Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
10.1098/ rsta.2003.1196
`Order from disorder sprung’: recognition and regulation in the immune system By Ta k W. M a k Advanced Medical Discovery Institute, Department of Medical Biophysics and Department of Immunology, University of Toronto, 620 University Avenue, Toronto, Ontario M5G 2C1, Canada Published online 2 May 2003
Milton’s epic poem Paradise lost supplies a colourful metaphor for the immune system and its responses to pathogens. With the role of Satan played by pathogens seeking to destroy the paradise of human health, GOD intervenes and imposes order out of chaos. In this context, GOD means `generation of diversity’: the capacity of the innate and speci c immune responses to recognize and eliminate a universe of pathogens. Thus, the immune system can be thought of as an entity that selfassembles the elements required to combat bodily invasion and injury. In so doing, it brings to bear the power of speci c recognition: the ability to distinguish self from non-self, and the threatening from the benign. This ability to de ne and protect self is evolutionarily very old. Self-recognition and biochemical and barrier defences can be detected in primitive organisms, and elements of these mechanisms are built upon in an orderly way to establish the mammalian immune system. Innate immune responses depend on the use of a limited number of germline-encoded receptors to recognize conserved molecular patterns that occur on the surfaces of a broad range of pathogens. The B and T lymphocytes of the speci c immune response use complex gene-rearrangement machinery to generate a diversity of antigen receptors capable of recognizing any pathogen in the universe. Binding to receptors on both innate and speci c immune-system cells triggers intricate intracellular signalling pathways that lead to new gene transcription and e¬ector-cell activation. And yet, regulation is imposed on these responses so that Paradise is not lost to the turning of the immune system onto self-tissues, the spectre of autoimmunity. Lymphocyte activation requires multiple signals and intercellular interactions. Mechanisms exist to establish tolerance to self by the selection and elimination of cells recognizing selfantigens. Immune system cell populations are reduced by programmed cell death once the pathogen threat is resolved. Once Paradise has been regained, memory cells remain in the body to sharply reduce the impact of a second exposure to a pathogen. Vaccination programs take advantage of this capacity of the human immune system for immunological memory, sparing millions the su¬ering associated with disease scourges. Thus does the order of the immune response spring from the disorder of pathogen attacks, and thus is Paradise preserved. Keywords: immune recognition; immune activation; immune tolerance; homeostasis
One contribution of 18 to a Theme `Self-organization: the quest for the origin and evolution of structure’. Phil. Trans. R. Soc. Lond. A (2003) 361, 1235{1250
1235
° c 2003 The Royal Society
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
1236
T. W. Mak
1. Introduction The theme speci ed by the Nobel Symposium Committee was `self-organization’. My objective for my talk at the symposium was therefore to illustrate self-organization in the immune system, and show how that self-assembly is an integral part of the recognition capacity and regulation of immune responses. The title of this paper is borrowed from the epic poem Paradise lost by John Milton (1667). To my mind, this poetic line captures the nature of the immune system perfectly: orderly responses being marshalled from a chaos of e¬ector cells attacking an unfathomable universe of pathogens. Paradise lost describes the downfall of Adam and Eve in the Garden of Eden, the attempts by Satan and his followers to take over Paradise, and God’s e¬orts to reclaim his creation. In the context of the immune system, Paradise is good health, Satan is the army of pathogens that would deprive us of Paradise, and God is GOD, the `generation of diversity’ possible in immune-system cells. Because of this diversity, there is no pathogen that can escape recognition by one immunesystem cell or another. Recognition leads to response, and e¬ector cells alerted or generated by the responding cell can dispose of virtually any threat. The diversity of the immune system is generated randomly, so one might say it is a type of disorder, but it gives rise to the proliferation of perhaps the one cell that can speci cally recognize a particular pathogen and mobilize an immune response to eliminate it. Thus, order is indeed sprung from disorder, and GOD preserves Paradise against Satan. What is the nature of this guardian of our individual paradises? The immune system is just that: a system of e¬ector cells that acts in concert with soluble molecules called cytokines. Cytokines are key intercellular messenger molecules that promote the activation and di¬erentiation of immune-system cells. When the immune system is in balance and functioning correctly, it protects us against pathogens and the rise of cancer cells. When the immune system is out of balance, we can experience di¬erent detrimental health e¬ects. Inappropriate immune responses to normally innocuous substances cause allergies. Immune responses against life-saving transplants are normal, but certainly not desirable. Immune responses against our own body cells constitute autoimmunity, while immunode ciency, either congenital or acquired (as in AIDS), leads to weak or absent immune responses against pathogens that should be easily contained. Three aspects of the immune system contain elements of self-assembly: the innate immune response, the acquired immune response and the homeostasis of the immune system. The innate immune response, which is present at birth, is mediated by a collection of cells and body barriers that constitute the rst line of defence against pathogens. The recognition capacities of these cells and barriers are hard-wired and cannot be changed to counter a new threat. The innate response controls many pathogens that attack us, but cannot bring concentrated resources to bear if the attack gets out of control. That job falls to the acquired immune response, another collection of cells and cytokines whose recognition capacities are not hard-wired and are acquired over the life of the animal. It is in the acquired response that GOD takes e¬ect and ensures that new pathogens are recognized and completely eliminated. However, as the speci cities of the acquired immune response are generated randomly, it is likely that some will recognize epitopes on self-tissues. The cells directed against these epitopes must be silenced, a state called `immune tolerance’. Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
Recognition and regulation in the immune system
1237
Tolerance ensures that the resources of the immune system are not wasted in attacks on self rather than on pathogens. Finally, after the disorder of the battle between immune-system cells and pathogens, the body requires that order be restored and that the system return to baseline. This recovery of order is called the `maintenance of homeostasis’. Activated e¬ector cells must be shut down after they have eliminated the pathogen, lest they start attacking self tissues. The production of cytokines calling cells to battle must cease, and spent cells must be retired from the eld.
2. Self-assembly in innate immunity At its simplest level, immunity is the recognition of non-self. Rationally, anything found in the body that is not `self’ is likely to be harmful and should be removed. Recognition of self is not new from the point of view of evolution, because even creatures as lowly as the sponges have an awareness of self. If fragments of di¬erent species of sponges are mixed together, only members of the same colony stick together again. In higher organisms such as insects, self-recognition is translated into selfdefence. Immune responses in insects are innate and very basic. Recognition capacity is rigidly de ned and there is no ability to react to molecules outside those hard-wired in an insect cell’s DNA. Vertebrates also have innate immunity, but the function of these responses has evolved to encompass more than defence. Cytokines produced by cells of the innate immune system are crucial for supporting the activation and di¬erentiation of cells of the acquired response. Innate immunity is underpinned by a complex system of receptors and signalling pathways (Janeway & Medzhitov 2002). These elements have to be in balance and functioning correctly for the innate response to be able to control pathogens and support the acquired response. The innate response rests on the recognition of a limited collection of molecular patterns, some of which appear on the surfaces of whole classes of pathogens. Innate immune-system cells express pattern-recognition receptors (PRRs) that recognize these patterns and bind to them, trapping and then destroying the pathogens. One of the best-studied PRR families is the Toll-like receptor (TLR) family. Phagocytes such as macrophages express several di¬erent TLRs, each of which recognizes a di¬erent molecule expressed by almost all members of a pathogen class. For example, TLR2 recognizes the peptidoglycan present in the walls of all Gram-positive bacteria, while TLR4 binds lipopolysaccharide (LPS) present in the cell walls of all Gram-negative bacteria ( gure 1). TLRs work because no matter what the virulence or growth habit of, say, a Gram-negative bacterium, it will express LPS and thus may be detected and engulfed by the macrophage. Similarly, any pathogen with peptidoglycan on its surface becomes a candidate for disposal by any innate immune-system cell expressing TLR2. A large portion of the daily onslaught of pathogens our bodies experience is controlled in this way. When the extracellular portion of a TLR is activated by binding to its pathogen ligand, an orderly oligomerization event takes place in the membrane, which leads to conformational changes in TLR proteins. These changes trigger the self-assembly of the TLR signalling complex. First, an adaptor protein called MyD88 associates closely with the cytoplasmic tail of a TLR ( gure 2). MyD88 in turn recruits to the TLR tail a kinase called interleukin-1 receptor-associated kinase 1 (IRAK-1). (The TLRs and the receptor for the cytokine interleukin-1 are closely related in Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
T. W. Mak
1238
pathogen 1
peptidoglycan
pathogen 4
pathogen 2
TLR2
APC
TLR4
LPS pathogen 3
Figure 1. Pattern recognition in the innate immune response. Pathogens express di® erent molecular patterns, which are recognized by pattern-recognition receptors (PRRs) expressed by cells of the innate immune response. The Toll-like receptors (TLRs) are key among these PRRs. Any pathogen whose cell wall contains peptidoglycan can be bound (and engulfed) by immune-system cells expressing TLR2, while a pathogen with lipopolysaccharide (LPS) in its cell wall binds to TLR4.
structure and share signal transducers.) IRAK-1 becomes activated by phosphorylation, possibly mediated by another structurally related kinase called IRAK-4, and associates with the adaptor protein TRAF6. This complex then eventually activates the important mammalian transcription factor NF-µB. NF-µB is vital for the transcription of genes functioning in cellular activation and cytokine production. In the absence of a pathogen attack, NF-µB is held in an inactive form in the cytoplasm by the inhibitory binding protein IµB. Once a TLR signalling pathway is activated by a pathogen, the action of the IRAK-4/TRAF6 complex induces the degradation of IµB, freeing NF-µB to enter the nucleus, bind to speci c sites in the genome and initiate the transcription of cytokines. So fundamental is the innate immune response that the intracellular signalling pathways triggered by the activation of a PRR have been conserved throughout evolution. Indeed, the prototypical Toll receptor, a homologue of mammalian TLRs, was rst discovered on the surfaces of Drosophila cells. The cytoplasmic tail of the Toll receptor binds to Tube, a ®y adaptor protein homologous to mammalian MyD88. Signalling triggered by the engagement of Toll by a pathogen ligand is transduced via Tube to the Drosophila kinase SIIK, equivalent to IRAK-1 in mammalian cells. Activation of SIIK leads to the degradation of Cactus, the Drosophila homologue of IµB (Medzhitov & Janeway 1998). NF-µB activation in the course of an innate immune response leads to production of the pro-in®ammatory cytokines IL-6, tumour necrosis factor (TNF) and IL-1. Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
Recognition and regulation in the immune system
1239
The principal e¬ects of these cytokines are the induction of in®ammation and fever. Immune system cells congregate in the tissues at the site of infection and employ various defence strategies in an attempt to remove the source of the infection. While in®ammation is critical to the innate response, too much in®ammation can itself cause tissue damage. Cytokines have powerful e¬ects on cellular structures and blood vessels, and excessive cytokine production can lead to major medical problems such as septic shock. It is an excessive cytokine response to the LPS component of Gramnegative bacteria that causes septic shock, and not the bacterium itself. From a clinical point of view, understanding the signalling pathways that initiate and regulate in®ammatory cascades may allow us to reduce the incidence of disorders such as septic shock. One might think from the above discussion that IRAK-1 is critical to in®ammatory signalling, but genetically engineered mice in which only the IRAK-1 gene has been disrupted, known as IRAK-1 `knockout’ mice, are still able to mount in®ammatory responses (Kanakara j et al. 1998; Thomas et al. 1999). The critical proteins in this cascade turn out to be MyD88, IRAK-4 and TRAF6. If wild-type mice are treated with a lethal dose of LPS from Gram-negative bacteria, 100% of these animals die within 24 hours, due to the mouse equivalent of septic shock. The huge cascade of cytokines produced in response to TLR signalling triggered by the LPS severely damages the tissues of these animals. However, IRAK-4-knockout mice challenged with a similar dose of LPS can survive (Suzuki et al. 2002). In other words, the mutant animals cannot mount the excessive in®ammatory responses that lead to the demise of their wild-type counterparts. Concentrations of pro-in®ammatory cytokines in the blood of the mutant mice are very low and the animals are thus protected from tissue damage. Similar results have been obtained for TRAF6 (Lomaga et al . 1999; Kawai et al . 1999) and MyD88-knockout mice. How is the ®ood of pro-in®ammatory cytokines brought under control when the pathogen has been eliminated and the cytokines are no longer required? How is order restored and the resting state again achieved? As for any self-assembly system, the innate immune response comes equipped with inherent negative feedback controls that down-regulate cytokine synthesis. When LPS binds to the TLR, it triggers not only the transcription of cytokines but also the transcription of the inhibitory IRAK-M protein (Kobayashi et al . 2002). IRAK-M acts to speci cally down-regulate in®ammation by interfering with activation of IRAK-1. NF-µB is no longer activated and transcription subsides. LPS thus induces both the in®ammatory response and the means to shut o¬ that response when the need for it has passed (Mak & Yeh 2002).
3. Self-assembly in acquired immunity The acquired immune response depends on specialized cells called lymphocytes, which express antigen receptors on their surfaces. Within the population of lymphocytes, the DNA encoding these receptor genes is physically adjusted to encode molecules capable of recognizing an entirely new pathogen. These adjustments to the DNA are random events; however, the production of e¬ector cells ready to take on the pathogen is anything but random. Because of GOD, the acquired immune response has the means to go beyond the common molecular patterns recognized by PRRs and can generate antigen receptors of very diverse speci cities that can recognize even very rare pathogen molecules or pieces of molecules. An individual is Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
T. W. Mak
1240
toll-like R
IRAK-4
IRAK-M
MyD88
IRAK-1
P P
IRAK-1
TRAF6
P P
NFk B
downstream signaling
Figure 2. In° ammatory signalling pathway. The binding of a pathogen to a TLR triggers intracellular signalling mediated by the adaptor protein MyD88, kinases IRAK-1 and IRAK-4 and the adaptor TRAF6. The eventual outcome of this signal transduction is the activation of the transcription factor NF-µB and the induction of transcription of pro-in° ammatory genes. Transcription of IRAK-M, a negative feedback molecule which inhibits the activation of IRAK-1 required for NF-µB activation, is also induced by TLR engagement.
not born with all these receptors: they develop as lymphocytes develop in the host, and lymphocytes expressing these receptors are only present in su¯ cient numbers to eliminate a pathogen once those cells have been activated by the presence of the speci c molecule recognized by their antigen receptors. The secret of the acquired immune response lies in the fact that only the lymphocyte speci cally recognizing the foreign antigen present in a tissue responds to it. Lymphocytes nearby which do not recognize the antigen remain in the resting state, a concept called `clonal selection’ (Burnet 1991). Once activated by engagement of its antigen receptor, the selected lymphocyte undergoes explosive proliferation to produce a clonal army of e¬ector cells speci cally directed against that antigen and the pathogen expressing it. These e¬ector cells and protein products lead to a response that is more e¯ cient and e¬ective than an innate immune response and almost always result in the complete elimination of the pathogen. There are two main classes of lymphocytes, called T cells and B cells. B cells express antigen receptors called B-cell receptors (BCRs) that can directly recognize portions of intact macromolecules on pathogens. The small area of the macromolecule bound in the binding site of a BCR is called the epitope. Di¬erent B cells will respond to di¬erent epitopes, multiplying the number of B cells that can be activated by this pathogen. B cells produce antibodies that circulate in the blood and inactivate the pathogen throughout the body. However, B cells cannot be activated without help from T cells. There are two types of T cells: helper T cells and cytotoxic T cells. Helper T (Th) cells produce cytokines and other soluble factors that assist both B cells and cytotoxic T (Tc) cells in their functions ( gure 3). Pathogens Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
Recognition and regulation in the immune system TYPE OF PATHOGEN
1241
RESPONDING LYMPHOCYTES
extracellular pathogens
Th cell
soluble factors
antibody production B cell
intracellular pathogens infected cell death
soluble factors Th cell
Tc cell
Figure 3. Responses of lymphocytes to extra- and intracellular pathogens. Extracellular pathogens supply antigens in a way that activates helper T (Th) lymphocytes and B lymphocytes. The Th1 cells produce cytokines that activate B lymphocytes and assist them in producing antigen-speci¯c antibodies that eliminate the pathogen. Intracellular pathogens avoid antibodies by hiding within cells. Th cells responding to antigens derived from these pathogens produce cytokines that activate cytotoxic T (Tc) cells and assist them in lysing the infected cells.
that are extracellular, that is, do not access the interior of a cell, are prime targets for destruction by antibodies. However, pathogens that are intracellular in habit escape antibodies. Instead, a cell infected with this type of pathogen starts to display molecules on its cell surface that make it a target for destruction by Tc cells. Cytotoxic lymphocytes bind to infected cells and produce molecules that punch holes in the infected cell’s membrane. Both the infected cell and its pathogen burden die. Similarly, tumour cells express unusual antigens on their surfaces that attract the attention of Tc cells and they also are destroyed. Both Th and Tc cells are activated only following the binding of speci c antigens to their antigen receptors, called T-cell receptors (TCRs). A long journey of discovery encompassing the contributions of many scientists was required to elucidate the nature of the TCR. Early studies had shown that T cells recognized speci c antigens, but the precise composition of the receptor mediating that recognition was a mystery. Surprisingly, it was found that T cells did not recognize simple antigens as B cells did. Instead, Zinkernagel & Doherty (1974) showed that T cells recognized antigens derived from pathogen antigens on the surface of a cell called an antigen-presenting cell (APC). Furthermore, the molecule presenting that antigen on the APC had to belong to the major histocompatibility complex (MHC) family of molecules (Zinkernagel & Doherty 1974). Thus, T-cell activation depended on the simultaneous recognition of a peptide and an MHC molecule (Bjorkman et al . 1987). This exciting result spawned a number of interesting questions, the chief among them being: was there one receptor on a T cell that recognized peptide and another that recognized MHC, or did one receptor recognize both? The only way to de nitively answer this question was to clone the genes for the TCR. In mid-1980s, our laboratory, and that of Mark Davis at Standford University School of Medicine, Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
T. W. Mak
1242 CD4
CD4 Th
APC
peptide
MHC Class II
MHC Class I
HC
peptide
TCR
CD8
CD8 Tc
TCR
Figure 4. Antigen presentation by cells expressing MHC classes I and II. Unlike B cells, T cells recognize peptide antigens derived from pathogens in association with MHC molecules displayed on the surface of host cells. Certain cell types, called antigen-presenting cells (APCs), are capable of expressing MHC class II and presenting a complex of pathogen peptide plus MHC class II to Th cells expressing the CD4 co-receptor. Almost all host cells (HC) can express MHC class I and thus present pathogen peptide plus MHC class I to Tc cells expressing the CD8 co-receptor.
succeeded in cloning the genes of the T-cell antigen receptors (Yanagi et al. 1984; Hedrick et al. 1984). Transfection of both the a- and b-chains of these genes showed that the TCR is a single receptor that recognizes a combined epitope of peptide bound to MHC (pMHC) (Dembic et al. 1986). There are two types of MHC molecules that present peptides to T cells. Th cells, which express a co-receptor molecule called CD4, express TCRs that recognize peptide bound to MHC class-II molecules ( gure 4). MHC class II is expressed only on the surfaces of the few cell types that serve as `professional’ APCs. Tc cells, which express the CD8 co-receptor, express TCRs that recognize peptide bound to MHC class-I molecules (Bjorkman et al . 1987). MHC class I is expressed on the surface of most body cells. X-ray crystallography studies have demonstrated that, in both cases, a peptide nestles in a binding groove formed by domains of the MHC molecule, and that this pMHC structure interacts with binding site formed from domains of the TCR molecule. The co-receptor of a T cell binds to a site on the MHC molecule distinct from the peptide-binding groove. The interaction between TCR and pMHC determines whether the peptide in question comes from friend or foe, and thus whether an immune response is mounted. A T cell is not activated by engagement of its TCR alone, and it is not the activated T cell itself that disposes of a pathogen. An activated T cell must proliferate and di¬erentiate into daughter e¬ector cells that recognize the same pMHC and do the physical work of eliminating the pathogen. Which types of e¬ector cells differentiate depends on whether the activated T cell is a Th or Tc cell, and on the cytokines present in the immediate microenvironment. Consider the case of an activated Th cell, often called a Th0 cell in this context. If the cytokine milieu surrounding the activated Th cell contains an abundance of the cytokines IFNg and IL-12, the Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
Recognition and regulation in the immune system
1243
daughter e¬ector cells develop into Th1 cells. Th1 cells secrete additional cytokines that preferentially promote Tc activation and the destruction of tumour cells or cells infected with intracellular pathogens. If, however, the cytokines in the milieu surrounding the Th0 cell are dominated by IL-4, Th2 e¬ector cells di¬erentiate. Th2 cells secrete additional IL-4 that promotes B-cell activation and the production of antibodies designed to clear extracellular pathogens. There is an orderly balance here, in that the pathogen attacking the body spurs cells of the innate immune response to secrete the cytokines that will cause the di¬erentiation of the Th e¬ector cell bestsuited to assisting in the elimination of that pathogen. Moreover, cytokines secreted by Th1 cells shut down a Th2 response, and vice versa, conserving the resources of the immune system (Glimcher 2001). How is the antigenic speci city of the TCR generated? TCRs are heterodimeric proteins composed of a- and b-chains. These chains contain a variable region, which distinguishes among antigens, and a constant region. The TCRa- and b-chains are encoded by the TCRa- and TCRb genes, which are very complex. In each gene, there are multiple versions of three types of gene segments called the V, D and J sequences ( gure 5). There are also exons that encode di¬erent constant region sequences. As any one T lymphocyte develops, its TCR genes undergo rearrangement such that one V, one D and one J are randomly chosen and joined at the DNA level to form a variable exon. The variable exon is then paired with a constant region exon to form the nal a-chain or b-chain gene in that cell. It is the sequence of the VDJ part of the rearranged gene that determines the antigenic speci city of that T cell. Di¬erent V, D and J segments will be randomly chosen in di¬erent T cells (Toyonaga & Mak 1987), creating a vast array of di¬erent TCRs that can `see’ di¬erent parts of di¬erent pathogens. This, then, is the basis of the GOD emphasized at the beginning of this paper, and is a wonderful example of gene self-assembly sprung from the chaos of random gene rearrangement. If GOD produces randomly diverse TCRs, it is likely that some will not recognize self-MHC and thus will be useless, while others will recognize self-antigens rather than foreign ones. How are such cells eliminated? The answer lies in the processes of positive and negative selection that occur in the thymus. T lymphocytes start life as undi¬erentiated precursors in the bone marrow. These cells move to the thymus and become thymocytes, which start to express TCRs and co-receptors and to develop into mature T lymphocytes in this location. T cells recognize peptides bound only to self-MHC molecules and not to foreign MHC molecules. Stromal cells in the thymus express a multitude of self-peptide/self-MHC combinations. Developing thymocytes whose TCRs do not bind to any of the self-peptide/selfpeptide epitopes displayed by the thymic stromal cells do not survive. These thymocytes, which constitute the vast majority of developing cells, are said to `die of neglect’. At the other extreme are those rare thymocytes that bind with high a¯ nity to self-peptide/self-MHC. If allowed to mature, these thymocytes would become mature T cells that could attack self-tissues. The engagement of the TCRs of these thymocytes delivers a death signal that induces them to die in a process called negative selection. It is the removal of these potentially self-reactive thymocytes in the thymus that establishes `central tolerance’, the ability of an organism to restrain its immune system from attacking self cells. After neglect and negative selection, we are left with those extremely rare thymocytes that bind weakly to self-peptide/self-MHC. It is these cells that are most likely to bind well to foreign Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
T. W. Mak
1244 Va1 Va2 Va75
TC R a germline DNA Ja1 Ja2 Ja3 Ja4
Ja61
Ca1
5’
3’
rearranged aDNA Va1 Va2Ja4 Ja61 Ca1
5’
3’
transcription
translation
ab TC R heterodimer
T cell
transcription Vb1 V b2Db1Jb6 Cb1
Db 2
1
5’
Jb2 3 4
Db 2
6
5’
5’
2
Cb 2
6
5
3’
rearranged b DNA
Vb1 Vb2 Vb48 Db1Jb6 Cb1
Vb1
translation
Vb2 Vb48 Db1
1
2
Jb1 4
3
Jb2
1
Cb 1
5
2
3
Cb2
4
Db 2
6
TCR b germline DNA
6
5
3’
Jb2
1
2
3
Cb 2
4
5
6
3’
Figure 5. TCR gene rearrangement. TCR proteins are heterodimers composed of a- and b-chains. Each chain is encoded by a complex gene containing multiple V, D and J gene segments and a constant (C) region exon. One of each segment type is randomly selected in each developing T cell to be joined together to form the variable exon of the rearranged gene. A transcript of the combined variable and C exons is translated into the protein chain. Because the choice of V, D and J segments is di® erent in each T cell, each TCR has a di® erent antigenic speci¯city. It has been estimated that 1020 di® erent TCR heterodimers can be formed. (Adapted from Kuby (1997, ¯g. 11.6).)
peptide/self-MHC generated later in the course of a pathogen attack. These thymocytes are thought to escape the induction of cell death and to be positively selected; that is, they receive a survival signal that allows them to continue their maturation. From positively selected thymocytes develop the mature CD4+ and CD8+ Th and Tc cells that patrol our bodies and react to the presence of foreign antigens (Bevan 1997). Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
Recognition and regulation in the immune system
1245
APC
LFA-1 ICAM-1
RAFTs
signalling complex
pMHC TCR
CD4 Lck
e d
B7 CD28
g e z z
Fyn
Zap70
signalling
T cell
Figure 6. Immunological synapse formation. T-cell activation requires multiple intercellular interactions, the ¯rst of which is the binding of the adhesion molecule ICAM-1 on the T cell with its partner LFA-1 on the APC. This adhesion allows the TCR to examine the ¯t of its binding site to the peptide-MHC complex pro® ered by the APC. If the ¯t is a match, signals are delivered down the accessory CD3", ¯ and ® chains associated with the TCRa/b heterodimer that result in the recruitment of Lck kinase. Phosphorylation events lead to the recruitment of lipid rafts containing additional signalling molecules. A signalling complex is formed that eventually transduces signals activating the transcription of genes required for T-cell proliferation and di® erentiation. Complete T-cell activation also requires co-stimulatory CD28{B7 interaction, which initiates further Lck and Fyn kinase signalling.
Given the power of the T-cell response, one would expect that T-cell activation would be complex, tightly regulated and involve a lot of molecules. However, only a small number of proteins are crucial for the self-assembly of the TCR signalling apparatus that mediates downstream signalling and T-cell activation. Picture a mature T cell residing in a lymph node, inspecting the o¬erings of pMHC presented by surrounding APCs. To enable this inspection, the T cell uses the adhesion molecule ICAM-1 on its surface to transiently bind to the LFA-1 adhesion protein expressed on the surface of the APC. This binding allows the TCR to make contact with the pMHC on the APC and determine if a match has been made. If the binding is of low a¯ nity, as one would expect for a self-peptide, the cells drift apart again. If the binding is of higher a¯ nity, however, the stage is set for T-cell activation (Krummel & Davis 2002). As well as TCR-pMHC interaction, T-cell activation requires signal ampli cation facilitated by the interaction of the co-stimulatory molecule CD28 on the T cell with its binding partner B7 on the APC. I like to use the analogy of a radio: rst you have to plug in the radio (establish adhesion between the TCR and the APC), then you have to tune it (the TCR must recognize the speci c pMHC with su¯ cient a¯ nity), and nally you have to turn the volume up (co-stimulation must occur to amplify the signal). Let us examine T-cell activation step by step. Upon engagement by speci c pMHC, multiple TCRs on the T-cell surface aggregate close to the site of T-cell-APC contact ( gure 6). The intercellular links between the APC and T cell during T-cell Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
1246
T. W. Mak
activation constitute a structure called the `immunological synapse’. The synapse weighs the strength of the overall interaction between the TCRs and the pMHCs and determines whether the T cell should continue in its activation cycle. In the T cell membrane are ®oating lipid `rafts’ that have embedded within them molecules vital for downstream transduction of the signal triggered by the binding of pMHC to the TCR. Co-stimulation via CD28{B7 interaction induces the lipid rafts to draw nearer to the cytoplasmic tails of the signalling proteins that associate with the cytoplasmic tail of the TCR. The TCR receptor complex then assembles, and sites in the tails of the TCR signalling molecules are phosphorylated by kinases like Lck and Fyn brought in by the rafts with the assistance of the co-receptors (Drevot et al . 2002). This phosphorylation in turn sends signals downstream in the cell that activate the transcription of genes necessary for the T cell to start proliferating and di¬erentiating into e¬ector cells. The immunological synapse can be maintained for several hours, ensuring that TCR signalling endures long enough to complete T-cell activation. Cytokines gure prominently during T-cell activation, di¬erentiation and e¬ector action. Activated T cells secrete IL-2, which promotes the proliferation of daughter e¬ector cells. Other cytokines promote the migration of e¬ector T cells into sites of infection where they can join the fray against the pathogen. Still other cytokines induce other immune-system cells to produce anti-microbial molecules like hydrogen peroxide and nitrous oxide. Chaos and disorder abound at the site of infection as cytokines, Tc cells and antibodies all work in various ways to destroy the pathogen. However, once the pathogen is eliminated, the T cells must be reined in to restore homeostasis in the organism. To shut down the primary response, signalling leading to continued T-cell activation and proliferation must be shut o¬, and e¬ector T cells that are no longer needed must be induced to die. To understand how activation signalling is extinguished, we have to return to the start of T-cell activation. For the rst two days after contact between the TCR and pMHC, co-stimulatory signalling through CD28 ampli es the signal and promotes T-cell proliferation and e¬ector cell di¬erentiation. After three to four days, there are usually enough e¬ectors in the eld and there is no need for continued proliferation. At this point, a molecule called CTLA-4 abruptly shuts down the ampli cation of TCR signalling mediated by CD28 (Boise et al . 1995). In the radio analogy, CTLA-4 grabs the volume knob and turns it way down. CTLA-4 has an a¯ nity for B7 that is in the order of 20 times greater than that of CD28 for B7, so that CD28 can be physically displaced from the receptor complex. Furthermore, the cytoplasmic tail of CTLA-4 recruits the phosphatase SHP-2 to the complex (Marengere et al. 1996), and this enzyme dephosphorylates and deactivates residues needed for downstream signal transduction. Knockout mice lacking CTLA-4 only live for two to three weeks before they die with massive enlargements of the spleen due to the presence of excess lymphocytes (Waterhouse et al. 1995; Tivol et al. 1995). Thus, the shut-down of activated T cells is self-regulating, because the appearance of CTLA-4 on the cell surface is a late event induced only by activation itself. We can see here a parallel with IRAK-M function in the innate response: the appearance of the inhibitor of an intracellular signalling pathway depends on the activation of that pathway. Thus, T-cell activation is another example of a self-assembly system regulated by inherent negative feedback. As well as blocking the production of new e¬ectors, the original e¬ectors must be removed. This is done by inducing apoptosis, a programmed form of cell death Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
Recognition and regulation in the immune system
1247
that is neat and tidy and does not leave necrotic debris in the tissues. Again, a self-assembly system is in operation because the molecules required to initiate and execute apoptosis are induced by T-cell activation signalling. Death receptors are proteins that, when engaged by their ligands, induce intracellular signalling leading to apoptosis. Fas is a death receptor that occurs as preformed trimers in the membranes of many immune-system cells. Its ligand, FasL, is expressed in the membranes of many non-lymphoid cells (Nagata 1999). Upon activation, T cells up-regulate their expression of Fas and, in so doing, sow the seeds of their own destruction. Binding of Fas to FasL causes the self-assembly of yet another receptor complex on the T cell surface ( gure 7). The adaptor protein FADD recruits the protease caspase-8, inducing the latter to undergo a conformational change that leads to its activation. Activated caspase-8 in turn triggers a cascade of protein cleavages that results in induction of apoptosis. Again, the system is self-regulating because a regulatory protein called Casper, whose synthesis is induced by T-cell activation, interferes with the interaction of FADD and caspase-8 and puts a brake on apoptosis. FADD and Casper are essential for the control of signal transduction leading to apoptosis initiated by the stimulation of Fas and other death receptors. However, investigations of FADD-/- and Casper-/-knockout mice demonstrated a very surprising nding about the nature of molecules of the immune system. Knockout mice lacking FADD or Casper were expected to be viable in a pathogen-free environment because, in the absence of a microbial threat, many types of mutant mice can survive without intact immune systems. However, FADD- and Casper-knockout mice died during embryonic development, revealing an unanticipated function for these genes. In the absence of FADD or Casper, the development of the myocardium in the mouse heart was abnormal (Yeh et al. 1998, 2000). Thus, FADD, caspase-8 and Casper may represent a cassette of genes that has one function during embryonic development and another in the adult animal. The cassette may have evolved rst to build the myocardium and then may have been subverted into a function in the immune response. This is not the only example of the immune system assembling itself with borrowed components. The transcription factor NFATc was known to be involved in signalling downstream of TCR activation. However, knockout mice lacking the NFATc gene die during embryogenesis because of faulty heart-valve development (de la Pompa et al. 1998; Ranger et al . 1998). As well as activating T cells responsive to pathogen antigens, the TCR signalling complex is responsible for silencing self-reactive T cells that escaped negative selection in the thymus and made it to the periphery of the body. Without some mechanism to prevent activation of these cells, they could cause autoimmunity. As stated above, T-cell activation requires CD28 to amplify the TCR signal. APCs that present peptides derived from pathogens on their MHC molecules can complete the B7-CD28 circuit and the volume on the radio is turned up loud. However, most tissue cells do not express B7. If the T cell that escaped negative selection in the thymus expresses a TCR that binds to a self-peptide presented by an MHC molecule on a tissue cell, no CD28 signalling is possible due to the lack of B7. The radio is plugged in and tuned, but the volume remains low. Instead of activation, an unknown signal is delivered to the T cell that tells it to ignore this self pMHC epitope in the future. Thus, the next time this T cell encounters that particular self pMHC, even if it appears in the presence of B7, the T cell will fail to activate. This T cell is said to have become Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
T. W. Mak
1248
FasL
Fas
FADD
Caspase-8
Casper
death
Figure 7. Death receptor signalling. Activated T cells up-regulate the expression of the death receptor Fas on their surfaces. When the activated T cell comes into proximity to a non-lymphoid cell expressing FasL (the membrane-bound ligand of Fas), a signalling pathway is triggered that induces the T cells to recruit the adaptor protein FADD and undergo caspase-8-mediated apoptosis. T-cell activation also up-regulates the synthesis of Casper, which acts as a negative feedback regulator of caspase-8 activation.
anergic, and this type of tolerance is known as peripheral tolerance (Powell et al . 1998). TCR signalling also plays a role in the development of immunological memory. In innate immunity, every response is of the same magnitude. In acquired immunity, a second exposure to a pathogen engenders a response that is faster and stronger than the primary response. At the conclusion of the primary acquired response, remnants of the original T-cell clone are preserved as memory T cells. Memory T cells react faster and mount more vigorous responses than T cells in the primary response, so that more e¬ectors are generated in a second exposure and the horse race against pathogen multiplication is more easily won. Pathogens are eliminated before they can get a good foothold, the individual does not come down with disease and the overall survival of the species is enhanced. At the conclusion of the secondary response, increased numbers of memory cells are generated to protect against a third exposure. Modern medicine has taken advantage of immunological memory to design vaccination programmes to combat human diseases. Healthy individuals are immunized Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
Recognition and regulation in the immune system
1249
with inactivated versions of a pathogen that still contain the antigens necessary to activate T cells. Memory T cells generated in response to the vaccination can then defend the body in a subsequent exposure to the real pathogen, most often preventing disease entirely. Vaccination against smallpox has eradicated this virus in the wild, and poliovirus is rapidly on its way out. This is not to say that we should rest on our laurels. No e¬ective vaccines are yet available for such scourges as tuberculosis, malaria, HIV and hepatitis C (Forrest & Burgess 1996). The natural immune responses against these organisms are still not understood, and vaccine design is thus compromised. Another area where we need to increase our understanding of immune responses is in autoimmune disease. We still do not know why peripheral tolerance fails in some people and leads to disorders like rheumatoid arthritis and diabetes. Nonetheless, if one day we can decipher all the nuances of the initiation and cessation of immune responses, I am optimistic that we will be well on the way to restoring the Paradise of good health for all.
References Bevan, M. J. 1997 In thymic selection, peptide diversity gives and takes away. Immunity 7, 175{178. Bjorkman, P. J., Saper, M. A., Samraoui, B., Bennett, W. S., Strominger, J. L. & Wiley, D. C. 1987 The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329, 512{518. Boise, L. H., Noel, P. J. & Thompson, C. B. 1995 CD28 and apoptosis. Curr. Opin. Immunol. 7, 620{625. Burnet, F. M. 1991 Immunologic recognition of self. The Nobel Lectures in Immunology. The Nobel Prize for Physiology or Medicine, 1960. Scand. J. Immunol. 33, 3{13. de la Pompa, J. L. (and 11 others) 1998 Role for the NF-Atc transcription factor in morphogenesis of cardiac valves and septum. Nature 392, 182{186. Dembic, Z., Haas, W., Weiss, S., McCubrey, J., Kiefer, H., von Boehmer, H. & Steinmetz, M. 1986 Transfer of speci¯city by murine alpha and beta T-cell receptor genes. Nature 320, 232{238. Drevot, P., Langlet, C., Guo, X. J., Bernard, A. M., Colard, O., Chauvin, J. P., Lasserre, R. & He, H. T. 2002 TCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts. EMBO J. 21, 1899{1908. Forrest, J. M. & Burgess, M. A. 1996 Update on immunization. Curr. Opin. Pediatr. 8, 21{27. Glimcher, L. H. 2001 Lineage commitment in lymphocytes: controlling the immune response. J. Clin. Invest. 108, s25{s30. Hedrick, S. M., Cohen, D. I., Nielsen, E. A. & Davis, M. M. 1984 Isolation of cDNA clones encoding T cell-speci¯ c membrane-associated proteins. Nature 308, 149{153. Janeway Jr, C. A. & Medzhitov, R. 2002 Innate immune recognition. A. Rev. Immunol. 20, 197{216. Kanakaraj, P. (and 10 others) 1998 Inerleukin (IL)-1 receptor-associated kinase (IRAK) requirement for optimal induction of multiple IL-1 signalling pathways and IL-6 production. J. Exp. Med. 187, 2073{2079. Kawai, T., Adachi, O., Ogawa, T., Takeda, K. & Akira, S. 1999 Unresponsiveness of MyD88de¯cient mice to endotoxin. Immunity 11, 115{122. Kobayashi, K., Hernandez, L. D., Galan, J. E., Janeway Jr, C. A., Medzhitov, R. & Flavell, R. A. 2002 IRAK-M is a negative regulator of Toll-like receptor signalling. Cell 110, 191{202. Krummel, M. F. & Davis, M. M. 2002 Dynamics of the immunological synapse: ¯nding, establishing and solidifying a connection. Curr. Opin. Immunol. 14, 66{74. Phil. Trans. R. Soc. Lond. A (2003)
Downloaded from http://rsta.royalsocietypublishing.org/ on January 15, 2018
1250
T. W. Mak
Kuby, J. 1997 Immunology, 3rd edn. New York: W. H. Freeman. Lomaga, M. A. (and 22 others) 1999 TRAF6 de¯ciency results in osteopetrosis and defective interluekin-1, CD40, and LPS signalling. Genes Dev. 13, 1015{1024. Mak, T. W. & Yeh, W. C. 2002 Immunology: a block at the toll gate. Nature 418, 835{836. Marengere, L. E., Waterhouse, P., Duncan, G. S., Mittrucker, H. W., Feng, G. S. & Mak, T. W. 1996 Regulation of T cell receptor signalling by tyrosine phosphatase SYP association with CTLA-4. Science 272, 1170{1173. Medzhitov, R. & Janeway Jr, C. A. 1998 An ancient system of host defense. Curr. Opin. Immunol. 10, 12{15. Milton, J. 1667 Paradise lost. Omaha, NB: Renascence Editions. Nagata, S. 1999 Fas ligand-induced apoptosis. A. Rev. Genet. 33, 29{55. Powell, J. D., Ragheb, J. A., Kitagawa-Sakakida, S. & Schwartz, R. H. 1998 Molecular regulation of interleukin-2 expression by CD28 co-stimulation and anergy. Immunol. Rev. 165, 287{300. Ranger, A. M., Grusby, M. J., Hodge, M. R., Gravallese, E. M., de la Brousse, F. C., Hoey, T., Mickanin, C., Baldwin, H. S. & Glimcher, L. H. 1998 The transcription factor NF-Atc is essential for cardiac valve formation. Nature 392, 186{190. Suzuki, N. (and 14 others) 2002 Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4. Nature 416, 750{756. Thomas, J. A., Allen, J. L., Tsen, M., Dubnico® , T., Danao, J., Liao, X. C., Cao, Z. & Wasserman, S. A. 1999 Impaired cytokine signalling in mice lacking the IL-1 receptor-associated kinase. J. Immunol. 163, 978{984. Tivol, E. A., Borriello, F., Schweitzer, A. N., Lynch, W. P., Bluestone, J. A. & Sharpe, A. H. 1995 Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3, 541{547. Toyonaga, B. & Mak, T. W. 1987 Genes of the T-cell antigen receptor in normal and malignant T cells. A. Rev. Immunol. 5, 585{620. Waterhouse, P., Penninger, J. M., Timms, E., Wakeham, A., Shahinian, A., Lee, K. P., Thompson, C. B., Griesser, H. & Mak, T. W. 1995 Lymphoproliferative disorders with early lethality in mice de¯cient in CTLA-4. Science 270, 985{988. Yanagi, Y., Yoshikai, Y., Leggett, K., Clark, S. P., Aleksander, I. & Mak, T. W. 1984 A human T cell-speci¯ c cDNA clone encodes a protein having extensive homology to immunoglobulin chains. Nature 308, 145{149. Yeh, W. C. (and 13 others) 1998 FADD: essential for embryo development and signalling from some, but not all, inducers of apoptosis. Science 279, 1954{1958. Yeh, W. C. (and 10 others) 2000 Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development. Immunity 12, 633{642. Zinkernagel, R. M. & Doherty, P. C. 1974 Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system. Nature 48, 701{702.
Phil. Trans. R. Soc. Lond. A (2003)
