Table Of ContentCurrent Topics in
Microbiology
and Immunology
198
Editors
A. Capron, Lille . R.W. Compans, Atlanta/Georgia
M. Cooper, Birmingham/Alabama· H. Koprowski,
Philadelphia· I. McConnell, Edinburgh· F. Melchers, Basel
M. Oldstone, La Jolla/California· S. Olsnes, Oslo
M. Potter, Bethesda/Maryland· H. Saedler, Cologne
P.K. Vogt, Los Angeles· H. Wagner, Munich
I. Wilson, La Jolla/California
Pathways for Cytolysis
Edited by G. M. Griffiths and J. Tschopp
With 45 Figures
Springer-Verlag
Berlin Heidelberg New York
London Paris Tokyo
Hong Kong Barcelona
Budapest
M.
GILLIAN GRIFFITHS
Basel Institute of Immunology
Grenzacherstr. 487
4005 Basel
Switzerland
JURG TSCHOPP
University of Lausanne
Department of Biochemistry
Ch. Boveresses 155
1066 Epalinges/Lausanne
Switzerland
Cover Illustration: The cover shows a human cytotoxic T
lymphocyte (CTL) clone which has recognised an EBV
transformed B cell target. The lytic granules of the CTL are
stained with an antibody to granzyme A (red), and visualised
using confocal microscopy. Courtesy of S. Issaz and G.M.
Griffiths.
Cover design: KLinke/+Lopka Werbeagentur, IIvesheim
ISSN 0070-217X
ISBN-13:97B-3-642-79416-2 e-ISBN-13:97B-3-642-79414-B
001: 10.1007/97B-3-642-79414-B
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Preface
At first glance the destruction of a target cell by a killer cell
seems to be a simple endeavor. A closer look, however, reveals
the complex mechanisms underlying this task. Killer cells are
able to specifically recognize altered or infected cells. A transient
contract with target cells has to be established to allow the
delivery of lethal molecules or signals. The killer cell then
disengages from the damaged cell and moves away to kill other
target cells. After the eradication of the target cells, the number
or activity of activated killer cells has to be reduced to avoid
nonspecific killing of innocent cells.
In 1992, Herman Eisen concluded, in his introductory remarks
in the most recent volume on lymphocyte cytotoxicity (EISEN
1993): "Given the immense amount of effort made in the past
decade to understand these (lytic) mechanisms, it seems
surprising that a consensus about the principal mechanisms has
not been reached." Since that time, advances made in the field
of cell-mediated cytolysis are so significant that our knowledge
regarding the lytic mechanisms has been considerably
augmented. Much of this is due to studies with transfectants
and mutants-either naturally occurring or generated by gene
targeting.
It is now clear that there are two predominant pathways
which act hand in hand and lead to efficienttarget cell destruction.
These pathways can be distinguished by the molecules which
mediate them, i.e., Fas, a member of the tumor necerosis fac
tor (TNF) receptor family, and perforin. The two pathways are
complementary, one requiring cell-cell contact and using a
regulated ligand able to lyse only Fas receptor-bearing cells, and
a second, perforin-granzyme-based, mechanism whose lytic
activity is dependent on the presence of phosphorylcholine,
thus allowing killing of virtually any target cell. It is possible that,
during evolution, the two cytolytic systems may have converged
in the same cytolytic cell to render it optimally equipped to lyse
all possible target cells.
VI Preface
The aim of this volume was to collect reviews covering the
different pathways for cytolysis: from the importance of
coreceptors in generating cytotoxic T lymphocytes (CTLs) to the
molecular details of the effects of lytic proteins on the cell cycle
which lead to apoptosis. The factors which induce the different
pathways and the cell biology of thier storage and release are
outlined. The results of the perforin knock-out mouse, the
biology of the TN F family of surface proteins, and the interactions
of the different lytic proteins, and their roles in contributing to an
efficient killing mechanism are discussed. What is known about
the mode of action of the different effector molecules on the
target cells is presented and the potential of the different
pathways in various disease and pathological states is explored.
We have attempted to present the reader with a current,
comprehensive overview of the different factors which contribute
to killing. It was definitely a gratifying task to put this volume
together in view of the new important findings described herein.
G.M. GRIFFITHS and J. TSCHOPP
Reference
Eisen H (1993) Introduction. In: Sitkowsky MV, Heukart PA (eds) Cytotoxic cells:
recognition, effector function, generation and methods. Birkhiiuser, Boston.
List of Contents
B. LOWIN, M.C. PEITSCH, and J. TSCHOPP:
Perforin and Granzymes:
Crucial Effector Molecules in Cytolytic T Lymphocyte
and Natural Killer Cell-Mediated Cytotoxicity ....
P. GOLSTEIN: Fas-Based T Cell-Mediated Cytotoxicity 25
G.M. GRIFFITHS and Y. ARGON:
Structure and Biogenesis of Lytic Granules 39
M. AZUMA and L.L. LANIER:
The Role of CD28 Costimulation Cytotoxic
in the Generation of T Lymphocytes .... 59
P.A HENKART, M.S. WILLIAMS, and H. NAKAJIMA:
Degranulating Cytotoxic Lymphocytes Inflict Multiple
Damage Pathways on Target Cells. . . . . . . . . . 75
AH. GREENBERG and D.W. LITCHFIELD:
Granzymes and Apoptosis: Targeting the Cell Cycle 95
E.R. PODACK: Perforin, Killer Cells and Gene Transfer
Immunotherapy for Cancer. . . . . . . . . . . . . . 121
P. ANDERSON: TIA-1 : Structural and Functional Studies on
a New Class of Cytolytic Effector Molecule . . . . . . . 131
R.C. BLEACKLEY, E.A ATKINSON, K. BURNS, and M. MiCHALAK:
Calreticulin: A Granule-Protein by Default or Design?. 145
H.G. MANNHERZ, M.C. PEITSCH, S. ZANoni, R. PADDENBERG,
and B. POLZAR: A New Function for an Old Enzyme:
The Role of DNase I in Apoptosis. . . . . . . . . . .. 161
VIII List of Contents
C.F. WARE, T.L. VANARSDALE, P.D. CROWE, and
J.L. BROWNING: The Ligands and Receptors of the
Lymphotoxin System 175
Subject Index . . . . 219
List of Contributors
(Their addresses can be found at the beginning of their respec
tive chapters.)
ANDERSON P. ....... 131 LOWIN B ............. .
ARGON Y. . . . ....... 39
MANNHERZ H.G. . . . . . .. 161
ATKINSON EA ...... 145
MICHALAK M. . . . . . . . .. 145
AZUMA M. ......... 59
NAKAJIMA H. .......... 75
BLEACKLEY R.C. ..... 145
BROWNING J.L. . . . . 175 PADDENBERG R. ........ 161
BURNS K.. . . . . .. ... 145 PEITSCH M.C.. . . . . . .. .. 1
PODACK E.R. .......... 121
CROWE P.O. . . . . . . .. 175
POLZAR B. ............ 161
GOLSTEIN P. . . . . . . . . 25
TSCHOPP J. . ......... .
GREENBERG A.H. . . . .. 95
GRIFFITHS G.M.. . . . . . 39 VANARSDALE T. L. ....... 175
HENKART PA . . . . . .. 75 WARE C.F.. . . . . . . . . . .. 175
WILLIAMS M.S. ........ 75
LANIER L.L. ........ 59
LITCHFIELD D.W. . . . . . 95 ZANoni S. . . . . . . . . . . .. 161
Perforin and Granzymes: Crucial Effector
Molecules in Cytolytic T Lymphocyte
and Natural Killer Cell-Mediated Cytotoxicity
B. LOWIN, M.C. PEITSCH*, and J. TSCHOPP
Lymphocytes and Natural Killer Cells .............. .
2 Target Cell Death ......................... . 3
3 The Perforin/Granule Exocytosis Model 3
4 Perforin ............... . 5
4.1 Structure.. . ....... . 6
4.2 Expression ............ . 9
4.3 Role in the Killing Process .. 10
4.3.1 Perforin Knock-Out Mice 10
5 Granzymes ............................................. . 12
5.1 Structure .. . 12
5.2 Substrate Specificities .... 13
5.3 Expression 16
5.4 Function 16
6 Perspectives 20
References . 20
1 Lymphocytes and Natural Killer Cells
The killing mediated by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells
represents an important mechanism in the immune defense against tumors,
virus-infected cells, parasites and other foreign invaders. The deleterious effects
observed in the absence of a properly working cellular immune response have
been characterized extensively in immunodeficient patients (for a review see
MATSUMOTO et al. 1992) and naturally occurring animal models such as SCID
(severe combined immunodeficiency) or nude mice. In general, immunodeficient
individuals are predisposed to develop severe opportunistic infections which
ultimately can lead to their death. Recently, gene targeting technology has been
used repeatedly to generate mice with defined mutations in genes implicated in
T and NK cell function (for a review see YEUNG et al. 1993). in an attempt to dissect
the individual pathways involved in the lymphocyte-mediated immune response.
Institute of Biochemistry. University of Lausanne, Ch. des Boveresses, 155, 1066 Epalinges,
Switzerland
* Present address: Glaxo Institute for Molecular Biology, 1021 Plan-Les-Ouates, Switzerland
2 B. Lowin et al.
Both ~2-microglobulin-deficient mice (KOLLER et al. 1990; ZIJLSTRA et al. 1990) and
CD8-deficient mice (FUNG-LEUNG et al. 1991) clearly showed the importance of
CDW T cells in virus clearance via their cytotoxic activity. In the initiation and
development of immunopathological disease processes, as in autoimmune
diseases, CD4+ T cells playa crucial role. In addition, CD8+ lymphocytes are also
found in autoimmune lesions. Their role and significance in the development of
autoimmunity, however, is still controversial; however, using mice lacking CD8
molecules, PENNINGER et al. (1993) demonstrated that CD8+ T cells may regulate
the disease severity in experimental autoimmune myocarditis.
T lymphocytes recognize antigens by means of T cell receptor (TCR)
glycoprotein complexes, in which the antigen-binding chains are encoded for by
rearranging gene families. Approximately 95% of blood T cells express the a/~
TCR, up to 5% have the y/b TCR. For T cells, the TCR/CD3 complex is necessary
yet not sufficient for antigen recognition. Another set of receptors, namely CD4
and CD8, is required. The generation of mice, deficient either in CD8 (FUNG-LEUNG
et al. 1991) or CD4 (RAHEMTULLA et al. 1991), has shown convincingly that CTLs,
which respond to antigenic challenge by lysis of their target, are recruited
predominantly from the CDW pool. CTLs are acquired by the host within a week
after infection and form a secondary immune defense. They playa central role in
antiviral immunity, but may also be of major importance in many bacterial and
protozoan infections. CD8-mediated protection has been shown for Listeria
monocytogenes, Mycobacterium tuberculosis, Plasmodium spp., Trypanosoma
cruzi and Leishmania major. It is, however, not yet fully understood if protection
is always associated with cytolytic activity or if it is provided through Interferon-y
(IFN-y) which is produced by most CDW T cells. Allograft rejection is another
situation in which T cell-mediated mechanisms are involved. A number of
experiments suggest a central role for CD4+ T lymphocytes in the rejection of
vascularized organ and skin allografts. Since endocrine cells, such as islet
allografts, do not express M HC class II molecules, their rejection is thought to be
dependent on CDW-mediated mechanisms (DESAI et al. 1993).
NK cells are a subpopulation of lymphocytes distinct from T and B cells that
have the ability to mediate cytotoxicity and cyt'lkine production. They represent
5%-15% of lymphocytes in the blood and are associated with a particular
morphology, that is, the large granular lymphocyte phenotype. NK cells are CD3-
S
(except for the chain), and they do not productively rearrange and express TCR
genes (for a review see TRINCHIERI 1989). They express the CD16 Fc receptor for
IgG on their cell surface, through which they can mediate ADCC (antibody
dependent cell cytotoxicity) of antibody-coated target cells. In vivo, NK cells
mediate rejection of transplanted tumor cells which is well reflected in in vitro
assays. NK cells kill a variety of tumor targets, while normal cells are usually
resistant. NK cells can also reject nonautologous bone marrow cells and
lymphocytes (Yu et al. 1992). The first phase of certain virus infections (such as
Cytomegalovirus) represents a third case in which NK cells can mediate natural
resistance in vivo. NK cells have also been implicated in resistance to certain
bacterial and parasite infections (TRINCHIERI 1989).
