CHAPTER 15 IMMUNE ACCESSORY MOLECULES AND SIGNAL TRANSDUCTION
CHAPTER 15 IMMUNE ACCESSORY MOLECULES AND SIGNAL TRANSDUCTION
THEODORE J. YUN
EDWARD A. CLARK
T-Cell-Dependent B-Cell Maturation
Accessory Molecules Involved in Immune Activation
Signal Transduction Through Extracellular Receptors
Antigen Receptor Signaling
Mitogen-Activated Protein Kinase Pathway
Signal Transducers and Activators of Transcription
Matters of Life and Death
Immunodeficiencies Due to Defects in Signal Transduction
This chapter describes the accessory molecules and signal transduction pathways that are important in lymphocyte function and turnover. In addition, this chapter provides a description of immune deficiency disorders that result from defects in the expression of signaling of immune accessory molecules.
Acronyms and abbreviations that appear in this chapter include: BCR, B-cell receptor; DAG, diacylglycerol; DD, death domain; DED, death effector domain; EBV, Epstein-Barr virus; GAS, gamma-activated site; GCs, germinal centers; IFN, interferon; Ig, immunoglobulin; IL-2, interleukin-2; IL-2R, IL-2 receptor; IREs, interferon response elements; ITAM, immunoreceptor tyrosine-based activation motif; JAK, Janus kinase; MAP, mitogen-activated protein; MAPK, MAP kinases; MAPKK, MAP kinase kinases; MAPKKK, MAP kinase kinase kinases; PH, Pleckstrin homology; PI3K, phosphatidylinositol-3 kinase; PLC-gc, phospholipase C-gc; PTK, protein tyrosine kinase; PY, phosphotyrosine; SAP, SLAM-associated protein; SH, src-homology; STATs, signal transducers and activators of transcription; TCR, T-cell receptor; TNF, tumor necrosis factor; TNFR, TNF receptor; V, Ig variable; XLP, X-linked chromosome lymphoproliferative disease.
The development, migration, activation, and maturation of lymphocytes are dependent on cell-cell interactions that are regulated and coordinated by cell surface accessory molecules and antigen receptors.1,2,3,4 and 5 During their development, lymphoid cells migrate to a number of sites in the body. Lymphocyte progenitors interact with accessory cells, such as epithelial cells in the thymus or stromal cells in the marrow. These cells nurture and instruct lymphoid precursor cells to express antigen-receptor genes, to proliferate, and to differentiate into mature cells. The newly formed lymphocytes then enter the blood and home to peripheral lymphoid tissues via cell-cell interactions with specialized endothelial cells in highly endothelial venules. Once in the spleen or lymph node, a naive B lymphocyte may encounter antigens and yet another set of cells, including T cells, dendritic cells, and, as germinal centers (GCs) are formed, follicular dendritic cells and activated T cells. In the presence of these cells and a T-cell-dependent antigen, the B lymphocyte is induced to become a memory B cell or a plasma cell.
Several families of surface molecules are involved in cell-cell interactions.6,7 These families include: (1) the integrin family, (2) the selectin family, (3) the type II C-lectin family, (4) the immunoglobulin (Ig) superfamily, (5) the tumor necrosis factor (TNF) family, (6) the TNF receptor (TNFR) family, (7) the complement control protein family, (8) the scavenger receptor family, and (9) the tetraspan, or transmembrane 4 pass, family.
T-CELL-DEPENDENT B-CELL MATURATION
After entering a lymph node, the B cells that have surface immunoglobulin receptors specific for foreign antigen trapped within the lymphoid tissue migrate into T-cell-rich paracortical regions of secondary lymphoid tissues (see Chap. 5). Here they may capture and process soluble antigen. Some T cells in this region already may have recognized antigenic peptides processed and presented in association with MHC class II molecules of potent antigen-presenting cells, such as extrafollicular dendritic cells (see Chap. 84). The activated T cells, and perhaps dendritic cells, then signal a few antigen-specific B cells to migrate to follicles and interact with folicular dendritic cells. The latter cells are large, spiny, nonhematopoietic cells that retain antigen-antibody immune complexes and induce B-cell proliferation and the formation of germinal centers. Somatic mutation of Ig variable (V) region genes occurs soon after antigenic stimulation during germinal center development (see Chap. 83).8 After somatic mutation occurs, B cells with high-affinity receptors for antigen are selected, and switching from IgM to another immunoglobulin class (“Ig class switching”) occurs. Four key cell types are required for this process: activated B cells, CD4+ T cells, GC dendritic cells, and follicular dendritic cells. Eventually germinal center B cells mature into memory B cells or plasmablasts.
Several cell-cell interaction receptor-ligand pairs are involved in T-cell-dependent B-cell maturation.8,9 and 10 In the paracortical regions, CD4+ T cells are activated by recognizing MHC class II molecules plus peptide on dendritic cells. Interdigitating dendritic cells are especially effective antigen-presenting cells because they express not only high levels of MHC class II and CD40 but also other key accessory molecules, such as CD54, CD58, CD80, and CD86. Since the affinity of the TCR for MHC class II + peptide is low, the binding of CD4 and CD2 ligands on T cells to their ligands, MHC class II and CD58 on antigen-presenting cells facilitates cell-cell interactions leading to T-cell activation. Cross-linking of the T cell receptor (TCR) and its coreceptors in turn leads to the rapid activation of an avid CD11a/18 complex,11 which then can bind to its ligand CD54. The fact that tissue dendritic cells, unlike resting B cells, express key accessory molecules, such as CD54, CD58, CD80, and CD86, may explain why naive T cells are activated by dendritic cells but not by resting B cells. Once B cells are activated initially by T cells and later by follicular dendritic cells, they express an array of surface accessory molecules that enable them to become efficient antigen-presenting cells.
ACCESSORY MOLECULES INVOLVED IN IMMUNE ACTIVATION
Two surface molecules of antigen-presenting cells, CD40 and CD80/CD86, have ligands found on activated or resting T cells. These receptor-ligand pairs are engaged during a cognate T-B dialogue, allowing T cells and B cells to sense each other’s state of activation and then respond appropriately.4
CD40, a member of the TNFR family, is expressed on antigen-presenting cells, such as B cells, dendritic cells, and activated macrophages. The ligand for CD40 is a TNF family member, CD154 (CD40L). It is expressed primarily on activated CD4+ T cells but not on resting T cells.10,12 When the CD154-CD40 interaction is blocked in vitro, B cells cannot proliferate or produce IgG in the presence of T cells.13 Patients with an X-linked chromosome defect in CD154 expression or function do not form germinal centers in response to antigen, and their B cells do not switch Ig classes in vivo.14 Thus, T cells and CD154-CD40 interactions are required for both germinal-center formation and Ig class switching.
While the CD154-CD40 interaction enables the B cell to respond to an activated T cell, a second critical receptor-ligand interaction through CD80 and CD28 allows peripheral T cells to respond to an activated B-cell partner, to divide, and to produce the cytokines required for T-cell differentiation. CD80 is expressed on activated B cells, macrophages, and some dendritic cells. It has two known ligands: CD28, found on both resting and activated T cells, and CD152 (CTLA-4), found on activated T cells. The binding of CD80 to CD28 on T cells, which previously were stimulated via the cross-linking of their TCRs, enhances interleukin-2 (IL-2) production and T-cell proliferation.15 Interference with the CD80 signal to T cells blocks T-cell proliferation and T-cell-dependent B-cell maturation. In contrast, weakly immunogenic tumors can be made immunogenic, i.e., able to induce a protective T-cell response, by transfecting them with the CD80 cDNA,16 demonstrating that the immunogenicity of a cellular antigen(s) is dependent on the coexpression of CD80.
Another ligand for CD28 and CD152 related to CD80 that has been identified on antigen-presenting cells is designated CD86.17,18 Monoclonal antibodies to CD86 inhibit CD28-dependent T-cell activation.17 Unlike CD80, CD86 is expressed on natural killer cells and is upregulated rapidly on antigen-presenting cells after activation.17,18 The fact that CD86, unlike CD80, is constitutively expressed on resting B cells suggests that it may provide one of the first costimulatory signals to T cells.
Once CD80/CD86 is expressed on an antigen-presenting cell, it in turn can signal the T cell via CD28 (see Chap. 84).10 There is an additional level of reciprocity in this T-B dialogue: cross-linking CD28 on activated T cells increases the expression of CD154,19 while cross-linking CD40 on B cells increases the expression of both CD80 and CD86.17,20 This “reciprocal dialogue” may occur in peripheral lymphoid T-cell zones and perhaps in the basal light zones of germinal centers, where T cells expressing CD154 have been identified.
Initiation of this sequential reciprocal dialogue must be carefully regulated to prevent the activation of autoreactive or bystander T cells or B cells. The presence of just CD40 and class II on resting B cells and the TCR/CD3 complex and CD28 on resting T cells is not sufficient for mutual T-B cell activation to commence, since resting B cells do not activate resting T cells. Rather, antigen-specific resting B cells induce tolerance in T cells to protein antigens.9 Similarly, activated T cells can provide contact-dependent helper activity for B cells, but resting T cells cannot.9 However, a reciprocal dialogue may ensue if either the T cell or the B cell has been activated. If the activated T cell expresses CD154, it is able to induce resting B cells to express CD80.20 Similarly, activated cells expressing CD80/CD86 are able to induce T cells to express CD154. In other words, the presence of either CD154 or CD80/CD86 enables both the T cell and the B cell to be activated in the presence of antigen. Theoretically, a number of agents that can stimulate the expression of CD80 on B cells, such as cross-linking of antigen receptors or class II, might promote a production dialogue. However, the antigen specificity of the T-B dialogue is maintained overall, since signaling through either CD40 on B cells, or CD28 on T cells, is most efficient after B or T cells have been stimulated through their antigen receptors.15
The CD40 signal to B cells and the CD28 signal to T cells have a number of similar and potentially reciprocal properties. Just as antibodies to CD28 promote the proliferation of T cells after TCR/CD3 cross-linking, antibodies to CD40 can promote the proliferation of B cells stimulated by immunoglobulin receptor cross-linking.10 Signaling T cells through their specific TCR alone without the CD28 signal from CD80 may lead to induction of T-cell tolerance or anergy. Cross-linking of CD28 not only can prevent anergy but also can prevent T cells from undergoing programmed cell death, or apoptosis.15 Similarly, apoptosis occurring in germinal center B cells, or induced by cross-linking surface IgM on immature B cells, can be blocked by cross-linking CD40. Thus, the presence or absence of T-cell help through CD40, or an antigen-presenting-cell signal through CD28, may determine whether antigen-stimulated B cells or T cells are activated or induced to die.
The reciprocal T-B cell signaling involving CD154-CD40 and CD28/CD152-CD80 is not the only means by which T cells and B cells interact. Other signaling pairs are involved in regulating reciprocal signaling. For example, the CD11a/18-CD54 integrin pair in particular is important in T-B cell cognate interactions. Furthermore, cross-linking CD40 on B cells promotes T-cell proliferation via CD11a/18-CD54-dependent interactions. Signaling through CD150 and a recently described member of the CD28/CD152 family, ICOS, can also promote T cell activation.
In addition, the binding affinities of the accessory molecules involved in cognate cell-cell interactions can be influenced by the activation states of the interacting cells. For example, cross-linking the TCR increases the affinity of the T cell’s CD11a/18 and CD2 surface molecules for their respective ligands, CD54 and CD58, on antigen-presenting cells.11,21,22 Furthermore, T-cell activation also induces increased expression of CD49d/29 (VLA-4), an integrin with binding activity for CD106 (VCAM-1) and fibronectin. The affinity of CD8 for its respective ligand, namely MHC class I proteins, also is increased after T-cell antigen recognition.23 The consequences of these increased affinities depend on the receptor-ligand pairs. For example, activated CD8, after avidly binding to MHC class I molecules, initiates hydrolysis of phosphatidylinositol, but adhesion of CD49d or CD49e to fibronectin does not23; the avid binding to fibronectin instead may amplify this signaling pathway. Cyclic AMP induces increased CD2 avidity to CD5822 but decreases avidity of CD11a/18 to CD54.11 This may be because activation induces the CD2 molecule to associate with tubulin in the cytoskeleton but induces CD11a/18 to interact with talin, and thus presumably with the actin cytoskeleton.
SIGNAL TRANSDUCTION THROUGH EXTRACELLULAR RECEPTORS
Although the key pathway for activating resting B and T lymphocytes is through their specific antigen receptors, certain other signals via accessory receptors or cytokine receptors may modulate or provide additional signals. The fate of antigen-receptor signaling depends on the context in which the interactions occur. In T-cell activation, signals that CD28 generates upon ligation by CD80/CD86 enhance the activation process. Likewise, signals through CD40 are as important in B-cell activation. Throughout T- or B-cell maturation, signals generated from costimulatory receptors or cytokine receptors influence the signal from the antigen receptor. The combination of all extracellular stimuli determines the fate of an individual cell. For example, antigen-receptor cross-linking may induce immature T cells or B cells to undergo apoptosis, affect stimulation of mature T cells, or rescue germinal center B cells from undergoing apoptosis.24,25 and 26
Understanding how a cell can integrate various extracellular stimuli and transmit these to the nucleus is an area of active research. Although a comprehensive description of all possible transduction pathways that a lymphocyte uses is beyond the scope of this chapter, we will focus on major pathways by which signals are transmitted from the membrane, through the cytosol and to the nucleus. Activation of other transcription factors, such as NF-kB and NF-AT follow similar principles.
ANTIGEN RECEPTOR SIGNALING
TCR or B cell receptor (BCR) complexes not only contain receptors for specific antigens but also have associated molecules that inform the cell interior that antigen has been recognized. Although Fig. 15-1 shows only a model for how the BCR may be activated, the BCR and TCR complexes have a number of similar features26,27 and thus likely have similar signaling mechanisms.3,28,29 and 30
FIGURE 15-1 Proximal signaling events after surface Ig stimulation in B cells. A simple model on the early kinases involved that initiate a cascade of effector molecules that exert a broad range of biological effects. In particular, tyrosine kinases such as Syk and Lyn can independently or cooperatively activate multiple substrates. Lipid-modifying enzymes, such as PLC-g and PI3-K, are activated by Syk or Lyn and can produce a variety of lipid products that activate the cell, such as DAG, IP3, and PIP3. The MAPK pathways can also be activated by these proximal events. This scheme generalizes the activation process, and in vivo this process is much more complex and mysterious. Other molecules, such as costimulatory molecules and cytokine receptors, are likely to be involved in modifying the signals transduced to the nucleus.
The TCR and BCR differ from the classic protein tyrosine kinase (PTK) receptors, such as the epidermal growth factor receptor or platelet-derived growth factor receptor, in that they do not themselves have protein tyrosine kinase activity. However, they are associated with potentially potent PTKs at the cell membrane, such as the src or Syk/ZAP-70 family kinases. These PTKs are inactive during the resting state, perhaps because they have been phosphorylated and are folded up upon themselves.31
After antigen-receptor cross-linking, a cell surface protein tyrosine phosphatase, CD45, apparently dephosphorylates inactive PTKs, freeing them to become active and to begin the signaling cascade.3,32 One initial event is the phosphorylation of TCR- or BCR-associated surface molecules on certain tyrosine residues. The phosphotyrosine residues are flanked by a characteristic set of amino acids making a phosphotyrosine motif, termed immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylated ITAMs can be recognized by proteins containing one or more src-homology (SH) region 2 domains, called SH2 domains (see below).33,34 The SH2 domains in signaling components, such as phospholipase C-g (PLC-g) or phosphatidylinositol-3 kinase (PI3K), bind with high affinity to certain phosphotyrosine (PY) motifs and thereby assemble to form a cytosolic signaling apparatus beneath the cell surface membrane. The tyrosine residues of the src-family kinases also are phosphorylated and contain an SH2 motif. As such, they may bind to each other or to other PY-containing proteins following activation (Fig. 15-1).
In T cells, src-family PTKs, probably Fyn and Lck, initiates phosphorylation on the TCR complex. Then, ZAP-70, in mature T cells, is recruited into the receptor complex by binding to tyrosine phosphorylated molecules. This adds an additional kinase specificity to the signaling pathway that, through tyrosine phosphorylation of substrates, can form additional distinctive signaling apparati. Together these apparati transmit distinct messages to the cell interior.
In B cells, the initial activation events are likely different from those in T cells (Fig. 15-1). Both Lyn, an src-family PTK, and Syk are excellent candidates for participation in the early events in receptor activation. Contrasting the sequential model of T-cell activation, BCR engagement can activate Syk and Lyn simultaneously, leading to parallel activation of both signaling pathways. This model is supported by the fact that the BCR can transmit signals in either Lyn- or Syk-deficient B cells.
One perplexing problem of signal transduction is the multiple substrate specificity of a given kinase. Also, multiple kinases can phosphorylate a single substrate. How does a cell achieve specificity by activating certain transduction pathways through a single receptor? A solution to this problem may be found in the emerging members of adapter proteins.
Adapter proteins are functionally defined as molecules that have no intrinsic kinase activity; however, these proteins contain “modules” that can specifically couple with other molecules, such as kinases.35,36 These modules, such as SH2, SH3, or PH domains, can recognize peptide motifs or phospholipids. Typically, adapter proteins will have multiple modules that will bind to and localize kinases and substrates (Fig. 15-2). Therefore, these adapter proteins are of critical importance to the cell’s signal transduction pathways. They serve to localize and organize specific pathways to certain receptors or subcellular sites.
FIGURE 15-2 Adapter molecules couple kinase to downstream molecules. In this example, a receptor tyrosine kinase epidermal growth factor receptor (EGFR) forms a dimer and becomes activated following ligand binding. A series of adapter molecules, Shc and Grb2, couple the kinase to Sos. Sos then activates Ras, which leads to a number of mitogenic signals, such as activation of the MAPK cascade.
Several different types of modules have been described. A module is typically 30 to 100 residues with a characteristic motif. The variable residues in the motif are thought to direct specificity with binding sites on target proteins. There are two types of SH domains that are commonly found in adapter proteins.34 SH2 domains often direct interaction with phosphotyrosine located in a specific motif. Some SH2 domains are also able to bind to certain phospholipids. Many receptor and nonreceptor tyrosine kinases become phosphorylated themselves. This may allow binding of SH2-containing adapter proteins to the kinases, which will functionally serve to couple the kinase with target molecules that are also bound to the adapter protein. SH3 domains have been described to interact with proline rich sequences in proteins, often containing the motif PXXP.
Pleckstrin homology (PH) domains can also be found in many signaling proteins.37 These modules can direct both protein-protein interactions and protein-phospholipid interactions. The interaction with phospholipid is especially important as these PH domains containing adapter proteins can cause recruitment of bound molecules to discrete regions of the cell membrane. Through protein-binding modules, adapter proteins can recruit and organize kinases and their targets. This can be a daunting task, especially in complex signaling apparatus such as lymphocyte antigen receptors, where multiple costimulatory and downregulatory molecules are within proximity to the antigen receptors, which themselves are associated with a number of kinases.38 The number of characterized adapter molecules is increasing, and soon it may be possible to delineate molecules that are involved in a particular transduction cascade initiating from the membrane and ending at the nucleus.
One result of cross-linking antigen receptors is that the enzymes PLC-g1 or PLC-g2 are activated to metabolize phosphatidylinositol bisphosphate into two second messengers, diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (Fig. 15-1). DAG acts to stabilize and activate membrane-associated protein kinase C, whereas inositol-1,4,5-trisphosphate induces release of calcium from the endoplasmic reticulum. The latter raises the intracellular calcium level (Ca2+]i) and activates calcium-dependent kinases, such as calcium/calmodulin-dependent kinase II or calcineurin, a Ca2+-dependent threonine/serine phosphatase that contributes to activation of genes encoding cytokines, such as interleukin-2. Protein kinase C and calcium/calmodulin-dependent kinase II in turn have a variety of cellular substrates that, when phosphorylated, promote cell activation, growth, or maturation. In particular, a set of kinases are activated that can function to regulate transcriptional processes through phosphorylation.39,40 The Ca2+-dependent pathway is blocked in part by cyclosporine. Cyclosporine forms a complex with the prolyl-isomerase, cyclophilin, that inhibits calcineurin’s phosphatase activity, thereby preventing downstream transcriptional activation.
PI3K is another important lipid-metabolizing enzyme which is activated upon receptor engagement, likely through an src-family kinase.41,42,43 and 44 Through a phosphorylation event by PI3K, phosphatidyl-inositol and its derivatives can form potent second messengers, PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P. Inhibition of PI3K enzymatic activity generally results in suppression of many cellular responses, such as cell survival or proliferation.45
Via SH2 or PH domains, certain proteins can specifically bind to distinct lipid products. These domains regulate protein-phospholipid and protein-phosphoprotein interactions. Through these domains, multiple kinases can be brought in proximity at the membrane. A number of examples exist. The SH2 domain of src has been demonstrated to bind to PtdIns(3,4,5)P3. Akt/PKB, a Ser/Thr kinase that has been implicated in cell survival, is downstream of PI3K.46 The PH domain of Akt/PKB has been shown to bind to PtdIns(3,4)P2, so Akt/PKB could be recruited to the membrane. In B-cell signaling, Btk may also be recruited to the membrane through its PH domain binding to PI3K lipid products. Btk is a critical regulator of B-lymphocyte signaling, since genetically Btk-deficient humans or mice lack functional B cells. In summary, following cellular stimulation, PI3K generates lipid products that mediate recruitment of upstream signaling molecules (Fig. 15-1).
MITOGEN-ACTIVATED PROTEIN KINASE PATHWAY
Mitogen-activated protein (MAP) kinase signaling cascades are a major means by which signals are transduced from the membrane to the nucleus. The cascade is composed of three analogous pathways (Fig. 15-3). At the core of each pathway there are three kinases that are categorized as MAP kinase kinase kinases (MAPKKK), MAP kinase kinases (MAPKK), or MAP kinases (MAPK). A straightforward model proposes that there is sequential activation of these kinases, i.e., one MAPKKK activates a MAPKK, which in turn activates a MAPK. These proteins are Ser/Thr kinases that are activated through mitogenic or stress-induced stimuli. The importance of this cascade is underscored by the fact that homologous pathways exist in diverse species such as yeast to human. The final outcome of activating the MAPK pathways can lead to a variety of biological effects, including cell proliferation, cell differentiation, and stress response induction. Currently, the ability of the kinases to cross-talk between other members of the parallel MAPK pathways is not known.
FIGURE 15-3 Depiction of the multiple, parallel pathways of the MAPK cascade. The MAPK cascade transduces signals from cell-surface-associated kinases to the nucleus via a series of Ser/Thr kinases. In each pathway, the MAPKKK family member activates its respective MAPKK, which then activates the MAPK. Activated MAPKs can then phosphorylate and activate a set of transcription factors that mediate a wide variety of biological effects.
Receptor activation is linked to activating the ERK MAPK by Ras. Ras is a guanine nucleotide binding protein that is involved in a variety of mitogenic signals. Although several mechanisms are known to exist that couple receptor activation to Ras activation, a general model can be proposed where receptor dimerization results in autophosphorylation and recruitment of SH2-containing adapter molecules, such as Grb2. Grb2 is able to recruit Sos, a guanine nucleotide exchange factor, which activates Ras by guanine nucleotide exchange (Fig. 15-2). Bound to GTP, Ras becomes a potent activator of signal transduction. The first “tier” of MAPKKK proteins in this cascade then become activated.47 Raf, the MAPKKK in the ERK2 pathway, is activated following membrane translocation and dimerization.
The activated MAPKKK phosphorylates MAPKKs on serine residues. Once activated, the MAPKK can then activate its respective MAPK family substrates (Fig. 15-3). The MAP kinases include ERK, JNK/SAPK (jun-N terminal or stress-activated protein kinase), and p38 MAPK/Hog. Finally, these three kinases can activate distinct but overlapping sets of transcription: ERK can activate Elk, Ets-1 and -2, and c-fos; JNK can activate Elk, c-jun, and ATF; and p38 can activate ATF2, Elk, CHOP, MEF2c, and Max (Fig. 15-3).48,49
Within the past decade, tremendous advances in understanding cytokine signaling have been made. In some ways, delineating the pathways of their signals has been as daunting as understanding antigen receptor signaling. Like antigen receptors, many cytokine receptors also lack intrinsic kinase activity. Furthermore, a cell can express multiple cytokine receptors, which are often multimeric complexes. Also, some cytokine receptor complexes share individual components. How can a cell integrate multiple signals originating from very similar receptors? Some cytokine receptors, such as the IL-2 receptor (IL-2R) complex,50 resemble the TCR, insofar as they may associate with src-family kinases such as p56lck. Different regions within the cytoplasmic tails of the IL-2Rb chain and IL-2Rg chain appear to link to distinct signaling pathways.50,51 Activation through the src-family protein tyrosine kinases requires a distal region of IL-2Rb chain’s cytoplasmic tail and the SH2 region in the tail of IL-2Rg chain. This protein tyrosine kinase group may be required for activation of the MAPK cascade, leading to expression of the c-fos and c-jun genes.
A region of IL-2Rg chain more proximal to the plasma cell membrane is required for activation of another protein tyrosine kinase that possibly is a member of the Janus kinase (JAK) family of protein kinases. Discovery of the JAK-STAT transduction pathway has begun to explain some of the mysteries regarding cytokine transduction.52 Janus kinases are a family of nonreceptor tyrosine kinases that can associate with some cytokine receptors. JAK activation can lead to recruitment of another family of molecules, signal transducers and activators of transcription (STATs), which, upon activation, translocate to the nucleus and bind a consensus motif called gamma-activated site (GAS) (Fig. 15-4).52 Much remains to be elucidated about this family, in terms of structure-function, regulation, and possible characterization of other family members. Homologous JAK-STAT pathways can be found in Drosophila and dichtostelium, emphasizing the importance of this pathway in eukaryotic cell differentiation and activation.
FIGURE 15-4 Diagram showing a general model of JAK-STAT activation. This figure illustrates a general model where cytokine binding to its appropriate receptor leads to cell activation or differentiation. Prior to cytokine binding, JAKs are associated with a chain of a cytokine receptor. Upon ligand binding, JAKs become activated, dissociate from the receptor, and phosphorylate the receptor chain. STATs then bind to the phosphorylated sites on the receptor. Next, bound STATs become phosphorylated, dissociated from the receptor, dimerize, and then translocate to the nucleus, where they bind to GAS sequences and activate transcription.
Currently, there are four mammalian JAKs: JAK1, JAK2, JAK3, and Tyk2.52 In terms of structure, JAKs have a catalytic domain, JH1, at the carboxy terminus. A pseudokinase domain, JH2, is also present. The proteins have further homologous domains termed JH3 to JH7; however, the function of these domains remain to be solved. The JH2 domain is also mysterious. Although it has homology to the kinase domain, it lacks catalytic activity. However, mutation or deletion of this domain affects JAK function. JAK1 and 2 are associated with multiple cytokine receptors, including receptors for interferons (IFN)-a and -b. JAK3 is more restricted in its associations. It binds to the gc receptor chain and has been reported to bind to CD40.53 Tyk2 binds to gp130 chain that is shared among several cytokine receptor complexes.
SIGNAL TRANSDUCERS AND ACTIVATORS OF TRANSCRIPTION
Additional specificity in cytokine signaling is achieved via the STAT family of transcription factors. STATs are cytoplasmic proteins which, when activated, translocate to the nucleus where they bind to GAS motif in interferon (IFN) response elements (IREs). Structurally, they have a DNA-binding domain, as well as SH2 and SH3 domains.52 The latter two domains most likely mediate protein-protein interactions. At the carboxy terminus is a transactivation domain not conserved between family members.
JAKs can phosphorylate key sites on receptors to which STATs are recruited and bound, probably via their SH2 domain (Fig. 15-4). Specificity for the receptor-STAT binding is mediated by the residues in the binding site and in the SH2 domain of the receptor and STAT respectively. Next, they become phosphorylated, which releases them from the receptor. Activated STATs then dimerize and translocate to the nucleus.
There are some variations to this general model of STAT activation. First, some STATs may be associated with some receptors without prior phosphorylation. Second, in some cases STATs may become activated without associating with a receptor. Third, STAT1 has a MAPK phosphorylation site, and some STATs may be phosphorylated on serine residues, raising the intriguing possibility that MAPK cascade may cross-talk with the JAK-STAT pathway. Once in the nucleus, STATs will bind to IREs. Different STAT family members can bind to different GAS sites. Although GAS motifs are similar in sequence, the exact base composition and spacing differs.
These signal-transduction pathways are regulated to prevent uncontrolled cell growth or differentiation. Lymphocyte protein tyrosine kinases are regulated by protein tyrosine phosphatases that inactivate the kinase substrates or the kinase itself. Some phosphatases even have SH2 motifs enabling them to bind to tyrosine-phosphorylated proteins and remove phosphates. Whether a cellular response ensues may depend on the balance of activities between tyrosine kinases and their competing phosphatases.54 Another way a signaling pathway may be regulated is via contact with another signaling pathway. For example, the MAPK cascade may be inhibited by protein kinase A, inactivating Raf-1.55
MATTERS OF LIFE AND DEATH
There are several ways to induce cells to undergo programmed cell death or apoptosis (see Chap. 12a).56,57 Cells may undergo apoptosis in response to a variety of stimuli, including withdrawal of essential growth factors, exposure to radiation or glucocorticoids, or following cross-linking of certain surface membrane receptors, such as Fas (CD95). Unlike necrosis, apoptosis requires new gene expression. Studies using homozygous null gene “knockout” mice have shown that expression of certain proteins is essential for inducing apoptosis or for preventing it from occurring.56,57 On the other hand, the c-myc protein is required for TCR-mediated apoptosis of some T cells. However, this or other proteins in other contexts can have the opposite or no effect. For example, the tumor suppressor p53 is required for irradiation-induced apoptosis of lymphocytes but does not play an apparent role in steroid-induced apoptosis of thymocytes. How different stimuli, such as those provided by TCR cross-linking, irradiation, or glucocorticoids, induce apoptosis is not yet defined.
Recent excitement has focused on defining molecules which are involved in cell survival or death.58 There appear to be two active pathways present in a cell that responds to extracellular signals which induce the apoptotic program, or an intrinsic signal when the cell “senses” to undergo cell death.59 An example of the former situation is signaling through the CD95/Fas receptor, a prototype TNFR family member generally associated with signaling cell death. Response to growth factor withdrawal exemplifies the latter case. These two pathways probably converge.
A tremendous advance in our understanding of apoptotic mechanisms comes from genetically defining three genes in Caenorhabditis elegans, a nematode. These genes, CED-3, CED-4, and CED-9, are involved in the development of C. elegans where 131 of 1030 somatic cells must undergo cell death in order for proper progression in development. Mammalian orthologs have been described for these three genes, and CED-3 encodes for a protease. In higher vertebrates, a family of CED-3-related molecules exist, called caspases, and form a proteolytic cascade. APAF is a gene that resembles CED-4; it functions as a scaffolding molecule that bridges CED-3 to CED-9. The mammalian homologs of CED-9, the bcl-2-related proteins, are a growing family of molecules that can either protect or activate apoptosis. Bcl-2 is a mitochondria protein with structural similarity to an ion channel; however, how this protein functions in mitochondria physiology and how this overall affects cell survival are still unanswered questions. Mice without the BCL-2 protooncogene display normal embryonic development and hematopoiesis. However, soon after birth these BCL-2-null mice undergo massive apoptotic involution in the thymus and spleen, demonstrating that BCL-2 plays a key role in preventing lymphocyte apoptosis.60
Although many TNFR family receptors can transduce an apoptotic signal, we will use CD95/Fas as the prototype example. Similar features of this cascade can be found in apoptotic cascades transduced by other receptors.
In the case of CD95, adapter molecules associate with the cytoplasmic tail via modules defined as death domains (DDs). Molecules containing DD motifs can potentially dimerize via these sequences (see Table 15-1). Although the homology among different DDs is low, the unique sequences mediate specificity of interactions. FADD is an adapter that associates with CD95.61,62 and 63 FADD contains a C-terminal DD and an N-terminal motif called a death effector domain (DED).63,64 Analogous to DDs, DEDs can associate, effectively bringing together molecules forming a death-inducing signaling complex.65 One critical DED-containing molecule in the CD95 apoptotic cascade is caspase-8.66 The entire molecule is actually an holoenzyme, and when activated, the procaspase-8 is cleaved into active p18 and p10 subunits. This initiates the apoptotic-signaling cascade that includes cleavage and activation of other proteases such as caspase-1, -3, -6 and -7, and kinases, such as Mst-1.
TABLE 15-1 MAMMALIAN DEATH PATHWAY-ASSOCIATED GENE FAMILIES
IMMUNODEFICIENCIES DUE TO DEFECTS IN SIGNAL TRANSDUCTION
Several X-linked chromosome immunodeficiency disorders are due to mutations in genes required for lymphocyte activation (see Chap. 88). Patients with the hyper-IgM syndrome have mutations in the CD40L gene (Xq25) such that their T cells either do not express CD154 or express a defective CD154 that is unable to bind CD40. Because of this, the T cells of these patients are unable to induce B cells to proliferate or switch immunoglobulin classes.14 As a result, these patients produce IgM only in response to antigens and do not form germinal centers. Patients with X-linked chromosome hypogammaglobulinemia (XLA) have a defective XLA gene (Xq22) encoding the B-cell tyrosine kinase critical for the development of B lymphocytes.67,68 Also, a group of patients with X-linked chromosome severe combined immunodeficiency have nonsense mutations in the gene encoding the g chain of the IL-2 receptor, IL-2RG (Xq13).69 These patients have reduced or absent levels of immature and mature T cells, demonstrating that IL-2Rg is essential for T-cell development. However, since the IL-2Rg chain also can associate with the receptors for IL-4 and IL-7 to form higher-affinity receptors for their respective cytokine, it is not clear whether the immunodeficiency of such patients is due solely to a defect in the IL-2R signaling. Another X-linked immunodeficiency results from mutations in an adapter molecule called SLAM-associated protein (SAP).70 This molecule associates with CD150, which is a costimulatory molecule that can modulate signals in T cells and B cells. Patients with mutation in SAP are susceptible to X-linked chromosome lymphoproliferative disease (XLP) after infection by Epstein-Barr virus (EBV). These patients have an uncontrolled proliferation of T and B cells that results in fatality in 51 percent of patients.71
On the other hand, defects that interfere with the regulation of lymphocyte cell growth or activation can lead to lymphoproliferative or autoimmune disease. The lpr mutation in mice interferes with expression of the Fas (CD95) antigen.72 The lymphocytes of mice that are homozygous for this mutation do not express Fas and, consequently, have prolonged and inappropriate life spans, resulting in lymphocyte accumulation and eventual lymphoproliferative disease. Homozygous motheaten mice (me/me) have a defect in a protein tyrosine phosphatase, designated the SHP-1 phosphatase.73 This defect impairs T-cell development and results in an autoimmune disease syndrome.
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Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn