The Thymus in Immunity

Immunobiology
PRINCIPLES OF MEDICAL BIOLOGY
A Multi-Volume Work, Volume 6
Editors: E. EDWARD BITTAR, Department of Physiology,
University of Wisconsin, Madison
NEVILLE BITTAR, Department of Medicine,
University of Wisconsin, Madison
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Principles of IVIe
A Multi-Volume Work
Edited by E, Edward Bittar, Department of Physiology,
University of Wisconsin, Madison and
Neville Bittar, Department of Medicine
University of Wisconsin, Madison
This work provides:
* A holistic treatment of the main medical disciplines. The basic sciences including most of the achievements in cell and molecular
biology have been blended with pathology and clinical medicine.
Thus, a special feature is that departmental barriers have been
overcome.
* The subject matter covered in preclinical and clinical courses has
been reduced by almost one-third without sacrificing any of the
essentials of a sound medical education. This information base
thus represents an integrated core curriculum.
* The movement towards reform in medical teaching calls for the
adoption of an integrated core curriculum involving small-group
teaching and the recognition of the student as an active learner.
* There are increasing indications that the traditional education system in which the teacher plays the role of expert and the student
that of a passive learner is undergoing reform in many medical
schools. The trend can only grow.
* Medical biology as the new profession has the power to simplify
the problem of reductionism.
* Over 700 internationally acclaimed medical scientists, pathologists, clinical investigators, clinicians and bioethicists are
participants in this undertaking.
This Page Intentionally Left Blank
Immunobiology
Edited by E. EDWARD BITTAR
Department of Physiology
University of Wisconsin
Madison, Wisconsin
NEVILLE BITTAR
Department of Medicine
University of Wisconsin
Madison, Wisconsin
( ^
Greenwich, Connecticut
jAI PRESS INC.
London, England
Library of Congress Cataloging-in-Publication Data
Immunobiology / edited by E. Edward Bittar, Neville Bittar.
p. cm.—(Principles of medical biology ; v. 6)
Includes index.
ISBN 1-55938-811-0
1. Immunology.
2. Molecular immunology.
I. Bittar, E. Edward.
II. Bittar, Neville.
III. Series.
[DNLM: 1. Immune System.
2. Immunity.
QW 50413223 1996]
QR181.I454
1996
616.07'9—dc20
DNLM/DLC
96-35160
for Library of Congress
CIP
Copyright © 1996 by JAI PRESS INC.
55 Old Post Road, No. 2
Greenwich, Connecticut 06836
JAI PRESS LTD.
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Covent Garden
London, England
All rights reserved. No part of this publication may be reproduced,
stored on a retrieval system, or transmitted in any form or by any
means, electronic, mechanical, photocopying, filming, recording, or
otherwise, without prior permission in writing from the publisher.
ISBN: 1-55938-811-0
Library of Congress Catalog No.: 96-35160
Manufactured in the United States of America
CONTENTS
List of Contributors
ix
Preface
£ Edward Bittar and Neville Bittar
Chapter 1
The Thymus in Immunity
J.FA.P. Miller
xii i
1
Chapter 2
The B-Cell in Immunity
David Tarlinton
21
Chapter 3
Cell-to-Cell Interactions in the Immune System
William A. Sewell and Ronald Penny
47
Chapter 4
Immunological Tolerance
J.FA.P. Miller
63
Chapter 5
The Generation of Diversity in the Immune
System
E.J. Steele and H.S. Rothenfluh
Chapter 6
The Antigen-Antibody Complex: Structure and
Recognition
P.M. Colman
vii
85
107
viii
CONTENTS
Chapter 7
The Major Histocompatibility Complex
Brian D.Tait
121
Chapter 8
B and T Cell Signaling at the Molecular Level
Tomas Mustelin and Paul Bum
137
Chapter 9
Cytokines in Immunology
Andrew J. Hapel and Shaun R. McColl
151
Chapter 10
Activation and Control of the Complement System
B.Paul Morgan
Chapter 11
Phagocytes in Immunity and Inflammation
Philip ISA. Murphy
171
197
Chapter 12
Anaphylaxis
Caiman Prussin and Michael Kaliner
231
Chapter 13
Autoimmunity and Autoimmune Disease
Sudershan K. Bhatia and Noel R. Rose
239
Chapter 14
Cell Death and the Immune System
R.M. Kluck and].W. Halliday
265
Chapter 15
Designer Antibodies
Andy Minn and Jose Quintans
281
Chapter 16
Psychoneuroimmunology
Ruth M. Benca
303
INDEX
315
LIST OF CONTRIBUTORS
Ruth M, Benca
Department of Psychiatry
University of Wisconsin
Madison, Wisconsin
Sudershan K. Bhatia
Department of Immunology and
Infectious Diseases
The John Hopkins University School of
Hygiene and Public Health
Baltimore, Maryland
Paul Bum
Department of Biology
Hoffmann-La Roche Inc.
Nutley, Ne Jersey
P.M. Colman
CSIRO Division of Biomolecular
Engineering
Parkville, Victoria, Australia
J.W. Halliday
Liver Unit
Queensland Institute for Medical
Research
Queensland, Australia
Andrew J. Hapel
Experimental Haematology Group
John Curtin School of Medical Research
Australian National University
Canberra, Australian Capital
Territory, Australia
Michael
Institute for Asthma and Allergy
Washington, D.C.
Kaliner
R.M. Kluck
Liver Unit
Queensland Institute for Medical
Research
Queensland, Australia
IX
LIST OF CONTRIBUTORS
J.FA,P. Miller
The Walter and Eliza Hall Institute of
Medical Research
Royal Melbourne Hospital
Melbourne, Victoria, Australia
Andy Minn
Department of Pathology
The University of Chicago
Chicago, Illinois
B. Paul Morgan
Department of Medical Biochemistry
University of Wales College of Medicine
Heath Park, Cardiff, Wales
Philip M. Murphy
The Laboratory of Host Defenses
National Institute of Allergy and
Infectious Diseases
National Institutes of Health
Bethesda, Maryland
Tomas Mustelin
La Jolla Institute for Allergy and
Immunology
La Jolla, California
Ronald Penny
Centre for Immunology
St. Vincent's Hospital and University of
New South Wales
Sydney, New South Wales, Australia
Caiman Prussin
National Institute of Allergy and
Infectious Diseases
National Institutes of Health
Bethesda, Maryland
Jose Quintans
Department of Pathology
The University of Chicago
Chicago, Illinois
Noel R. Rose
Department of Immunology and
Infectious Diseases
The Johns Hopkins University School of
Hygiene and Public Health
Baltimore, Maryland
List of Contributors
XI
H.S. Rothenfluh
Division of Immunology and Cell Biology
The John Curtin School of Medical
Research
Australian National University
Canberra, Australia
William A. Sewell
Centre for Immunology
St. Vincent's Hospital and University of
New South Wales
Sydney, New South Wales, Australia
E.I. Steele
Department of Biological Sciences
University of Wollongong
Wollongong, New South Wales, Australia
David
The Walter and Eliza Hall Institute of
Medial Research
Royal Melbourne Hospital
Melbourne, Victoria, Australia
Tarlinton
Brian D. Tail
Tissue Typing Laboratories
Royal Melbourne Hospital
Parkville, Melbourne, Australia
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PREFACE
As this volume demonstrates, immunobiology is a young science which is undergoing explosive growth. Judged by results, it is already an elaborate discipline
which cuts across every other area in biomedical research and even has its own
vocabulary (e.g., the “veto” effect). Rather than inculcate the habit of superficial
learning by having the student go through a maze of details, we have sought to
gather together sixteen essays that range from T-cells to psychoneuroimmunology.
This is in keeping with the growing understanding that the student is expected to
read and think far more for herselfhimself.
Next to nothing is known about innate immunity. However, recent evidence
suggests that collectins might bridge the gap between innate immunity and specific
clonal immune responses. Collectins are soluble effector proteins that include
serum mannose-binding protein, and lung surfactants A and D. They are considered
to be ante-antibodies.
Our most grateful thanks are due to the contributors who have made this volume
possible. They are also due to Ms. Lauren Manjoney and the production staff of
JAI Press for their skill and courtesy.
E. EDWARD BITTAR
NEVILLE BITTAR
xiii
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Chapter 1
The Thymus in Immunity
J.F.A.P. MILLER
Introduction
Historical Background
Antigen Recognition and the Major Histocompatibility Complex (MHC)
Peripheral T Cell Subsets
T Cell Migration
Recirculation of Naive T Cells
Tissue-Selective Homing of Activated and Memory T Cells
Intrathymic Events
The Thymus in Disease States
Summary
Recommended Readings
Principles of Medical Biology, Volume 6
Immunobiology, pages 1-20.
Copyright © 1996 by JAI Press Inc.
All rights of reproduction in any form reserved.
ISBN: 1-55938-811-0
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2
J.F.A.P. MILLER
INTRODUCTION
The cells in the immune system responsible for specifically targeting and causing
the removal of foreign material or antigen are known as lymphocytes. They
circulate in blood and lymph and populate areas of the body known as lymphoid
tissues which include the spleen, lymph nodes, thymus, tonsils, adenoids, and
Peyer's patches, the last three being located along the alimentary tract.
The thymus in mammals is situated in the upper part of the thoracic cavity where
it overlies the heart and some of the major blood vessels (Figure 1). It is unique
among lymphoid tissues, both as regards structure and function. Relatively large
in the infant, its maximum size is reached at the time of puberty, after which it
regresses slowly, becoming reduced to little more than a vestigial structure in old
age. It is divided into lobules each with a central part or medulla and a peripheral
part or cortex (Figure 2). The main types of cells are the lymphocytes and the
so-called stromal cells which include the cells of the epithelial framework and of
the dendritic-macrophage lineages. T cell precursors (derived from fetal liver or
later from bone marrow) enter from vessels at the cortico-meduUary junction and
first associate with macrophages. Two or three days later they are found m the
subcapsular cortex. They eventually give rise to more differentiated thymus lymphocytes.
The cell composition of the thymus may be divided into three distinct layers. (1)
In the outer cortex, beneath the capsule, is a layer of dividing primitive lymphocytes
(lymphoblasts), which constitute 5 to 15% of the total thymic lymphocyte population. Some lymphoblasts interact with specialized epithelial cells, the "nurse cells,"
which promote their proliferation and differentiation to more mature smaller forms.
(2) The newly derived lymphocytes migrate from the cortex towards the medulla.
In the deep cortex are three major classes of cells: small lymphocytes, dendritic
cortical epithelial cells and macrophages. The lymphocytes have a thin rim of
cytoplasm, make up about 80 to 85% of the thymic lymphocyte population and are
Thyroid
Figure 1. The location of the thymus in the chest.
The Thymus in Immunity
capsule
subcapsular blasts - c O .
nurse cells
small
cortical
thymus
lymphocytes
cortical
dendritic
epithelial
cell
medullary
epithelial
cells
Q
MEDULLA
O
interdigitating
dendritic cells
medullary
thymus
lymphocytes
Figure 2. Structure of the thymus. Diagram to show cellular architecture of the
thymus (see text).
in intimate contact with the dendritic epithelial cells. These have long processes
and are connected to one another by junctions known as desmosomes. They may
be involved in selecting the T cell repertoire (see later and Figure 11). Interspersed
among the network of dendritic epithelial cells are macrophages which engulf the
many lymphocytes that have died or are destined to die. On the medullary side of
the cortico-meduUary junction lie structures called Hassall's corpuscles which
constitute the final graveyards for the massive numbers of dying lymphocytes. (3)
The medulla contains medium sized thymic lymphocytes, macrophages, spatulate
medullary epithelial cells and bone-marrow derived interdigitating dendritic cells.
The latter are most conspicuous near the cortico-meduUary junction and are
involved in negative selection of those lymphocytes which have the potential to
inflict damage on the body's own tissues, the so-called self-reactive lymphocytes
(see below). Some medullary mature T cells may be derived partly from the
intrathymic maturation process and partly from extrathymic circulating T cells.
The proportion of lymphocytes undergoing cell division (mitosis) is much higher
in the thymus than in any other lymphoid tissues throughout the life of the
individual. Furthermore, thymus lymphocyte mitotic activity, unlike similar activity elsewhere, is not dependent on antigenic stimulation but is preprogrammed and
hence controlled intrinsically (from within the thymus).
During development, the thymus, unlike other lymphoid tissues, is a purely
epithelial organ. Lymphocytes first appear in the epithelial network at about 10
4
J.F.A.P. MILLER
weeks of gestation in the human and 12 days in the mouse. They are derived by
differentiation of hemopoietic ancestral or stem cells which enter from the blood
stream. It is only much later that lymphocytes make their appearance in other
lymphoid organs. The thymus is thus often referred to as a primary or central
lymphoid organ and the other lymphoid tissues as secondary or peripheral.
When animals are immunized by antigen, characteristic cellular changes occur
in lymph nodes and spleen. For example, small lymphocytes enlarge to larger
"blast" cells which stain with a particular RNA-staining dye (methyl green pyronin). These undergo mitosis and antibody forming "plasma cells" accumulate in
certain areas. None of these antigen-induced changes have ever been found in the
intact thymus of immunized animals under normal conditions. These findings
raised questions as to whether the thymus played any role in immunity.
HISTORICAL BACKGROUND
Prior to I960, the functions of the thymus and its lymphocytes were obscure. By
contrast, the circulating small lymphocytes, as found in blood, lymph and lymphoid
tissues, were proven to be immunologically competent by the work that Gowans
and his collaborators performed in the late fifties and early sixties (Gowans, 1961).
Yet although the thymus was known to be a lymphocyte-producing organ, immunologists did not consider it to have any immunological function. This may have
been because some investigators, for example. Good and his collaborators
(MacLean et al., 1957), concluded from experiments, in which the thymus was
removed from adult rabbits, that they had obtained "evidence that the thymus gland
does not participate in the control of the immune response." In the early sixties,
Medawar (1963) even suggested that "we shall come to regard the presence of
lymphocytes in the thymus as an evolutionary accident of no very great significance." What then was responsible for reversing the tide?
In the late fifties and early sixties, Miller, then working with a leukemogenic
virus of mice, surgically removed the thymus (thymectomized) of newborn (neonatal) mice to determine whether the virus, when introduced at birth, had first to
multiply in thymus tissue. He found that neonatally thymectomized mice died
prematurely from causes unrelated to leukemia induction and suggested "that the
thymus at birth may be essential to life" (Miller, 1961a). Further experiments
showed clearly that mice thymectomized at one day of age, but not later, were
highly susceptible to infections, had a marked deficiency of lymphocytes in the
circulation and in lymphoid tissues and were unable to reject skin grafts taken from
incompatible mice of other strains (Miller, 196 lb). These results led to the hypothesis that "during embryogenesis the thymus would produce the originators of
immunologically competent cells many of which would have migrated to other sites
at about the time of birth. This would suggest that lymphocytes leaving the thymus
are specially selected cells" (Miller, 1961b). In adult mice, thymectomy had for
long been known not to have any untoward effects. Miller (1962a), however.
The Thymus in Immunity
exposed adult thymectomized mice to total body irradiation which partially destroyed the lymphoid system and was able to show that the recovery of lymphoid
and immune functions was thymus-dependent. Implanting thymus tissue into
neonatally thymectomized or adult thymectomized and irradiated mice allowed a
normal immune system to develop. When the thymus graft came from a foreign
strain, the neonatally thymectomized recipients failed to reject skin from mice of
the strain that had donated the thymus, although they could reject skin graft from
other incompatible strains. This led to the suggestion that "when one is inducing a
state of immunological tolerance in a newly born animal," for example by the
classical technique of injecting foreign bone marrow cells at birth (see Chapter 4),
"one is in effect performing a selective or immunological thymectomy" (Miller,
1962b). Thus, lymphocytes developing in the thymus in the presence of foreign
cells would be deleted, implying that the thymus should be the seat where tolerance
to the body's own tissues (self tolerance) is imposed. Some of these findings were
soon confirmed by groups working independently, notably those headed by
Waksman and by Good (Amason et al., 1962; Martinez et al., 1962).
In the late fifties and early sixties, only a single variety of lymphocyte was
believed to be involved in performing all types of immune responses in mammalian
species. In birds, however, it seemed that two distinct subsets of lymphocytes
performed those immune responses mediated by antibody (the "humoral" immune
responses) and those in which cells, but not antibody, were involved (the "cellmediated" immune responses). The latter include transplant rejection, delayedhypersensitivity reactions such as tuberculin sensitivity, and killing or "lysis" of
target cells. The finding of a division of labor among avian lymphocytes was first
reported by Szenberg and Warner (1962) using newly hatched chicks: surgical
removal of the bursa (an organ found only in birds and analogous to the thymus but
situated near the cloaca) soon after hatching was associated with defects in antibody
formation and early thymectomy with defects in cellular immune responses. Since
mice do not have a bursa and since neonatal thymectomy in that species prevented
both cellular and most humoral immune responses, it was widely believed that the
mammalian thymus fulfilled the fimctions of both the avian thymus and bursa. A
hint that two distinct lymphocyte subsets may indeed be involved in immune
responses in mice, however, came from the experiments of Claman and his
colleagues in 1966. They showed that irradiated mice receiving a mixed population
of marrow and thymus cells produced far more antibody than when given either
cell source alone. Having no genetic markers on their cells, they could not, however,
determine whether the antibody-forming cells were derived from the thymus or the
marrow. In independent investigations, (Miller and Mitchell, 1967,1968; Mitchell
and Miller, 1968) introduced genetically marked cells into neonatally thymectomized or thymectomized irradiated hosts and established beyond doubt and for the
first time that antibody-forming cell precursors (subsequently known as B cells)
5
6
J.F.A.P. MILLER
were derived from bone marrow, and that thymus-derived cells (now called T cells)
were essential to help B cells to respond to antigen by producing antibody.
The existence of two distinct lymphocyte subsets, T and B cells, was not only
confirmed but led to a re-investigation of numerous immunological phenomena
including memory, tolerance, autoimmunity, and genetically determined unresponsive states. T cells were clearly responsible for the "cell-mediated" immunities, and
T cells were themselves soon subdivided into subsets based on function, cell surface
markers and secreted products or "lymphokines."
In 1957, prior to the discovery of T and B cells, Burnet postulated that lymphocytes had predetermined reactivities. A cell with a receptor that best fitted a given
antigenic determinant is selected by that antigen and activated to divide producing
a clone of daughter cells, all with the same specificity (Figure 3). The antigen
receptor on the membrane of these progeny cells would be identical in its binding
site to the antibody eventually secreted by members of the clone. The theory has
stood the test of time and for B cells, it was clear that the antigen recognition unit
or receptor was an accurate sample of the antibody or immunoglobulin (Ig) which
that cell would produce after successfiil antigenic stimulation. It was also found
that a small proportion of naive B lymphocytes could specifically bind labeled
antigen and that this binding could be blocked by antibody directed against the
immunoglobulin receptor itself Yet T cells could never be shown to bind antigen
Clonal Selection
lymphocytes
^^R
antigen
^A
^^
lymphocyte-antigen
interaction
lymphocyte proliferation
and differentiation
clone
antibody
k 1 i 'A^
'
Jli\
Figure 3. Burnet's clonal selection theory. The antigen-specific receptor is unique on
mature lymphocytes. A cell with a receptor into which a given antigenic determinant
can be accommodated is selected by the antigen to divide and produce a clone of
daughter cells, each with the same antigen specificity. In the case of B cells, as shown
in this diagram, the membrane receptor is identical in its binding site to the antibody
which members of the stimulated clone will eventually secrete.
The Thymus in Immunity
7
and great controversy raged for many years over the nature of the antigen receptor
on T cells.
ANTIGEN RECOGNITION AND THE MAJOR
HISTOCOMPATIBILITY COMPLEX (MHC)
Unlike B cells, T cells perceive, not naked antigen, but antigen presented on the
surface of other cells. Highly visible to T cells are molecules encoded by the major
histocompatibility complex (MHC), a series of genes which code for molecules on
the surface of a variety of cell types (see Chapter 7). They provoke violent rejection
reactions on the part of responding T cells and are perfect targets for killer or
cytotoxic T cells. Following virus infection and virus entry into cells, as first shown
by Zinkemagel and Doherty in 1974, T cells recognize not just the virus derived
antigenic determinants, but these in association with MHC-encoded molecules on
CLASS
peptide groove
CLASS II
peptide groove
^2^
Figure 4, Class I and class II MHC molecules. The class I molecule is composed of
two polypeptide chains. The heavy chain has 3 external domains, a l , a2 and a3, a
transmembrane portion (TM) and a cytoplasmic tail (CY). It is associated in its
extracellular portion with the light chain, p2-microglobulin (p2m), a molecule not
encoded by the MHC gene locus. The polymorphic regions of the class 1 heavy chain
are those where the amino acid sequences of the polypeptide chain differ among
unrelated individuals. They are situated in the a1 and a2 domains which form a groove
that can accommodate peptide fragments derived from the processing of proteins
synthesized within the cell (e.g., self proteins or virus-derived proteins in virus infected
cells). The class II molecules are composed of two polypeptide chains, a and p, both
encoded by the MHC gene locus. Each chain spans the membrane and hence has a
transmembrane region, a cytoplasmic tail and an extracellular portion. Both the a and
the p chains have two external domains, a1, a2, and p i , p2. The polymorphic regions
lie in the a1 and pi portions which also form a groove into which can be accommodated peptides generally derived from proteins taken up by the cell from the external
milieu (see also Figure 5). A separate class of antigens known as "superantigens'' (e.g.,
certain bacterial toxins) bind not to the groove of the class II molecules but to the
external face of the domains and to the p chain of the TCR.
J.F.A.P. MILLER
8
the cell surface. This phenomenon became known as MHC restriction and the MHC
molecules involved as restriction elements.
The MHC molecules that serve as targets of T cell responses occur in two major
forms, termed class I and II (Figure 4). The former are found on most tissue cells
and are composed of two noncovalently linked polypeptide chains—SL heavy one
(molecular weight 45 kD) spanning the cell membrane and having three extracellular portions or domains ( a l , a2 and a3), and a lighter chain termed p2-microglobulin. This does not span the membrane and is encoded by a gene distinct from
the MHC genes. The class II molecules consist of two noncovalently linked a (28
kD) and P (34 kD) chains, both encoded by the MHC and both having two
extracellular domains. The distribution of class II molecules is restricted mostly to
B cells, macrophages, and dendritic cells. Both class I and II molecules exhibit a
striking degree of structural variation or polymorphism within individuals of the
same species. The polymorphic regions of the molecules, where there are differEndogenous
pathway
r
peptides O
degradation
class I
I
proteins
\ \
endogenous synthesis
Exogenous
pathway
JL
r
t
[o]*-(cO**—O exogenous
antigen
endocytosis
class 11
Figure 5. Antigen-presenting cell (APC) and processing pathways. Professional ARC
present processed antigen in association with MHC class I and ii molecules. Two
pathways of antigen processing operate: they are referred to as endogenous and
exogenous. (1) Some proteins synthesized by the APC are chopped into fragments
(degraded into peptides) by cellular enzymes. Most newly synthesized class I molecules are unstable unless peptide is associated with them. The binding of peptides to
M H C class I molecules occurs in an intracellular compartment known as the endoplasmic reticulum and the peptide-MHC complex can then be transported to the surface.
This particular route is known as the endogenous pathway. (2) External antigens taken
up by the APC ("endocytosis") are degraded In compartments known as endocytic
vesicles which fuse with other vesicles that contain class ii but not class I molecules.
This type of transport is referred to as the exogenous pathway.
The Thymus in Immunity
9
ences in amino acid sequences among unrelated individuals, are situated in the a l
and a2 domains of the class I molecules and the a l and pi domains of the class II
molecules. These domains form a groove or pocket capable of binding fragments
derived from the enzymatic degradation or processing of self or foreign components
(Bjorkman et al., 1987; Brown et al., 1993). Such fragments derived from protein
antigens are known as peptides and are made up of short sequences of amino acids
with a carboxyl end or "terminus" and an amino terminus. Cells which perform the
processing task and transport the peptide-MHC complex to their cell membrane
where T lymphocytes can examine them, are termed professional antigen-presenting cells (APC) (Figure 5).
The antigen-specific receptor on T cells (the "TCR") has specificity for both the
peptide and the external surface of the MHC molecule which accommodates the
peptide (Davis and Bjorkman, 1988). Most TCRs are composed of two disulfidelinked polypeptide chains, a and p (Figure 6), although less common TCR use other
chains termed y and 5. Each chain has a constant amino acid sequence in its carboxyl
terminus (C) and a variable sequence in its amino terminus (V). Other molecules
intimately associated with the TCR are the CD3 complex composed of three
polypeptide chains (y, 5, and s) and the so-called q-q "homodimer" composed of a
pair of identical polypeptide chains. The CDS and q-q molecules are essential for
TCR
extracellular
membrane
cytoplasm
Figure 6. The antigen-specific T cell receptor (TCR) and associated CD3 and q-q
complexes. The antigen-specific TCR is composed of two disulfide-linked polypeptide
chains, a and |3. Each chain has a constant amino acid sequence in its carboxyl
terminus (C) and a variable sequence in its amino terminus (V). The CD3 complex,
composed of three polypeptide chains y, 6, and 8, and the homodimer q-q are
intimately associated with the TCR and are involved in TCR assembly and signal
transduction when the TCR has bound a peptide-MHC complex.
J.F.A.P. MILLER
10
the assembly and transport to the cell surface of the TCR and play a role in
transducing signals after occupation of the TCR by a peptide-MHC complex.
The TCR chains are encoded by several genes which rearrange during T cell
development and contribute to the great diversity of specificities associated with
TCRs (Davis and Bjorkman, 1989). This is described in detail in Chapter 5. Briefly,
individuals inherit from their parents sets of "germline genes" which code for the
combining site of antigen-specific receptors on both T and B cells. A variety of
mechanisms then operate during T and B cell differentiation to rearrange and join
together the germline elements and eventually give rise to the active gene which is
a mosaic of these units. Hence, an enormous diversity can be generated and a great
variety of antigen-specific receptors is made available to ensure that lymphocytes
can recognize an infinite number of antigenic determinants.
PERIPHERAL T CELL SUBSETS
Although almost all T cells bear the Thy-1 marker, they are heterogeneous with
respect to function and other cell surface markers. The two major subsets of T cells
are termed CD4'" and CD8"^ T cells (Figure 7). The former are characterized by the
presence on the membrane of the CD4 molecule and act as "helper" cells by
assistmg B cells in producing certam types of antibody. The collaboration between
T and B cells is described in Chapter 3, The CDS"^ T cells have CDS molecules on
Figure 7. The CD4 and CDS co-receptor molecules on T cells. The CD4 and CD8
molecules characterize mature peripheral T cells which recognize peptides m association with MHC class II and class I molecules, respectively. The CD8 molecule has
an affinity for specific sites on the a3 domain of the class I molecule and the CD4 for
some sequences on the nonpolymorphic portion of the class 11 molecule.
The Thymus in Immunity
11
their surface and, after direct contact with their target cells, act as killer or cytotoxic
cells destroying foreign cells and cells infected by viruses. In some situations CDS"^
T cells require help from 004"" T cells for cytotoxic activity. The CD4 and CDS
molecules are "coreceptors" as they act m concert with the TCR. The CD4
co-receptor has a binding site specific for a portion of the MHC class II molecule
and the CDS co-receptor has one specific for a part of the a3 region of the class I
molecule. Co-aggregation of CD4 or CDS molecules with the CD3-(;-c; complex
and the TCR, once bound to its specific peptide-MHC complex, initiates a signaling
cascade to "activate" the T cell, turning on its functional and lymphokine-secretion
machinery (Janeway, 1992). T cell-derived lymphokines control the differentiation
of a wide variety of cells of the hemopoietic and lymphoid systems and are active
in initiating inflammatory responses such as delayed-type hypersensitivity.
CD4'^ T helper (Th) cells are themselves heterogeneous in terms of their lymphokine release pattern. Although the naive CD4 cell can synthesize a variety of
lymphokines immediately after activation, the way in which antigen is presented
by different cells eventually restricts the secretion pattern. Thus Th cells can be
divided into ThO (which can secrete various lymphokines) and into further differentiated forms known as Thl and Th2 cells (Mosmann and Coffman, 1989).
Although both these cells can secrete the lymphokines IL-3, GM-CSF and TNF-a,
they differ in their pattern of release of other lymphokines. Thus, Thl cells produce
interferon-y and interleukin-2 while Th2 cells secrete interleukin-4, 5, 6, 10, and
13. Thl and Th2 can antagonize each other thus playing a role in immunoregulation
(see also Chapters 4 and 9).
Under certain conditions, T cells can suppress the responsiveness of other
lymphocytes. Whether such "suppressor T cells" exist as a distinct subset or reflect
the production of inhibitory cytokines, such as TGF-(3 (A. Miller et al., 1992), has
yet to be established.
T cells can also be subdivided according to their previous antigenic experience.
Those which have not met antigen are termed "naive" or "virgin" cells and are
characterized by the presence of distinct molecules on their surface. For example,
they express the high molecular weight forms of the CD45 molecule (notably
CD45RA), low levels of the molecule known as CD44 and high levels of L-selectin
(also called MEL-14). Those T cells which have been stimulated by antigen are the
progeny of naive T cells and are large "blasts" known as effector or activated T
cells. They may become small "memory" cells whose existence may depend on
continuous antigenic stimulation. Both activated and memory T cells exhibit on
their cell membrane the CD45RO molecules, high levels of CD44, low levels of
L-selectin and various adhesion molecules such as LFA-1 and CD2. All these
molecules are involved in various T cell functions including intracellular signaling,
adhesion to APC or to cells lining blood vessels ("endothelial cells") (Mackay,
1993; Sprent, 1993). Some of the molecular interactions occurring between specific
T cells and professional APC are shown in Figure S.
J.F.A.P. MILLER
12
APC or
target cell
Figure 8. Adhesion and co-stimulatory molecules involved during the interaction of
T cells with TCR specific for an MHC-peptide complex presented by a professional
APC. Cell surface molecules expressed on T cells may play a role in immune responses
by functioning as receptors for cell surface molecules expressed on APC. The
interaction of such molecules may strengthen the binding between the T cell and the
APC and may be Involved in transmembrane signals initiated by TCR occupancy or
independent of the TCR. The B7 molecule is characteristically expressed by professional APCs and its binding to the T cell's CD28 molecule produces a powerful
co-stimulator signal to ensure that the T cell becomes fully activated following the
binding of its own TCR to the MHC-peptide complex presented by the APC.
As stated above, most T cells utilize TCR a and P genes but a smaller subset use
the genes y and 6. Some 76 cells exist in certain epithelial environments and, unlike
aP T cells, exhibit a highly restricted TCR specificity. They may thus express
invariant TCRs and perform totally different tasks. Much remains to be learned
about their functions.
T CELL MIGRATION
The total number of cells released from the thymus is small being of the order of 1
to 2 million per day in young mice. Output is maximal at an early age and declines
when thymic atrophy sets in, reaching very low levels in old age. The cells leaving
the thymus are typical CD4^ and CDS"*" T cells which may have to undergo a further
period of maturation during several days. The emigrants migrate non-randomly
along well defined routes, the actual pathway depending on whether the T cells are
naive or activated.
The Thymus in Immunity
13
Recirculation of Naive T Cells
Naive small T cells recirculate from blood through lymphoid tissues and back to
blood directly or via the lymph. They have a long lifespan and do not divide unless
stimulated by antigen. Recirculation allows naive T cells to patrol the body and
home in on sites in lymph nodes and spleen which have trapped antigens and
invading micro-organisms.
Naive T cells have specialized receptors ("L-selectin") which allow them to bind
to distinct molecules on the surface of endothelial cells lining specialized venules
in the lymph nodes known as "post-capillary" or "high endothelial" venules (HEV)
(Figure 9). They then enter lymph nodes through a region known as the paracortex
which contains a network of specialized APCs including the so-called dendritic
cells. This area of the node is known as the T-cell dependent area and antigens
which provoke cellular immunity produce histological changes in this area, the
small T cells enlarging to large blasts which divide. Other areas of the lymph nodes,
including the follicles are known as the B-cell dependent areas (see Chapter 2).
After traversing the paracortex, the recirculating T cells enter the medulla of the
nodes and leave by efferent lymphatics which drain into other lymph nodes or end
up in the thoracic duct. This large vessel empties into a major blood vessel in the
neck.
The spleen is divided into red and white pulps (Figure 10), the former containing
many hemopoietic cells such as red blood cells, the latter being populated by
lymphocytes. The spleen does not have significant lymphatics and T cells enter via
the splenic artery which terminates in a loose network of vessels in the red pulp.
The T cells migrate to the area of the white pulp around the arterioles (the
"periarteriolar lymphocyte sheath" or PALS), a T-cell dependent area rich in APCs
afferent lymphatic
subcapsular sinus
primary follicle
(B area)
cortex
medulla
medullary cord
(B area)
medullary sinus
efferent lymphatic
Figure 9, Microanatomy of a lymph node (see text).
I.F.A.P. MILLER
14
red pulp
marginal
sinus
central
arteriole
t
primary
follicle
(B area)
marginal
zone
(B area)
Figure 10. Microanatomy of a section of the spleen (see text).
including dendritic cells, and the B cells migrate to the follicles in the white pulp.
T cells leave the spleen by going to the red pulp and entering tributaries of the
splenic vein.
If recirculating T cells encounter antigen presented by APCs in the T-cell
dependent areas of lymph nodes or spleen, those T cells with TCR specific for the
antigen are sequestered from the circulating pool and activated to proliferate and
to produce effector cells which perform the cell-mediated immune responses
(Sprent et al., 1971).
Tissue-Selective Homing of Activated and Memory T Cells
T cells homing to the alimentary canal ("gut-tropic cells") are either activated
blasts or smaller CD45RO"^ memory-type cells. After antigen activation, T cells
downregulate the expression of L-selectin which was present on naive T cells and
instead express the a4(37 adhesion molecule ("integrin"). This allows them to bind
to specific molecules on endothelial cells found in gut mucosa and gut-associated
lymphoid tissues and thus to enter such tissues.
The skin represents a major entry point for microorganisms. T cells that home to
the skin are almost exclusively of the memory type and express the a4pi integrin.
This serves as an adhesion receptor for the molecule E-selectin which is found in
inflamed skin.
The Thymus in Immunity
15
Memory-type T cells predominantly localize to inflamed sites. The inflammatory
response can also affect lymph nodes and antigen stimulation induces the expression of defined vascular adhesion molecules on the endothelial cells of HEV. This
in turn allows a marked increase in the migration of memory-type T cells to the
node.
The different migration behavior of naive and effector or memory T cells ensures
that the immune system provides a most economical way of displaying its resources
to fight against dangerous intruders.
INTRATHYMIC EVENTS
Most T cells arise in the thymus as a result of the programmed differentiation of
incoming stem cells. There is little evidence for extra-thymic production of T cells.
Since T cells are specialized to recognize antigenic determinants in association with
self MHC molecules, the thymus must provide a repertoire of T cells by selecting
those cells with TCR that have some degree of specificity for these MHC molecules.
But because TCR specificities are randomly generated and there is extensive MHC
polymorphism among individuals of a particular species, the specificities of T cells
in the preselected pool of differentiating thymus lymphocytes must by chance be
directed to all the MHC molecules expressed in the species. Most thymocytes will
therefore lack the correct specificity and hence will be unsuitable. This presumably
accounts for the vast numbers of thymocytes generated each day (about 10^ in
mice), the massive rate of cell death and the small number exported to the periphery
(about 10^ per day).
The earliest T cell precursor derived from stem cells entering the thymus is
characterized by the surface expression of the molecule CD44 and very low levels
of CD4. Soon after, these early cells lose the CD4 marker and transiently express,
in addition to CD44, the CD25 molecule (which is a receptor for the interleukin,
IL-2). At this stage, the cells are referred to as "double negative (DN) cells" because
they lack expression of both CD4 and CDS which characterize mature T cells. The
DN cells then lose CD44 expression and begin to rearrange and express the (3 chain
genes of the TCR. This is followed by rearrangement of the TCR a locus,
expression of low levels of the aP TCR on the surface and loss of CD25. A separate
subset of DN cells rearrange the y and 8 locus of the TCR to express a y8 TCR.
The early thymocytes account for less than 3% of the cells in a mouse thymus.
They proliferate extensively in the subcapsular cortical zone presumably as a result
of interacting with cortical epithelial cells which produce a number of factors or
"cytokines" influencing cell proliferation. The DN cells then migrate to the deeper
cortex. A few can go out to the periphery without expressing the CD4 or CDS
coreceptor molecules, but most give rise to cells that express both CD4 and CDS
(termed double positive cells) and low to intermediate levels of the TCR. They are
now subjected to stringent selection tests (Figure 11).
16
J.F.A.P. MILLER
Random expression
of ap TCR genes
Positive selection
by MHC molecules Negative selection
towa/S
TCR expression
high a/S
TCR expression
cortical
low TCR a/?
macr^hages
I medullary epithefium
^
(7)<
Ni
(^
high a/S
TCR expression
t dendritic ceHs
epithelium
precursor
Mature T
cell pool
J
f 4* j < no interaction — • T 4* J<
no engagement
> programmed cell death
Figure 11. Differentiation of thymus lymphocytes and repertoire selection. Pre-T cells
entering the thymus from the circulation pass through a number of differentiation steps
that lead either to death or to maturity. The diagram shows the stages at which the
cells express the TCR, acquire or lose the coreceptors CD4 and CDS and are subjected
to positive and negative selection forces. The timing of the selection processes depends
on a variety of factors including the combined avidity of the TCR and coreceptors for
the presented antigen and the intra-thymic localization of the selecting cells (see text).
Positive selection ensures that T cells expressing TCRs that have some degree of
binding for polymorphic regions of the MHC molecules displayed on cortical
epithelial cells are selected for survival. The binding is presumed to protect the cells
from "programmed cell death" vv^hich affects the bulk of the double positive
thymocytes that express TCRs unable to bind to MHC molecules expressed
intrathymically. Positive selection by MHC class I or II molecules involves
concomitant engagement of either CDS or CD4, respectively, and dow^nregulation
of the unengaged coreceptor. The single positive cells express high levels of TCR
and are allowed to emigrate if they pass the negative selection test (see belovv^).
Positive selection is thus a mechanism that allows the CD8"^ T cell to respond to peptides
associated with the individual's own self MHC class I molecules, and the CD4'^ T cell
to recognize peptides complexed with self class II molecules. It is therefore the basis
of the phenomenon of MHC restriction, but it does not prevent the differentiation of
cells with high affinity receptors for self peptides presented in association with MHC
molecules. A negative selection mechanism is therefore required.
The Thymus in Immunity
17
Developing T cells expressing TCRs able to bind strongly to peptide-MHC
complexes presented by cells of the dendritic-macrophage lineages or by some
medullary epithelial cells are deleted. This negative selection process ensures that
T cells will not react to self antigens, at least to those existing in the thymus. It is
thus essential for the induction of self-tolerance (Chapter 4). The timing and exact
intrathymic site of this process depends on many factors among which are the
accessibility of differentiating T cells to self-antigens, the combined avidity (bmding strength) of the TCR and coreceptors for the self-MHC-peptide complexes and
the intra-thymic location of the deleting cells.
If both positive and negative selection involve recognition by the TCR of the
same self peptide-MHC complexes, how do all T cells avoid elimination? Several
possibilities have been suggested to explain this paradox. For example, low affinity
interactions of the TCR with self peptide MHC complexes may be sufficient to
trigger positive selection but insufficient to induce deletion. Furthermore, T cells
at different stages of maturation or in different intrathymic locations ("microenvironments") may exhibit a difference in the structure of their TCR which does not
affect antigen specificity but does influence the outcome of intracellular signaling.
It is also clear that while high affinity interactions between the TCR and antigen
leads to negative selection in the thymus, such interactions in fully mature T cells
outside the thymus can lead to activation and a powerful immune response.
THE THYMUS IN DISEASE STATES
Congenital immune deficiency states arise when the thymus does not develop
normally during gestation as, for example, in the diGeorge syndrome. Deletion or
mutations affecting the gene coding for the enzyme, adenosine deaminase (ADA),
are associated with severe immune deficiencies. ADA is an important enzyme in
the purine catabolic pathway. If it is lacking, the normal catabolism of purines does
not occur and this leads to the accumulation of metabolic products that are
especially toxic to lymphocytes. Defective DNA repair and anomaly of the recombinase enzymes essential for the rearrangement of genes which code for the T and
B cell antigen-receptors, will also result in severe immunodeficiencies.
Malignancy of various types can arise in the thymus, either from the epithelium
when they are termed thymomas, or from the lymphocytes as in acute lymphoblastic
leukemia or lymphoma. The virus, HTLV-1, infects and causes malignant transformation of mature T cells. It is the cause of some adult T cell leukemias.
The human retrovirus, HIV, is the cause of Acquired Immunodeficiency Syndrome or AIDS. The CD4 molecule is the receptor for virus entry into cells and
hence CD4"^ T cells are the principal targets for the virus. As CD4 is also present
on monocytes, macrophages, dendritic cells and some brain cells (microglia), some
of these cells also become infected and act as reservoirs for the virus. There follows
a relentless loss of CD4'^ T cells and disruption of the architecture of lymph nodes
18
J.F.A.P. MILLER
and of the thymus, itself. This leads to extreme susceptibility to infections with
other micro-organisms.
Autoimmune diseases often arise as the result of the activation of T cells that
have the potential to respond to autoantigens as described in Chapter 4.
SUMMARY
The thymus is a lymphoid organ which differs from other lymphoid tissues such as
spleen and lymph nodes both with regards to structure and function. It is within the
thymus that T lymphocytes differentiate from incoming stem cells, express genes
which code for the specific antigen receptor (TCR), and are subjected to stringent
selection procedures. These ensure that the mature T cell that emigrates from the
thymus will not be able to react strongly to the body's own tissues and will
recognize and be activated by foreign antigenic determinants only if these can
associate with cell surface molecules encoded by the major histocompatibility
complex (MHC) presented on the surface of so-called "professional" antigenpresenting cells (APC). Two distinct subsets of mature T cells exist as distinguished
by the expression of the co-receptor molecules CD4 and CDS, the CDS'*" T cells
able to perceive antigen in association with MHC class I molecules and the CD4"^
T cells recognizing antigen complexed with class II molecules. The migration of T
cells follows well-defined routes, naive (non-activated) T cells recirculating as
non-dividing cells from blood through certain "T-cell dependent" areas of the
lymphoid tissues, to lymph and back to blood. Once successfully stimulated by
antigen, naive T cells become effector or memory T cells, can secrete products
known as lymphokmes, perform immunoregulatory and cytotoxic functions and
express on their surface distinct molecules allowing them to migrate into nonlymphoid tissues and into areas where inflammation has occurred. Immune deficiency
diseases occur when the thymus or the T cells derived from it either fail to develop
normally or are the targets of infection by various retroviruses, such as HIV which
causes AIDS.
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hypersensitive reactions. Nature 194, 99-100.
Bjorkman, P.J., Saper, M.A., Samraoui, B., Bennett, W.S., Strominger, J.L., & Wiley, D.C. (1987).
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Brown, J.H., Jardetzky, T.S., Gorga, J.C, Stem, L.J., Urban, R.G., Strominger, J.L., & Wiley, D.C.
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Burnet, F.M. (1959). The Clonal Selection Theory of Acquired Immunity. Cambridge University Press,
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Claman, H.N., Chaperon, E.A., & Triplett, R.F. (1966). Thymus-marrow cell combinations—synergism
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Davis, M.M., & Bjorkman, P.J. (1988). T-cell antigen receptor genes and T-cell recognition. Nature
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Miller, A., Lider, O., Roberts, A.B., Spom, M.B., & Wiener, H.L. (1992). Suppressor T cells generated
by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses
by the release of transforming growth factor p after antigen-specific triggering. Proc. Natl. Acad.
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Miller, J.F.A.P. (1961a). Analysis of the thymus influence in leukaemogenesis. Nature 191, 248-249.
Miller, J.F.A.P. (1961b). Immunological function of the thymus. Lancet 2, 74^749.
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Miller, J.F.A.P. (1962b). Effect of neonatal thymectomy on the immunological responsiveness of the
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Mitchell, G.F., & Miller, J.F.A.P. (1968). Cell to cell interaction in the immune response. II. The source
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RECOMMENDED READINGS
Boyd, R.L., & Hugo, P. (1991). Towards an integrated view of thymopoiesis. Immunol. Today 12,
71-79.
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Kelso, A. (1989). Cytokines: Structure, function, and synthesis. Current Opinion Immunol. 2,215-225.
Klem, J., & Nagy, Z.A. (1982). MHC restriction and Ir genes. Adv. Cancer Res. 37, 233-317.
Miller, J.F.A.P. (1992). The Croonian Lecture. The key role of the thymus in the body's defence
strategies. Phil. Trans. Roy. Soc. 337B, 105-124.
Chapter 2
The B-Cell in Immunity
DAVID TARLINTON
Introduction
B Cell Development in the Bone Marrow
B Cell Migration
B Cell Responses to Antigen
T Cell Dependent Responses
A Secondary Response
T Cell Independent Responses
Other Types of B Cells
B Cell Deficiencies
X-Linked Agammaglobulinemia (X-LA)
X-LA with Hyper-IgM
Selective Immunoglobulin Isotype Deficiencies
Common Variable Immunodeficiency (CVID)
B-Cell Development and Function Analyzed by Gene Targeting
Summary
Recommended Readings
Principles of Medical Biology, Volume 6
Immunobiology, pages 21-45.
Copyright © 1996 by JAI Press Inc.
All rights of reproduction in any form reserved.
ISBN: 1-55938-811-0
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