each
clone to recognize and respond to only very few of the millions of different
antigens to which an individual animal can respond. The enormous diver-
sity of B- and T-cell receptors arises from the many germline gene seg-
ments that encode them; as each lymphocyte matures, different combina-
tions of these segments are joined (combinatorial diversity) and additional
variations in sequence are introduced at the junctures (junctional diver-
sity), leading to an immense number of variable domain sequences.
Despite extensive similarities in amino acid sequence between Ig and
TCR and in the organization and recombination of the gene segments that
encode them, these receptors differ remarkably in the universe of antigens
they recognize. The antigens recognized by antibodies (or their membrane-
bound form on B cells) vary enormously: Physically they may be soluble,
colloidal, particulate, or parts of virions or microbial or eukaryotic cells,
and chemically they may be proteins, peptides, carbohydrates, lipids, nucleic
acids, or any of a limitless number of diverse small organic molecules. In
contrast, the TCR reviewed here normally recognize and respond only to
complexes formed between small peptides and a specialized set of pro-
teins encoded by the major histocompatibility complex (MHC). Because
MHC molecules (sometimes called histocompatibility antigens) are integral
membrane proteins, these complexes (termed pepMHC) are confined to
cell surfaces, and the TCR of a T cell is therefore normally able to recog-
nize antigenic complexes only on the surfaces of other cells, called antigen
presenting cells or target cells. Inasmuch as both TCR and their natural
pepMHC ligands are embedded in cell surface membranes, analysis of
their interaction at the molecular level poses a major challenge. Later in
this review we focus on how this challenge is being met and emphasize recent
results that illuminate the way in which TCR react with (or “recognize”)
their natural pepMHC ligands, particularly the TCR on those T cells (called
cytotoxic T lymphocytes or CTL) that destroy other cells (termed target cells).
ANTIGEN-SPECIFIC T-CELL RECEPTORS 3
11. HISTORAYND BACKGROUND
The immune system has been under study for about 100 years, but only
in the past 30 years have T and B cells been distinguished and only in the
past 12 years have TCR molecules and the genes encoding them been
identified. Nevertheless, for decades before T cells and TCR emerged as
recognized entities, their existence was foreshadowed by certain antigen-
specific inflammatory responses produced by injecting antigens into the
skin of individuals with previous exposure to such antigens, either through
natural infection of deliberate inoculation (immunization). Around 1890,
Robert Koch showed that the injection of tubercle bacilli (or a mixture of
proteins called “tuberculin” from supernatants of tubercle bacilli cultures)
elicited an intense inflammatory response in guinea pigs if they had been
previously infected with these microorganisms. The responses appeared
1248h r following antigen injection, and similar delayed-type hypersensi-
tivity (DTH) responses, always specific for the original inciting antigen,
were subsequently demonstrated with crude protein mixtures from many
other microbes (bacteria, fungi, and later, viruses). Responses sometimes
occurred following deliberate immunization with purified proteins (e.g.,
ovalbumin) or even with small organic molecules applied to the skin, pro-
viding they reacted in situ to form covalent derivatives of skin proteins (as
with 2,4-dinitrochlorobenzene,o r a catechol in the case of poison ivy).
In contrast to the late appearance of DTH responses, many other
antigen-specific skin responses appear almost immediately, e.g., within 1 min
or sometimes 2-3 hr. Because the transfer of serum antibodies from an
immunized to a nonimmunized (naive) individual confers on the recipient
the same prompt antigen-specific responses, it was clear that these rapid
or immediate-type responses were mediated by antibodies. But serum
failed to transfer the delayed-type responses (e.g., to tuberculin). Although
efforts were made to reconcile these failures with a role for special
antibodies, it came to be widely believed that antigen-specific cells rather
than soluble antibody molecules were the direct mediators of DTH
responses. This belief was supported by the finding that DTH responses
could be transferred to naive recipients with inflammatory cells from im-
munized donors, although the transferred cells were complex mixtures of
leukocytes that probably included some antibody-forming cells. The reso-
lution of all doubt came after T cells were distinguished from B cells,
largely through studies involving extirpation of the thymus from newborn
mice. These studies led to the establishment of a clear dichotomy between
those lymphocytes that develop from immature precursors in the thymus
(T cells) and those that develop to maturity in the bone marrow (B cells).
B cells were shown to be the source of Ig and antibodies, and T cells were
4 HERMAN N. EISEN ET AL
shown to be required for the optimal production of antibodies. In addi-
tion, highly purified populations of T cells could transfer DTH responses,
suggesting that T cells bear receptors that recognize antigens.
Early studies on the nature of the antigen-recognizing receptors on
T cells were marked by intense disagreements arising largely, it appears in
retrospect, because the T-cell populations studied were often contami-
nated by small numbers of B cells and because reliance was placed almost
exclusively on serological analyses using anti-Ig antisera. Antigen-specific
molecules on T cells were variously suggested to be Ig molecules firmly
attached to T cells, to be some other (non-Ig) type of cell-associated pro-
tein, or to be some other type of nonprotein informational macromole-
cule. The debate was resolved by the development of monoclonal antibody
(MAb) technology and the ability to grow T-cell clones in culture. Using
MAb raised against T-cell clones, one a malignant lymphoma and the
others normal T cells, three independent studies succeeded in immuno-
precipitating a T-cell surface protein that was unique to cells of the immu-
nizing clone (Allison et al., 1982; Meuer et al., 1983; Haskins et al., 1983).
The clonally specific (or clonotypic) protein was in each case a disulfide-
linked heterodimer (-90 kDa) consisting of a relatively acidic membrane-
bound a chain and a more basic membrane-bound p chain.
The idea that these clone-specific heterodimers were antigen-specific
receptors was strengthened by two findings. First, antibodies against them
could block antigen-driven responses of the corresponding T-cell clones
(Lancki et al., 1983). Second, amino acid sequences of proteolytic fragments
from isolated a and p subunits suggested that, like Ig heavy and light
chains, they had some regions where amino acid sequences varied from
clone to clone and others where these sequences were invariant (Kappler
et al., 1983; Meuer et al., 1984; McIntyre and Allison, 1983). The isolation
and sequencing of cDNA clones for the b subunit (Yanagi et al., 1984;
Hedrick et al., 1984; Saito et al., 1984a) and then for the a subunit (Saito
et al., 1984b; Chien et al., 1984) finally demonstrated unambiguously that
the clonally diverse heterodimers greatly resembled Ig and had all the
characteristics expected of cell surface integral membrane proteins serv-
ing as antigen-specific receptors. Moreover, introduction of genes for both
the a and /3 subunits into a T-cell clone having unrelated specificity trans-
ferred the antigen-specific responsiveness of the donor cell to the recipient
cell (Dembic et al., 1986; Saito et al., 1987).
In the course of searching a T-cell cDNA library for clones for the a and
p subunits, cDNA for a third related subunit was found (Saito et al., 1984a).
Termed y, the third gene turned out to encode an Ig-like chain that paired
with the Ig-like product of a fourth gene, 6, to form a y6 heterodimer
(Chien et al., 1984). Closely similar to the ab TCR, y6 heterodimers are
ANTIGEN-SPECIFIC T-CELL RECEPTORS 5
another type of antigen-specific receptor found on a subset of T cells
+
located primarily in epithelia. yd cells constitute about 1-5% of periph-
eral T cells in mice and humans; these TCR will not be considered further
here, as their natural ligands are not as well-defined as those recognized
by aB TCR and the hnction of yd T cells is still not clear.
Ten years before the molecular identity of the ab TCR began to take
shape, several observations pointed to the special character of its natural
ligands. By transferring T cells to athymic (nude) mice, Kindred and
Shreffler (1972) saw that these cells reacted only when the T cells and the
recipients had the same MHC type. The ineffectiveness of MHC-dispartate
T cells could not be attributed to their immune elimination in the recipi-
ent because athymic mice do not reject allografts (see Alloaggression,
Section IX). It was therefore concluded that the MHC “must play an active
role in ensuring cooperation between B and T cells.”
Shortly thereafter, in vitro studies of T-cell enhancement of the produc-
tion of antibodies by B cells (Katz et al., 1973) and of T-cell responses to
antigens presented by macrophages (Rosenthal and Shevach, 1973) clearly
indicated that successful T-cell responses required the responding T and B
cells, or T cells and macrophages, to have the same MHC type. Then, in a
seminal publication, Zinkernagel and Doherty (19 74) reported that virus-
infected target cells were lysed by T cells from a mouse infected by that virus
only if the target cells expressed the same MHC products as the infected
animal. Work with different antigens revealed similarly that antigen-
specific lysis of antigen-bearing target cells by cytotoxic T cells depended
on target cell expression of a proper MHC protein (Shearer, 1974; Bevan,
1975; Gordon et al., 1975). This dual requirement of antigen recognition,
referred to as “MHC restriction” (Zinkernagel and Doherty, 1979), has
since been found to be characteristic of all a/?T CR-mediated reactions.
To explain MHC restriction, two models were proposed. In the two-
receptor model, T cells had one receptor for an MHC product and an-
other for antigen, and both had to be occupied for a successful T-cell
response (Cohn and Epstein, 1978). According to the alternative one-
receptor model, each T cell had a single type of receptor that recognized
an antigen-MHC complex. The one-receptor model was shown to be cor-
rect by various approaches. In one, a hybridoma resulting from the fusion
of two cells recognizing antigen A and MHC X or antigen B and MHC Y
+ +
responded specifically to cells expressing either A X or B Y but not to
+ +
those expressing A Y or B X, indicating that the T cell did not see the
antigen and the MHC product separately (Kappler et al., 1981). The
antigeneMHC complex was sometimes referred to as “altered self’ be-
cause in this context the restricting MHC is indigenous (self) with respect
to the responding T cells and is therefore nonimmunogenic, while the
6 HERMAN N. EISEN ET AL
antigen is foreign (nonself) and somehow alters the character of self MHC,
imposing “foreignness” and ability to elicit a reaction.
Mature T cells bearing ap TCR fall into two groups, each marked by
one of two cell surface glycoproteins. Termed CD4 and CD8 (formerly
called Lyt-1 and Lyt-2,3 in the mouse), both are present on,immature
(double positive) T cells in the thymus, and one of them is lost by what
appears to be a stochastic process (Corbella et al., 1994) to yield single
positive (CD4+ or CD8+) mature T cells. Some mature CD4+ T cells,
termed helper (Th2) cells, are required for optimal B-cell responses, while
other helper cells (Thl) produce cytokines that cause inflammation, as in
DTH responses. In contrast, CD8+T cells, which also produce some cyto-
kines, behave primarily as CTL, lysing target cells that bear appropriate
antigenic MHC complexes.
The CD4/CD8 T-cell dichotomy also extends to the types of MHC pro-
tein that restrict antigen recognition. Class I MHC proteins (MHC-I) restrict
antigen recognition by CD8+ T cells, and class I1 MHC proteins (MHC-11)
restrict antigen recognition by CD4+ T cells (Table I). Virtually all cells
express MHC-I and can present peptides to CD8+ cells, whereas only
specialized cells (principally macrophages, dendritic cells, and B cells) ex-
press MHC-I1 for interactions with CD4+ cells. In common usage, cells
that present pepMHC-I1 complexes to CD4+ cells are called antigen pre-
senting cells (APC), whereas those that present pepMHC-I complexes to
CD8+ CTL are termed target cells.
Once it became established that a single heterodimeric receptor on
T cells reacts with an antigen displayed on the surface of another cell and
TABLE I
Peptide Binding to MHC-I and MHC-II Molecules
Parameter MHC-I MHC-I1
Distribution All nucleated cells Specialized APC
Domain structure az, a3 + Pzm al, a2 + PI,P Z
Accessory molecule CD8 CD4
Typical T-cell response Cytolytic activity B-cell help, DTH
Origin of most bound Cytosolic (endogenous) Extracellular and membranous
peptides proteins
-
Length of bound peptides Usually 8-9 residues Variable, 12-25 residues
Pockets in MHC groove Yes Yes
Peptide N and C termini Buried in groove May extend outside groove
Many critical contacts Peptide side chains Peptide backbone
-
Equilibrium constant, Ka -104-109 M-I measured 106-1 OH h.T’ measured
