Immunology_Janeway

Immunology_Janeway. T cells, Syk is broadly expressed in all leukocyte subsets; in contrast, ZAP-70 has been found only in T cells and NK cells. Several viral pathogens seem to have acquired ITAM-containing receptors from their hosts. These include the Epstein–Barr virus (EBV), whose LMP2A gene encodes a membrane protein with a cytoplasmic tail containing an ITAM. This enables EBV to trigger B-cell proliferation by the signaling pathways discussed in Section 7-20, an important step in the development of EBV-induced malignancies. Another virus that expresses an ITAM-containing protein is the Kaposi sarcoma herpesvirus (KSHV or HHV8), which causes malignant transformation and proliferation of the cells it infects. 7-12 Activated ZAP-70 phosphorylates scaffold proteins and promotes PI 3-kinase activation.


Immunology_Janeway

Immunology_Janeway. 7-12 Activated ZAP-70 phosphorylates scaffold proteins and promotes PI 3-kinase activation. As described in Section 7-10, phosphorylation of the tyrosines in the T-cell receptor ITAMs leads to the recruitment and activation of ZAP-70. This provides ZAP-70 proximity to the cell membrane, where it phosphorylates the scaffold protein LAT (linker for activated T cells), a transmembrane protein with a large cytoplasmic domain (Fig. 7.15). ZAP-70 also phosphorylates another adaptor protein, SLP-76. LAT and SLP-76 can be linked by the adaptor protein Gads; this three-protein complex, referred to as the LAT:Gads:SLP-76 complex, plays a central role in T-cell activation. This is illustrated by the profound TCR signaling and T-cell development defects seen in mice lacking any MOVIE 7.1


Immunology_Janeway

Immunology_Janeway. Fig. 7.15 ZAP-70 phosphorylates LAT and SLP-76, initiating four downstream signaling modules. activated Zap-70 phosphorylates the scaffold proteins LaT and SLp-76 and recruits them to the activated T-cell receptor (TcR) complex. an adaptor protein, Gads, holds the tyrosinephosphorylated LaT and SLp-76 together. The multiple binding sites on these scaffold proteins recruit several additional adaptors and enzymes that initiate four essential downstream signaling modules. one key component required for several of these modules is the activation of pi 3-kinase, which phosphorylates pip2 in the plasma membrane to generate pip3. These four modules include the activation of the serine/ threonine kinase akt, which promotes enhanced cellular metabolic activity; the activation of pLc-γ, which leads to transcription factor activation; the activation of vav, which induces actin polymerization and cytoskeletal reorganization; and the recruitment of adap, an adaptor protein that promotes enhanced


Immunology_Janeway

Immunology_Janeway. to transcription factor activation; the activation of vav, which induces actin polymerization and cytoskeletal reorganization; and the recruitment of adap, an adaptor protein that promotes enhanced integrin adhesiveness and clustering.


Immunology_Janeway

Immunology_Janeway. Akt activation leads to increased cellular metabolic activity PIP3AktPHdomainADAP recruitment leads to enhanced integrin adhesiveness and clustering ADAPVav activation leads to actin polymerization and cytoskeletal reorganization PLC-˜activation leads to transcription factor activation PLC-˜GadsVavZAP-70LATSLP-76Activated ZAP-70 phosphorylates LAT and SLP-76, initiating four signaling modules


Immunology_Janeway

Immunology_Janeway. Fig. 7.16 The recruitment of phospholipase C-γ by LAT and SLP-76, and its phosphorylation and activation by the protein kinase Itk, are crucial steps in T-cell activation. Zap-70 phosphorylates the scaffold proteins LaT and SLp-76, which are brought together to form a complex at the activated T-cell receptor by the adaptor protein Gads. This complex also promotes the activation of pi 3-kinase, leading to the production of pip3 (formed by the phosphorylation of pip2 by pi 3-kinase). phospholipase c-γ (pLc-γ) is recruited to the membrane by its ph domain binding to pip3 and then binds to phosphorylated sites in LaT and to the proline-rich domain of SLp-76. To be activated, pLc-γ must be phosphorylated by the Tec-family kinase itk. itk is recruited to the membrane by interaction of its ph domain with pip3, and by interactions with phosphorylated SLp-76. once phosphorylated by itk, phospholipase c-γ is active.


Immunology_Janeway

Immunology_Janeway. one of these components, and in humans lacking ZAP-70. A second essential event that occurs rapidly following ZAP-70 activation is the recruitment and activation of the enzyme PI 3-kinase (see Section 7.4); while the detailed mechanism linking PI 3-kinase activation to T-cell receptor stimulation is not well understood, current evidence suggests a role for the small GTPase Ras. In this case, Ras may be activated by recruitment of the Ras-GEF Sos to LAT via Sos binding to the small adaptor protein Grb2, forming a second three-protein complex containing LAT and Sos, bridged by Grb2.


Immunology_Janeway

Immunology_Janeway. Following formation of the LAT:Gads:SLP-76 complex and the activation of PI 3-kinase, the T-cell receptor signaling pathway branches into several downstream modules, each of which induces cellular changes that contribute to optimal T-cell activation (see Figure 7.15). Each module is initiated by the recruitment of a key intermediate to the active signaling complexes, either via binding to the LAT:Gads:SLP-76 complex, to the PIP3 generated by PI 3-kinase, or to both. In brief, these modules lead to the activation of phospholipase C-γ (PLC-γ), which affects transcription; the activation of the serine/threonine kinase Akt, which affects metabolism, among other things; the recruitment of the adaptor protein ADAP, which upregulates cell adhesion; and the activation of the protein Vav, which initiates actin polymerization. Each of these modules will be described in detail in the sections below.


Immunology_Janeway

Immunology_Janeway. 7-13 Activated PLC-γ generates the second messengers diacylglycerol and inositol trisphosphate that lead to transcription factor activation. One important module of T-cell receptor signaling is the activation of the enzyme phospholipase C-γ (PLC-γ). First, PLC-γ is brought to the inner face of the plasma membrane by the binding of its PH domain to the PIP3 that has been formed by the phosphorylation of PIP2 by PI 3-kinase; it then binds to phosphorylated LAT and SLP-76. The actions of PLC-γ produce two second messengers that activate three distinct terminal branches of the T-cell receptor pathway leading to transcription factor activation.


Immunology_Janeway

Immunology_Janeway. Due to this crucial role in T-cell activation, PLC-γ activation is controlled at several different levels. Recruitment to the membrane, while necessary, is not sufficient to activate PLC-γ. PLC-γ activation requires phosphorylation by Itk, a member of the Tec family of cytoplasmic tyrosine kinases. Like PLC-γ, Tec kinases contain PH, SH2, and SH3 domains and are recruited to the plasma membrane by interactions via these domains; specifically, the PH domain interacts with PIP3 on the inner face of the membrane (Fig. 7.16), and the SH2 and SH3 domains interact with SLP-76. These interactions serve to localize Itk in close proximity to its substrate, PLC-γ.


Immunology_Janeway

Immunology_Janeway. Once PLC-γ has been recruited to the inner face of the plasma membrane and has been activated, it can catalyze the breakdown of the membrane lipid PIP2 (see Section 7-4 and Fig. 7.5) to generate two products, the membrane lipid diacylglycerol (DAG) and the diffusible second messenger inositol 1,4,5-trisphosphate (IP3) (not to be confused with the membrane lipid PIP3) (Fig. 7.17). DAG is confined to the membrane, but diffuses in the plane of the membrane and serves as a molecular target that recruits other signaling molecules to the membrane. IP3 diffuses into the cytosol and binds to IP3 receptors on the endoplasmic reticulum (ER) membrane. These receptors are Ca2+ channels, which open and release calcium stored in the ER into the cytosol. The consequent low levels of calcium in the ER then cause a conformational change in the transmembrane protein STIM1, promoting its clustering within the ER membrane. The STIM1 oligomers bind to the plasma membrane, where they interact directly with


Immunology_Janeway

Immunology_Janeway. cause a conformational change in the transmembrane protein STIM1, promoting its clustering within the ER membrane. The STIM1 oligomers bind to the plasma membrane, where they interact directly with ORAI1, the plasma membrane calcium channel (also known as the CRAC channel: calcium release-activated calcium channel). Binding of STIM1 to ORAI1 triggers calcium channel opening, allowing extracellular calcium to flow into the cell to activate further signaling pathways and to replenish ER calcium stores.


Immunology_Janeway

Immunology_Janeway. The activation of PLC-γ marks an important step in T-cell activation, because after this point the PLC-γ signaling module splits into three distinct branches— the stimulation of Ca2+ entry, the activation of Ras, and the activation of protein kinase C-θ (PKC-θ)—each of which ends in the activation of a different transcription factor. These signaling pathways are utilized by many cell types, in addition to lymphocytes. Their importance in T-cell activation is shown by the observation that treatment of T cells with phorbol myristate acetate (an analog of DAG) and ionomycin (a pore-forming drug that allows extracellular calcium to flow into the cell) can reconstitute many of the effects of T-cell receptor stimulation. Additionally, deficiencies in several of these signaling components, including Lck, Zap-70, Itk, CD45, Carma1, and ORAI1, have been found to be mutated in cases of severe combined immunodeficiency (SCID). 7-14 Ca2+ entry activates the transcription factor NFAT.


Immunology_Janeway

Immunology_Janeway. 7-14 Ca2+ entry activates the transcription factor NFAT. One of the three signaling pathways leading from PLC-γ results in an influx of calcium ions into the cytosol. An important outcome of the increased cytosolic Ca2+ resulting from T-cell receptor signaling via PLC-γ is the activation of a family of transcription factors called NFAT (nuclear factor of activated T cells). NFAT is something of a misnomer, because the five members of this family are expressed in many different tissues. In the absence of activating


Immunology_Janeway

Immunology_Janeway. Fig. 7.17 Phospholipase C-γ cleaves inositol phospholipids to generate two important signaling molecules. Top panel: phosphatidylinositol bisphosphate (pip2) is a component of the inner leaflet of the plasma membrane. When pLc-γ is activated by phosphorylation, it cleaves pip2 into two parts: inositol trisphosphate (ip3), which diffuses away from the membrane into the cytosol, and diacylglycerol (daG), which stays in the membrane. Both of these molecules are important in signaling. middle panel: there are two phases of calcium release. ip3 binds to a receptor in the endoplasmic reticulum (eR) membrane, opening calcium channels (yellow) and allowing the early phase of calcium ions (ca2+) to enter the cytosol from the eR. The depletion of ca2+ stores in the eR causes the aggregation of an eR calcium sensor, STim1. Bottom panel: aggregated STim1 stimulates the second phase of calcium entry by binding to and opening calcium channels called oRai1 in the plasma membrane. This further


Immunology_Janeway

Immunology_Janeway. an eR calcium sensor, STim1. Bottom panel: aggregated STim1 stimulates the second phase of calcium entry by binding to and opening calcium channels called oRai1 in the plasma membrane. This further increases cytosolic calcium and restores eR ca2+ stores. daG binds and recruits signaling proteins to the membrane, most importantly the Ras-Gef called RasGRp and a serine/threonine kinase called protein kinase c-θ (pKc-θ). Recruitment of RasGRp to the plasma membrane activates Ras, and pKc-θ activation results in the activation of the transcription factor nfκB.


Immunology_Janeway

Immunology_Janeway. Left panel: nfaT is maintained in the cytoplasm by phosphorylation on serine and threonine residues. center panel: after antigen receptor stimulation, calcium enters the cytosol, first from the endoplasmic reticulum (not shown; see fig. 7.17) and later from the extracellular space (shown). after entering the cytosol, calcium binds to calmodulin, and the ca2+:calmodulin complex binds to the serine/threonine phosphatase calcineurin, activating it to dephosphorylate nfaT. Right panel: once dephosphorylated, nfaT moves into the nucleus, where it binds to promoter elements and activates the transcription of various genes.


Immunology_Janeway

Immunology_Janeway. MOVIE 7.2 signals, NFAT is kept in the cytosol of resting T cells by phosphorylation on serine/threonine residues. This phosphorylation is mediated by serine/threonine kinases, including glycogen synthase kinase 3 (GSK3) and casein kinase 2 (CK2). When phosphorylated, the nuclear localization sequence of NFAT is not recognized by nuclear transporters, and NFAT is unable to enter into the nucleus (Fig. 7.18).


Immunology_Janeway

Immunology_Janeway. The cytoplasmic Ca2+ resulting from T-cell receptor signaling binds to and induces a conformational change in a protein called calmodulin, which is then able to bind to and activate a wide range of different target enzymes. In T cells, an important target of calmodulin is calcineurin, a protein phosphatase that acts on NFAT. Dephosphorylation of NFAT by calcineurin allows the nuclear localization sequence to be recognized by nuclear transporters, and NFAT enters the nucleus (see Fig. 7.18). There it participates in turning on many of the genes crucial for T-cell activation, such as the gene for the cytokine interleukin-2 (IL-2).


Immunology_Janeway

Immunology_Janeway. The importance of NFAT in T-cell activation is illustrated by the effects of selective inhibitors of calcineurin called cyclosporin A (CsA) and tacrolimus (also known as FK506). CsA forms a complex with the protein cyclophilin A, and this complex inhibits calcineurin. Tacrolimus binds a different protein, FK-binding protein (FKBP), making a complex that similarly inhibits calcineurin. By inhibiting calcineurin, these drugs prevent the formation of active NFAT. T cells express low levels of calcineurin, so they are more sensitive to inhibition of this pathway than are many other cell types. Both cyclosporin A and tacrolimus thus act as effective immunosuppressants, and are widely used to prevent the rejection of organ transplants (see Chapter 16, Section 16-3). 7-15 Ras activation stimulates the mitogen-activated protein kinase (MAPK) relay and induces expression of the transcription factor AP-1.


Immunology_Janeway

Immunology_Janeway. A second branch of the PLC-γ signaling module is the activation of the small GTPase Ras. This can occur by various means. The most efficient mechanism for Ras activation in T cells is via the DAG generated by PLC-γ, which diffuses in the plasma membrane and activates a variety of proteins. One of these is the protein RasGRP, which is a guanine-nucleotide exchange factor that specifically activates Ras. RasGRP contains a protein-interaction module called a C1 domain that binds to DAG. This interaction recruits RasGRP to the membrane near active signaling complexes (Fig. 7.19), where it activates Ras by promoting the exchange of GDP for GTP. Ras is also activated in the T-cell receptor signaling pathway by the guanine-exchange factor Sos, which is recruited by the adaptor protein Grb2 (see Sections 7-2 and 7-3), which has itself been recruited by binding to phosphorylated LAT and SLP-76.


Immunology_Janeway

Immunology_Janeway. Ras is initially inactive. TCR signaling produces DAG, which recruits RasGRP to the membrane where it activates Ras Ras activates Raf, which phosphorylates Mek, which phosphorylates Erk Activated Erk enters the nucleus and activates transcription factors such as Elk-1 inactive Ras RasGRP active Ras DAG Raf transcription factor nucleus cytoplasm Mek Erk Activated Ras then triggers a three-kinase relay that ends in the activation of a serine/threonine kinase known as a mitogen-activated protein kinase or MAP kinase (MAPK) (see Fig. 7.19). In the case of antigen receptor signaling, the first member of the relay is a MAPK kinase kinase (MAP3K) called Raf. Raf is a serine/threonine kinase that phosphorylates the next member of the series, a MAPK kinase (MAP2K) called MEK1. MEK1 is a dual-specificity protein kinase that phosphorylates a tyrosine and a threonine residue on the last member of the relay, a MAPK which in T cells and B cells is Erk (extracellular signal-related kinase).


Immunology_Janeway

Immunology_Janeway. Signaling by MAPK cascades is facilitated by specialized scaffold proteins that bind to all three kinases in a particular MAPK relay, thereby accelerating their interactions. The scaffold protein kinase suppressor of Ras (KSR) functions in the Raf/MEK1/Erk pathway. During T-cell receptor signaling, KSR associates with Raf, MEK1, and Erk and localizes itself and its cargo to the membrane. In that location, activated Ras can engage with the Raf bound to KSR and trigger the kinase relay (see Fig. 7.19). An important function of MAPKs is to phosphorylate and activate transcription factors that can then induce new gene expression. Erk acts indirectly to generate the transcription factor AP-1, which is a heterodimer composed of one monomer each from the Fos and Jun families of transcription factors (Fig. 7.20). Active Erk phosphorylates the transcription factor Elk-1, which


Immunology_Janeway

Immunology_Janeway. Fig. 7.19 DAG activates MAPK cascades, leading to transcription factor activation. all mapK cascades are initiated by the activation of small G proteins, such as Ras in this example. Ras is switched from an inactive state (first panel) to an active state (second panel) by a guanine-nucleotide exchange factor (Gef), RasGRp, which is recruited to the membrane by daG. Ras activates the first enzyme of the cascade, a protein kinase called Raf, a mapK kinase kinase (map3K) (third panel). Raf phosphorylates mek, a map2K, which in turn phosphorylates and activates erk, a mapK. The scaffold protein KSR associates with Raf, mek, and erk to facilitate their efficient interactions (not shown). phosphorylation and activation of erk releases it from the complex so that it can diffuse within the cell and enter the nucleus (fourth panel). phosphorylation of transcription factors by erk results in new gene transcription.


Immunology_Janeway

Immunology_Janeway. Fig. 7.20 The transcription factor AP-1 is formed as a result of the Ras/ MAPK signaling pathway. Left panel: phosphorylation of the mapK erk activated as a result of the Ras–mapK cascade allows erk to enter the nucleus, where it phosphorylates the transcription factor elk-1. elk-1, along with serum response factor (SRf), binds to the serum response element (SRe) in the promoter of the gene (FOS) for the transcription factor c-fos, stimulating its transcription. Right panel: the protein kinase pKc-θ can induce the phosphorylation of another mapK called Jun kinase (JnK). This enables JnK to enter the nucleus and phosphorylate the transcription factor c-Jun, which forms a dimer with c-fos. The phosphorylated c-Jun/fos dimer is an active ap-1 transcription factor that binds to ap-1 sites and promotes transcription of many target genes.


Immunology_Janeway

Immunology_Janeway. cooperates with a transcription factor called serum response factor to initiate transcription of the FOS gene. Fos protein then associates with Jun to form the AP-1 heterodimer, but this remains transcriptionally inactive until another MAPK called Jun kinase (JNK) phosphorylates Jun. Similar to NFAT, AP-1 participates in turning on transcription of many genes important for T-cell activation, including the gene encoding the cytokine IL-2. 7-16 Protein kinase C activates the transcription factors NFκB and AP-1.


Immunology_Janeway

Immunology_Janeway. 7-16 Protein kinase C activates the transcription factors NFκB and AP-1. The third signaling pathway leading from PLC-γ results in the activation of PKC-θ, an isoform of protein kinase C that is restricted to T cells and muscle. Mice lacking PKC-θ develop T cells in the thymus, but their mature T cells have a defect in the activation of two crucial transcription factors, NFκB and AP-1, in response to signaling by the T-cell receptor and CD28. These transcription factors also participate in turning on genes required for T-cell activation. For example, transcription of the gene for IL-2 requires NFκB in addition to NFAT and AP-1, making PKC-θ activation an important component of T-cell activation.


Immunology_Janeway

Immunology_Janeway. PKC-θ has a C1 domain and is recruited to the membrane when DAG is generated by activated PLC-γ (see Fig. 7.17). In this location, the kinase activity of PKC-θ initiates a series of steps that results in the activation of NFκB (Fig. 7.21). PKC-θ phosphorylates the large membrane-localized scaffold protein CARMA1, causing it to oligomerize and form a multisubunit complex with other proteins. This complex recruits and activates TRAF-6, the same protein that we encountered in Chapter 3 in its role in activating NFκB in the TLR signaling pathway (see Fig. 3.13). NFκB is the general name for a member of a family of homoand heterodimeric transcription factors made up of the Rel family of proteins. The most common NFκB activated in lymphocytes is a heterodimer of p50:p65Rel.


Immunology_Janeway

Immunology_Janeway. DAG recruits PKC-˜to the membrane where it phosphorylates CARMA1 Phosphorylated CARMA1 creates a scaffold with BCL10 and MALT1 TRAF-6 is recruited and makes a polyubiquitin scaffold on itself and on NEMO TAK1, recruited by TAB1/2, phosphorylates IKK-°, which phosphorylates I˛B, inducing I˛B ubiquitination and degradation Degradation of I˛B releases NF˛B, which activates transcription in the nucleus PKC-˜CARMA1I°BdegradedI°B˛˝ ˙(NEMO)IKKTAB1/2TAK1ubiquitinBCL10MALT1TRAF-6NF°B


Immunology_Janeway

Immunology_Janeway. Fig. 7.21 Activation of the transcription factor NFκB by antigen receptors is mediated by protein kinase C. diacylglycerol (daG), produced as a result of T-cell receptor signaling activating pLc-γ, recruits a protein kinase c (pKc-θ) to the membrane, where it phosphorylates a scaffold protein called caRma1. This forms a complex with BcL10 and maLT1 that recruits the e3 ubiquitin ligase TRaf-6. as described in fig. 3.13, the kinase TaK1 is recruited by the polyubiquitin scaffold produced by TRaf-6 and phosphorylates the iκB kinase (iKK) complex [iKKα:iKKβ:iKKγ (nemo)]. iKK phosphorylates iκB, stimulating its ubiquitination, which targets iκB for degradation by the proteasome. This releases nfκB to enter the nucleus and stimulate transcription of its target genes. a defect in nemo that prevents nfκB activation causes immunodeficiency that results in susceptibility to extracellular bacterial infections, along with a skin disease known as ectodermal dysplasia.


Immunology_Janeway

Immunology_Janeway. The dimer is held in an inactive state in the cytoplasm by binding to an inhibitory protein called inhibitor of κB (IκB). As described for TLR signaling (see Fig. 3.13), TRAF-6 stimulates the degradation of IκB by first activating the kinase TAK1, which activates a complex of serine kinases, IκB kinase (IKK). IKK phosphorylates IκB, causing its ubiquitination and subsequent degradation, and the consequent release and entry into the nucleus of active NFκB. Inherited deficiency of the IKKγ subunit (also called NEMO) leads to a syndrome known as X-linked hypohidrotic ectodermal dysplasia and immunodeficiency, which is characterized by developmental defects in ectodermal structures such as skin and teeth, as well as immunodeficiency.


Immunology_Janeway

Immunology_Janeway. PKC-θ can also activate JNK, and might be able to activate the transcription factor AP-1 by this route. However, T cells lacking PKC-θ have a defect in AP-1 activation in addition to their defect in NFκB activation, but no defect in JNK activation, indicating that our understanding of this pathway is still incomplete. 7-17 PI 3-kinase activation upregulates cellular metabolic pathways via the serine/threonine kinase Akt.


Immunology_Janeway

Immunology_Janeway. While the activation of transcription factors is an important outcome of antigen receptor signaling, a productive T-cell response also requires substantial changes in cellular metabolism, needed to accommodate the energetic and macromolecular demands of rapidly dividing cells. The PI 3-kinase pathway plays a central role in this response via the recruitment and activation of the second important signaling module, initiated by the serine/threonine kinase Akt (also known as protein kinase B). Akt, via its PH domain, binds to PIP3 in the membrane, which is generated by PI 3-kinase (Fig. 7.22; see also Fig. 7.5). In that location, Akt is phosphorylated by PDK1, and once activated, phosphorylates a variety of downstream proteins. One of its effects is to promote cell survival by inhibiting cell death via multiple mechanisms. A major mechanism is the phosphorylation of the pro-apoptotic protein Bad. When phosphorylated, Bad can no longer bind to and inhibit the antiapoptotic (pro-survival)


Immunology_Janeway

Immunology_Janeway. death via multiple mechanisms. A major mechanism is the phosphorylation of the pro-apoptotic protein Bad. When phosphorylated, Bad can no longer bind to and inhibit the antiapoptotic (pro-survival) protein Bcl-2 (see Fig. 7.22). Another effect of activated Akt is to regulate the expression of homing and adhesion receptors that orchestrate the migratory properties of activated T cells (discussed in detail in Chapters 9 and 11). Activated Akt also functions to stimulate the cell’s metabolism by increasing the utilization of glucose; this is mediated by increasing the activity of glycolytic enzymes and by inducing the upregulation of nutrient transporters on the T-cell membrane.


Immunology_Janeway

Immunology_Janeway. Fig. 7.22 The serine/threonine kinase Akt is activated by TCR signaling and promotes cell survival and enhanced metabolic activity via mTOR. panel one: T-cell receptor (TcR) signaling activates pi 3-kinase (not shown), generating pip3 in the plasma membrane; pip3 recruits and activates the kinase pdK1. akt, a second serine/threonine kinase, binds to pip3 via its ph domain, and is phosphorylated and activated by pdK1. panel two: active akt phosphorylates the pro-apoptotic protein Bad, which is binding to and inhibiting the anti-apoptotic protein Bcl-2 at the mitochondrial membrane. panel three: phosphorylated Bad binds to 14-3-3, releasing Bcl-2 to promote cell survival. panel four: a second function of active akt is to phosphorylate the TSc1/2 complex, a Gap for the small GTpase Rheb. panel five: When TSc1/2 is phosphorylated, it releases the inactive Rheb protein, leading to Rheb activation. active Rheb binds to and activates mToR (mammalian target of rapamycin). once activated, mToR


Immunology_Janeway

Immunology_Janeway. When TSc1/2 is phosphorylated, it releases the inactive Rheb protein, leading to Rheb activation. active Rheb binds to and activates mToR (mammalian target of rapamycin). once activated, mToR acts on multiple pathways that lead to increased lipid production, ribosome biosynthesis, mRna synthesis, and protein translation.


Immunology_Janeway

Immunology_Janeway. Akt Bcl-2 14-3-3 inactive Rheb active RhebTSC1/2 Bad outer mitochondrial membrane inner mitochondrial membrane matrix mTOR PDK1 lipid production ribosome biosynthesis mRNA synthesis protein translation PIP3 recruits PDK1 and Akt, allowing PDK1 to phosphorylate and activate Akt Active Akt phosphorylates Bad, releasing Bad from Bcl-2 Upon release, phosphorylated Bad binds 14-3-3, allowing Bcl-2 to promote cell survival Active Akt phosphorylates the TSC1/2 complex, a GAP for the small GTPase, Rheb Phosphorylated TSC1/2 releases Rheb, allowing active Rheb to bind mTOR Fig. 7.23 Recruitment of ADAP to the LAT:Gads:SLP-76 complex activates integrin adhesion and aggregation.


Immunology_Janeway

Immunology_Janeway. Fig. 7.23 Recruitment of ADAP to the LAT:Gads:SLP-76 complex activates integrin adhesion and aggregation. Left panel: prior to T-cell receptor (TcR) signaling, the integrin Lfa-1 is present on the T-cell membrane in a low-affinity conformation that binds weakly to icam-1 on antigen-presenting-cells. middle panel: following TcR signaling, the adaptor protein adap is recruited to the LaT:Gads:SLp-76 complex by an interaction between tyrosine-phosphorylated adap and the Sh2 domain of SLp-76. adap then recruits a complex of SKap and Riam (Rap1-GTpinteracting adaptor molecule), activating the small GTpase Rap1. Right panel: active Rap1 induces aggregation of Lfa-1 and a conformational change in Lfa-1 that leads to high-affinity binding to icam-1.


Immunology_Janeway

Immunology_Janeway. Yet another important function of activated Akt is to stimulate the mTOR (mammalian target of rapamycin) pathway, a key regulator of macromolecular biosynthesis (see Fig. 7.22). In this case, Akt phosphorylates and inactivates the TSC complex, a GAP for the small GTPase Rheb. This leads to Rheb activation, and in turn, to the activation of mTOR. The mTOR pathway has multiple effects on cellular metabolism; collectively, these changes are essential to provide the raw materials needed to carry out the increased gene expression, protein production, and cell division that accompany T-cell activation. Specifically, mTOR activation leads to increased lipid production, ribosome biosynthesis, mRNA synthesis, and protein translation. 7-18 T-cell receptor signaling leads to enhanced integrin-mediated cell adhesion.


Immunology_Janeway

Immunology_Janeway. The third signaling module induced by TCR stimulation leads to enhanced integrin adhesion. Together with cytoskeletal changes (discussed in the next section), this process promotes stability of the T cell-APC interaction and localizes active signaling complexes into a structure known as the ‘immune synapse’ described below in Section 7-19 (and see Fig. 7.25). The immune synapse, the region of the T-cell membrane that is in direct and stable contact with the APC or target cell, is formed within minutes of T-cell receptor recognition of MHC/peptide ligands. One important component of this is increased adhesiveness of the T-cell integrin LFA-1. On nonstimulated T cells, LFA-1 resides in a low-affinity state and is well dispersed on the T-cell membrane, resulting in weak binding to its ligand, ICAM-1. Following T-cell receptor stimulation, LFA-1 molecules aggregate at the synapse, and also undergo a conformational change that converts each LFA-1 molecule into a high-affinity binding


Immunology_Janeway

Immunology_Janeway. ICAM-1. Following T-cell receptor stimulation, LFA-1 molecules aggregate at the synapse, and also undergo a conformational change that converts each LFA-1 molecule into a high-affinity binding partner for ICAM-1. Together, these changes lead to enhanced adhesion between the T cell and the APC, and to stabilization of this cell–cell interaction. These effects on LFA-1 are induced by the recruitment of the adaptor protein ADAP to the LAT:Gads:SLP-76 scaffold complex (Fig. 7.23). In turn, ADAP recruits two additional proteins, SKAP55 and RIAM. The ADAP:SKAP55:RIAM complex binds to the small GTPase Rap1, activating Rap1 at the site of T-cell receptor signaling. GTP-bound Rap1 then promotes LFA-1 aggregation and the conformational change that converts LFA-1 into a high-affinity binding partner for ICAM-1. The importance of this pathway is underscored by the finding that ADAP-deficient T cells show impaired proliferation and cytokine production following T-cell receptor stimulation.


Immunology_Janeway

Immunology_Janeway. 7-19 T-cell receptor signaling induces cytoskeletal reorganization by activating the small GTPase Cdc42.


Immunology_Janeway

Immunology_Janeway. The fourth TCR signaling module, also involved in the formation of a stable immune synapse, leads to reorganization of the actin cytoskeleton. Without this process, integrin aggregation would not occur, interactions between the T cell and the APC would not stabilize, and in fact, T-cell activation would completely fail. A major component of this T-cell receptor signal is transduced by Vav, a GEF that activates Rho-family GTPases, including Cdc42. Like PLC-γand Itk, Vav is recruited to the site of receptor activation by interactions of the Vav PH domain with PIP3 and of the Vav SH2 domain with the LAT:Gads:SLP-76 scaffold complex (Fig. 7.24). When Cdc42 is activated by Vav, the GTP-bound Cdc42 induces a conformational change in the protein WASp (Wiskott– Aldrich syndrome protein), which is also recruited to the LAT:Gads:SLP-76 scaffold complex by binding to the small adaptor protein Nck. This active form of WASp binds to WIP, and together, these proteins recruit Arp2/3, leading to


Immunology_Janeway

Immunology_Janeway. is also recruited to the LAT:Gads:SLP-76 scaffold complex by binding to the small adaptor protein Nck. This active form of WASp binds to WIP, and together, these proteins recruit Arp2/3, leading to actin polymerization. The importance of this pathway is underscored by the fact that defects in WASp are the basis for the immunodeficiency disease Wiskott–Aldrich syndrome. Due to the widespread expression of WASp, individuals suffering from this disease have impairments in multiple immune cell types, all of which depend on WASp-dependent actin polymerization for their functions. One major defect in this disease is in T cell-dependent antibody responses, due to the requirement for actin polymerization to ensure effective interactions between CD4 T cells and B cells. Thus, the failure of WASpdeficient T cells to provide adequate ‘help’ to B cells most likely results from a defect in the formation of the immune synapse, which is normally required to ensure directed secretion of cytokines


Immunology_Janeway

Immunology_Janeway. T cells to provide adequate ‘help’ to B cells most likely results from a defect in the formation of the immune synapse, which is normally required to ensure directed secretion of cytokines from the T cell onto the B-cell membrane (Fig. 7.25).


Immunology_Janeway

Immunology_Janeway. 7-20 The logic of B-cell receptor signaling is similar to that of T-cell receptor signaling, but some of the signaling components are specific to B cells. There are many similarities between signaling from T-cell receptors and signaling from B-cell receptors. As with the T-cell receptor, the B-cell receptor is composed of antigen-specific chains associated with ITAM-containing signaling chains, in this case Igα and Igβ (see Fig. 7.10). In B cells, three protein tyrosine kinases of the Src family—Fyn, Blk, and Lyn—are thought to be responsible for phosphorylation of the ITAMs (Fig. 7.26). These kinases associate with


Immunology_Janeway

Immunology_Janeway. Fig. 7.24 Recruitment of Vav to the LAT:Gads:SLP-76 complex induces activation of Cdc42, leading to actin polymerization. Left panel: vav, a guanine-nucleotide exchange factor (Gef) for the small GTpase cdc42, is recruited to the activated T-cell receptor (TcR) complex by binding via its ph domain to pip3 in the membrane and by binding to phosphorylated SLp-76. The small adaptor protein nck binds to an adjacent phosphorylated tyrosine on SLp-76 and recruits the inactive form of the protein WaSp. middle panel: vav activates cdc42, which binds to and activates WaSp. Right panel: active WaSp binds to Wip, recruiting arp2/3 and inducing actin polymerization. The importance of this pathway is illustrated by the discovery of WaSp as the protein encoded by the gene responsible for the human immunodeficiency disease Wiskott– aldrich syndrome.


Immunology_Janeway

Immunology_Janeway. Fig. 7.25 The immune synapse provides a structure for directed secretion of T-cell cytokines. When the T-cell receptors (TcRs) on a T cell recognize peptide:mhc on an antigen-presenting cell, a process of receptor reorganization takes place on the plasma membranes of the two interacting cells. Left panel: when a cd4+ T cell recognizes its peptide:mhc ligand on a B cell, the immune synapse functions to direct T-cell-secreted cytokines onto the B-cell surface at the site of closest contact between the plasma membranes of the two cells. Right panel: confocal microscopy images of the TcR/peptide:mhc (red) and the Lfa-1/icam-1 (green) proteins 30 minutes after the initiation of signaling show a central accumulation of the TcR/peptide:mhc complexes and a peripheral ring of the Lfa-1/icam-1 complexes. These structures have been called the central supermolecular activation complex (cSmac, red) and the peripheral supermolecular activation complex (pSmac, green). The combined structure is known


Immunology_Janeway

Immunology_Janeway. These structures have been called the central supermolecular activation complex (cSmac, red) and the peripheral supermolecular activation complex (pSmac, green). The combined structure is known as the immune synapse. photograph courtesy of y. Kaizuka.


Immunology_Janeway

Immunology_Janeway. resting receptors via a low-affinity interaction with unphosphorylated ITAMs in Igα and Igβ. After the receptors have bound a multivalent antigen, which cross-links them, the receptor-associated kinases are activated and phosphorylate the tyrosine residues in the ITAMs. B cells do not express ZAP-70; instead, a closely related tyrosine kinase, Syk, containing two SH2 domains, is recruited to the phosphorylated ITAM. In contrast to ZAP-70, which requires additional Lck phosphorylation for activation, Syk is activated simply by its binding to the phosphorylated site.


Immunology_Janeway

Immunology_Janeway. For B cells, the co-receptor and co-stimulatory receptor functions are combined into one accessory receptor that is a complex of cell-surface proteins—CD19, CD21, and CD81—often referred to as the B-cell co-receptor (Fig. 7.27). As with T cells, antigen-dependent signaling from the B-cell receptor is enhanced if the B-cell co-receptor is simultaneously bound by its ligand and clusters with the antigen receptor. CD21 (also known as complement receptor 2, CR2) is a receptor for the C3dg fragment of complement. This means that antigens such as bacterial pathogens on which C3dg is bound (see Fig. 7.27) can cross-link the B-cell receptor with the CD21:CD19:CD81 complex. This induces phosphorylation of the cytoplasmic tail of CD19 by B-cell receptor-associated tyrosine kinases, which in turn leads to the binding of additional Src-family kinases, the augmentation of signaling through the B-cell receptor itself, and the recruitment of PI 3-kinase (see Section 7-4). PI 3-kinase


Immunology_Janeway

Immunology_Janeway. Fig. 7.26 Src-family kinases are associated with B-cell receptors and phosphorylate the tyrosines in ITAMs to create binding sites for Syk and Syk activation via transphosphorylation. The membrane-bound Src-family kinases fyn, Blk, and Lyn associate with the B-cell antigen receptor by binding to iTams, either (as shown in the figure) through their amino-terminal domains or by binding a single phosphorylated tyrosine through their Sh2 domains. after ligand binding and receptor clustering, the associated kinases phosphorylate tyrosines in the iTams on the cytoplasmic tails of igα and igβ. Subsequently, Syk binds to the phosphorylated iTams of the igβ chain. Because there are at least two receptor complexes in each cluster, Syk molecules become bound in close proximity and can activate each other by transphosphorylation, thus initiating further signaling.


Immunology_Janeway

Immunology_Janeway. initiates an additional signaling pathway leading from the B-cell receptor (see Fig. 7.27). Thus, the B-cell co-receptor serves to strengthen the signal resulting from antigen recognition. The role of the third component of the B-cell receptor complex, CD81 (TAPA-1), is as yet unknown.


Immunology_Janeway

Immunology_Janeway. Once activated, Syk phosphorylates the scaffold protein SLP-65 (also known as BLNK). Like LAT and SLP-76 in T cells, SLP-65 functions as a composite of these two proteins, providing multiple sites for tyrosine phosphorylation and recruiting a variety of SH2-containing proteins, including enzymes and adaptor proteins, to form several distinct multiprotein signaling complexes that can act in concert. As in T cells, a key signaling protein is the phospholipase PLC-γ, which is activated with the aid of the B cell-specific Tec kinase Bruton’s tyrosine kinase (Btk) and hydrolyzes PIP2 to form DAG and IP3 (see Fig. 7.27). As discussed for the T-cell receptor, signaling by calcium and DAG leads to the activation of downstream transcription factors. A deficiency in Btk (which is encoded by a gene on the X chromosome) prevents the development and functioning of B cells, resulting in the disease X-linked agammaglobulinemia, which is characterized by a lack of antibodies. Besides Btk, mutations


Immunology_Janeway

Immunology_Janeway. on the X chromosome) prevents the development and functioning of B cells, resulting in the disease X-linked agammaglobulinemia, which is characterized by a lack of antibodies. Besides Btk, mutations in other signaling molecules in B cells, including receptor chains and SLP-65, have been linked to B-cell immunodeficiencies (see Chapter 8).


Immunology_Janeway

Immunology_Janeway. Several other downstream pathways described for TCR signaling are also shared with BCR signaling, and are dependent on the adaptor protein SLP-65. These include the Vav-dependent induction of actin polymerization by Cdc42 and WASp, and the recruitment and activation of small GTPases that promote integrin adhesion (see Fig. 7.27). In the case of B-cell recognition of


Immunology_Janeway

Immunology_Janeway. B-cell receptor (BcR) signaling is greatly enhanced when the antigen is tagged by complement fragments, engaging the B-cell co-receptor together with the B-cell antigen receptor. cleavage of the antigen-bound complement component c3 to c3dg (see fig. 2.30) allows the tagged antigen to bind to the cell-surface protein cd21 (complement receptor 2, cR2), a component of the B-cell co-receptor complex, which also includes cd19 and cd81 (Tapa-1). cross-linking and clustering of the co-receptor with the antigen receptor result in the phosphorylation of tyrosine residues in the iTam sequences of the cytoplasmic domains of the BcR signaling subunits, igα and igβ. The Src-family kinase also phosphorylates tyrosine residues in the cytoplasmic domain of cd19. The phosphorylated iTams in igα and igβ recruit and activate the tyrosine kinase Syk, which functions similarly to Zap-70 in T cells. The phosphorylated tail of cd19 recruits pi 3-kinase, leading to pip3 generation in the plasma membrane.


Immunology_Janeway

Immunology_Janeway. recruit and activate the tyrosine kinase Syk, which functions similarly to Zap-70 in T cells. The phosphorylated tail of cd19 recruits pi 3-kinase, leading to pip3 generation in the plasma membrane. activated Syk phosphorylates the membrane-associated scaffold protein SLp-65, which associates with the plasma membrane by binding cin85. pip3 recruits pdK1 and akt, leading to akt activation. phosphorylated SLp-65 and pip3 recruit the Tec-family tyrosine kinase Btk and pLc-γ, leading to Btk phosphorylation and activation of pLc-γ. phosphorylated SLp-65 and pip3 also recruit vav, nck, and inactive WaSp. vav activates small GTpases that activate WaSp, leading to actin polymerization; the activated GTpases also induce integrin aggregation and conversion of Lfa-1 to the high-affinity binding state.


Immunology_Janeway

Immunology_Janeway. ˜˜°˛˝˙˙ˆmIgMTCR Ig°Ig˛Tyrosine kinases Adaptors and scaffold proteins Phospholipases and lipid kinases GTPases, serine/threonine kinases, and phosphatases Transcription factors, cytoskeletal changes, adhesion, metabolism


Immunology_Janeway

Immunology_Janeway. Fig. 7.28 Summary of antigen receptor signaling pathways. as outlined in this section, the signal transduction pathways downstream of the T-cell and B-cell receptors occur in an orchestrated series of stages involving many categories of proteins that produce widespread changes in the cells. The first detectable events following antigen receptor stimulation are the activation of tyrosine kinases. following this, adaptor proteins and scaffolds are modified, recruiting phospholipases and lipid kinases to the activated receptor complexes. The next level of signaling amplifies these earlier stages by activating multiple small GTpases, serine/threonine kinases, and protein phosphatases. Together, these lead to transcription factor activation, cytoskeletal changes, and increases in cellular adhesion and metabolism, all of which contribute to T-and B-cell activation.


Immunology_Janeway

Immunology_Janeway. membrane-bound antigen, B-cell receptor signaling also produces an immune synapse that localizes signaling complexes to the cell–cell interface. One key function of the immune synapse in B cells is to promote antigen uptake by the B cell, a prerequisite to presenting that antigen in the form of peptide:MHC complexes to CD4 T cells. B-cell receptor signaling also induces metabolic changes in activated B cells. As is the case for T cells, this response is dependent on the action of PI 3-kinase, whose activation leads to formation of the membrane phosphoinositide PI(3,4,5)P3 at the site of the activated B-cell receptor. This response is augmented by combined signaling through the B-cell receptor plus the B-cell co-receptor complex of CD19/CD21/CD81. PI(3,4,5)P3 recruits Akt, which is then phosphorylated and activated, leading to downstream activation of mTOR, as well as additional Akt-dependent pathways promoting cell survival and proliferation (see Fig. 7.27). Summary.


Immunology_Janeway

Immunology_Janeway. The antigen receptors on the surface of lymphocytes are multiprotein complexes in which the antigen-binding chains interact with additional proteins that are responsible for signaling from the receptor. These protein chains carry tyrosine-containing signaling motifs known as ITAMs. Signaling chains containing ITAM motifs are widely used by activating receptors in many immune cell types in addition to lymphocytes. In lymphocytes, activation of the receptors by antigen results in a series of biochemical events that are broadly outlined in Fig. 7.28. This signaling cascade is initiated by the phosphorylation of the ITAMs by Src-family kinases. The phosphorylated ITAM then recruits another tyrosine kinase, ZAP-70 in T cells and Syk in B cells. Activation of ZAP-70 or Syk results in the phosphorylation of scaffolds called LAT and SLP-76 in T cells, and SLP-65 in B cells, and in the activation of PI 3-kinase. Multiple signaling proteins are recruited and activated by these phosphorylated


Immunology_Janeway

Immunology_Janeway. of scaffolds called LAT and SLP-76 in T cells, and SLP-65 in B cells, and in the activation of PI 3-kinase. Multiple signaling proteins are recruited and activated by these phosphorylated scaffolds, including phospholipase C-γ, ADAP, and Vav, whereas Akt is recruited and activated by the action of PI 3-kinase generating PIP3 at the plasma membrane. PLC-γ generates inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 has an important role in inducing changes in intracellular calcium concentrations, and DAG is involved in activating protein kinase C-θ and the small G protein Ras. In T cells, these pathways ultimately result in the activation of three transcription factors, namely, AP-1, NFAT, and NFκB; together, these transcription factors induce transcription of the cytokine IL-2, which is essential for the proliferation and further differentiation of the activated lymphocyte. In addition to transcription factor activation, antigen receptor signaling in both T cells and B cells


Immunology_Janeway

Immunology_Janeway. is essential for the proliferation and further differentiation of the activated lymphocyte. In addition to transcription factor activation, antigen receptor signaling in both T cells and B cells leads to enhanced cell survival, metabolic activity, adhesiveness, and cytoskeletal reorganization. Signaling by antigen receptors is facilitated by co-receptors that become engaged as a result of receptor–antigen binding. These co-receptors are the MHC-binding CD4 and CD8 transmembrane proteins in T cells and the complement-binding B-cell co-receptor complex containing CD19 in B cells.


Immunology_Janeway

Immunology_Janeway. Co-stimulatory and inhibitory receptors modulate antigen receptor signaling in T and B lymphocytes.


Immunology_Janeway

Immunology_Janeway. Signals initiated by the T-cell and B-cell antigen receptors are essential for lymphocyte activation, and determine the specificity of the adaptive immune response that is initiated. However, signaling from the antigen receptor is not on its own sufficient to activate a naive T cell or B cell. These naive lymphocytes require additional signals to achieve full activation. Receptors on T cells and B cells that can provide this necessary second signal are called costimulatory receptors, and are members of either the CD28 family of proteins or of the TNF receptor superfamily. While naive T cells primarily utilize CD28 as the co-stimulatory receptor, naive B cells use the TNF receptor family member CD40. The overall function of signaling through these co-stimulatory receptors is to enhance the antigen receptor signals that induce transcription factor activation and PI 3-kinase activation, thereby ensuring activation of the T cell or B cell. In contrast to these activating co-stimulatory


Immunology_Janeway

Immunology_Janeway. antigen receptor signals that induce transcription factor activation and PI 3-kinase activation, thereby ensuring activation of the T cell or B cell. In contrast to these activating co-stimulatory receptor signals, other cell-surface receptors on T cells and B cells function to downregulate activation signals. These inhibitory receptors are important in preventing excessive immune responses that can lead to destructive inflammatory or autoimmune conditions, particularly in the case of chronic infections that are inefficiently controlled by the immune system.


Immunology_Janeway

Immunology_Janeway. 7-21 The cell-surface protein CD28 is a required co-stimulatory signaling receptor for naive T-cell activation. The signaling through the T-cell receptor complex described in the previous sections is not by itself sufficient to activate a naive T cell. As noted in Chapter 1, antigen-presenting cells that can activate naive T cells bear cell-surface proteins known as co-stimulatory molecules or co-stimulatory ligands. These interact with cell-surface receptors, known as co-stimulatory receptors, on the naive T cell to transmit a signal that is required, along with antigen stimulation, for T-cell activation—this signal is sometimes called ‘signal 2.’ We discuss the immunological consequences of this requirement for co-stimulation in detail in Chapter 9.


Immunology_Janeway

Immunology_Janeway. The best understood of these co-stimulatory receptors is the cell-surface protein CD28. CD28 is present on the surface of all naive T cells and binds the co-stimulatory ligands B7.1 (CD80) and B7.2 (CD86), which are expressed mainly on specialized antigen-presenting cells such as dendritic cells. To become activated, the naive lymphocyte must engage both antigen and a co-stimulatory ligand on the same antigen-presenting cell. CD28 signaling aids antigen-dependent T-cell activation mainly by promoting T-cell proliferation, cytokine production, and cell survival. All these effects are mediated by signaling motifs present in the cytoplasmic domain of CD28.


Immunology_Janeway

Immunology_Janeway. After engagement by B7 molecules, CD28 becomes tyrosine phosphorylated by Lck in its cytoplasmic domain on tyrosine residues in a YXN motif that can recruit the adaptor protein Grb2, and in a non-ITAM motif YMNM. The cytoplasmic tail of CD28 also carries a proline-rich motif (PXXP) that binds the SH3 domains of Lck and Itk. Although the details are uncertain, a major effect of CD28 phosphorylation is to activate PI 3-kinase to generate PIP3 (Fig. 7.29). By this mechanism, the co-stimulatory signal induced by CD28 cooperates with the T-cell receptor signal to ensure maximal activation of three of the four T-cell receptor signaling modules described above. Specifically, a high concentration of PIP3 recruits Itk to the membrane, where Lck can phosphorylate it, thereby enhancing PLC-γ activation. PIP3 also functions to recruit and activate Akt, which promotes cell survival and increased cellular metabolism (see Section 7.17). An additional function of Akt is to phosphorylate the


Immunology_Janeway

Immunology_Janeway. activation. PIP3 also functions to recruit and activate Akt, which promotes cell survival and increased cellular metabolism (see Section 7.17). An additional function of Akt is to phosphorylate the RNA-binding protein NF-90; when phosphorylated, NF-90 translocates from the nucleus to the cytoplasm and binds to and stabilizes the IL-2 mRNA, leading to increased IL-2 synthesis. Finally, PIP3 recruits Vav, leading to cytoskeletal reorganization (see Section 7.19). Thus, co-stimulatory signaling through CD28 functions to amplify most of the downstream responses to T-cell receptor stimulation (see Fig. 7.29).


Immunology_Janeway

Immunology_Janeway. MOVIE 7.3 CD28B7MHCPIP2PIP3PI3KPI3KPIP3 recruits PDK1 and Akt, allowing PDK1 to phosphorylate and activate Akt B7 binding induces CD28 phosphorylation, activating PI 3-kinase to produce PIP3 B7.1 and B7.2 are CD28 ligands expressed on specialized APCs PIP3 also recruits Itk, allowing it to phosphorylate PLC-˜PIP3 also recruits Vav, leading to Cdc24 activation SH2domainkinasedomainAktItkSLP-76LATPDK1PLC-˜activeCdc42inactiveCdc42Vav


Immunology_Janeway

Immunology_Janeway. Fig. 7.29 The T-cell co-stimulatory protein CD28 transduces signals that enhance antigen receptor signaling pathways. The ligands for cd28, namely B7.1 and B7.2, are expressed only on specialized antigen-presenting cells (apcs) such as dendritic cells (first panel). engagement of cd28 induces its tyrosine phosphorylation, which activates pi 3-kinase (pi3K), with subsequent production of pip3 that recruits several enzymes via their ph domains, thus bringing them together with their substrates in the membrane. The protein kinase akt, which becomes phosphorylated by phosphoinositide-dependent protein kinase-1 (pdK1), is activated and enhances cell survival and upregulates cell metabolism (see fig. 7.22). Recruitment of the kinase itk to the membrane is critical for the full activation of pLc-γ (see fig. 7.16). pip3 also recruits vav, leading to cdc42 activation and inducing actin polymerization (see fig. 7.24).


Immunology_Janeway

Immunology_Janeway. 7-22 Maximal activation of PLC-γ, which is important for transcription factor activation, requires a co-stimulatory signal induced by CD28. One important function of co-stimulatory signaling through CD28 is to promote the maximal activation of PLC-γ via the local production of PIP3. This recruits Itk by its PH domain, enhancing Itk phosphorylation by Lck. Activated Itk is then recruited to the phosphorylated LAT:Gads:SLP-76 complex by its SH2 and SH3 domains binding to SLP-76, where it phosphorylates and activates PLC-γ (see Fig. 7.16). Activated PLC-γ cleaves PIP2 to produce the two second messengers DAG and IP3, ultimately leading to the activation of transcription factors NFAT, AP-1, and NFκB. Thus, the full activation of PLC-γ leading to transcription factor activation requires signals emanating from both the T-cell receptor and CD28.


Immunology_Janeway

Immunology_Janeway. In T cells, one of the major functions of NFAT, AP-1, and NFκB is to act together to stimulate expression of the gene for the cytokine IL-2, which is essential for promoting T-cell proliferation and differentiation into effector cells. The promoter for the IL-2 gene contains multiple regulatory elements that must be bound by transcription factors to initiate IL2 expression. Some control sites are already bound by transcription factors, such as Oct1, that are produced constitutively in lymphocytes, but this is not sufficient to switch on IL-2. Only when AP-1, NFAT, and NFκB are all activated and are bound to their control sites in the IL-2 promoter is the gene expressed. NFAT and AP-1 bind to the promoter cooperatively and with higher affinity by forming a heterotrimer of NFAT, Jun, and Fos. In addition, CD28 co-stimulation further enhances IL-2 transcription by increasing NFκB activation. Thus, the IL-2 promoter integrates signals from both the T-cell receptor and CD28 signaling


Immunology_Janeway

Immunology_Janeway. Fos. In addition, CD28 co-stimulation further enhances IL-2 transcription by increasing NFκB activation. Thus, the IL-2 promoter integrates signals from both the T-cell receptor and CD28 signaling pathways to ensure that IL-2 is produced only in appropriate circumstances (Fig. 7.30). Together with the CD28-induced phosphorylation of NF-90 leading to increased IL-2 mRNA stability, CD28 co-stimulation leads to substantially increased production of IL-2 by activated T cells.


Immunology_Janeway

Immunology_Janeway. 7-23 TNF receptor superfamily members augment T-cell and B-cell activation. While naive Tand B-cell activation requires signaling through the antigen receptors on these cells, T-cell receptor or B-cell receptor signaling, Fig. 7.30 Simplified scheme depicting multiple signaling pathways that converge on the IL-2 promoter.


Immunology_Janeway

Immunology_Janeway. Fig. 7.30 Simplified scheme depicting multiple signaling pathways that converge on the IL-2 promoter. ap-1, nfaT, and nfκB binding to the promoter of the IL-2 gene integrate multiple signaling pathways emanating from the T-cell receptor (TcR) and cd28 into a single output, the production of the cytokine iL-2. The mapK pathway activates ap-1; calcium activates nfaT; and protein kinase c activates nfκB. all three pathways are required to stimulate IL-2 transcription. activation of the gene requires both the binding of nfaT and ap-1 to a specific promoter element, and the additional binding of ap-1 on its own to another site. oct1 is a transcription factor that is required for IL-2 transcription. unlike the other transcription factors, it is constitutively bound to the promoter and is therefore not regulated by T-cell receptor or cd28 signaling.


Immunology_Janeway

Immunology_Janeway. respectively, is not sufficient to activate the cells. For naive T cells, an additional co-stimulatory signal is required and, as discussed above (see Sections 7-21 and 7-22), is frequently provided by the CD28 receptor. For naive B cells, the additional activation signal can be contributed by direct interactions between the pathogen and an innate pattern recognition receptor (PRR), such as a TLR, on the B cell. However, for more effective B-cell activation leading to the production of all classes of antibodies and to the formation of memory B cells, additional B-cell activation signals are contributed by CD4 T cells. One component of this is the production of T-cell cytokines, which bind to and stimulate their receptors on the B-cell surface (see Chapter 10). The second and more essential component provided by the CD4 T cells is the stimulation of CD40 on the B cell by the CD40 ligand expressed on the T cell. The importance of the CD40–CD40-ligand interaction for B-cell responses to


Immunology_Janeway

Immunology_Janeway. component provided by the CD4 T cells is the stimulation of CD40 on the B cell by the CD40 ligand expressed on the T cell. The importance of the CD40–CD40-ligand interaction for B-cell responses to protein antigens is highlighted by the discovery that a severe immunodeficiency disease resulting from impaired antibody responses is caused by the absence of CD40 ligand expression on a patient’s CD4 T cells.


Immunology_Janeway

Immunology_Janeway. CD40 is a member of the large TNF receptor superfamily, which consists of more than 20 members. While some members of this family, such as Fas (see Chapter 11), are specialized to induce cell death, the majority of TNF receptor superfamily members, including CD40, activate both the NFκB and the PI 3-kinase pathways following receptor stimulation (Fig. 7.31). While NFκB activation leads to enhanced cell survival, the PI 3-kinase pathway has widespread and pleiotropic effects on B-cell physiology, and is a central feature of CD40 signaling. The major mediator of the PI 3-kinase signal is the serine/threonine kinase Akt, which is recruited and activated following the generation of PI(3,4,5)P3 at the B-cell membrane. Akt then stimulates multiple downstream pathways that induce cell survival, cell cycle progression, glucose uptake and metabolism, and mTOR activation, all of which are essential for the productive response of the activated B cell. In general terms, CD40 on the B cell


Immunology_Janeway

Immunology_Janeway. cell cycle progression, glucose uptake and metabolism, and mTOR activation, all of which are essential for the productive response of the activated B cell. In general terms, CD40 on the B cell functions in a manner analogous to CD28 on the T cell, as both receptors serve to enhance the levels of Akt activation induced by the B-cell receptor or T-cell receptor signaling pathways, respectively.


Immunology_Janeway

Immunology_Janeway. TNF receptor superfamily members, including CD40, signal by a mechanism distinct from that of antigen receptors, as it does not involve the activation of tyrosine kinases. Instead, stimulation of TNF receptors recruits adaptor proteins known as TRAFs (TNF receptor-associated factors). In addition to serving as simple adaptors that promote the assembly of multiprotein complexes, five


Immunology_Janeway

Immunology_Janeway. Fig. 7.31 The TNF receptor superfamily member CD40 is an important co-stimulatory molecule on B cells. Several members of the Tnf receptor superfamily are expressed on T cells and B cells. a key function of these receptors is the activation of nfκB, which occurs by a pathway distinct from the one initiated by antigen receptor stimulation and is often referred to as the non-canonical nfκB pathway. Tnf receptor superfamily members also activate pi 3-kinase signaling pathways. one important Tnf receptor superfamily member on B cells is cd40. prior to stimulation, TRaf molecules, which are ubiquitin ligases, are associated with ciap, another ubiquitin ligase, and the nfκB-inducing kinase niK. under these steady-state conditions, TRaf binding promotes ubiquitination and degradation of niK. When cd40 is stimulated by binding to cd40L, this complex is recruited to the intracellular domain of cd40. TRaf2 catalyzes K63-linked ubiquitination of ciap, leading to ciap-mediated K48-linked


Immunology_Janeway

Immunology_Janeway. niK. When cd40 is stimulated by binding to cd40L, this complex is recruited to the intracellular domain of cd40. TRaf2 catalyzes K63-linked ubiquitination of ciap, leading to ciap-mediated K48-linked ubiquitination of TRaf3. This leads to TRaf3 degradation, releasing niK, and allowing niK to phosphorylate and activate iκB kinase α (iKKα). iKKα phosphorylates the nfκB precursor protein p100, inducing its cleavage to form the active p52 subunit, which binds to relB to form the active nfκB transcription factor. cd40L stimulation of cd40 also activates pi 3-kinase, which leads to the activation of akt by pdK1.


Immunology_Janeway

Immunology_Janeway. of the six known TRAFs also function as E3 ubiquitin ligases. This activity contributes to the ability of most TNF receptor superfamily members to activate the NFκB pathway, using a pathway distinct from the one initiated by antigen receptor stimulation and often referred to as the non-canonical NFκB pathway (see Fig. 7.31). In contrast, the precise biochemical mechanism by which TNF receptors and TRAFs induce PI 3-kinase activation is not yet known.


Immunology_Janeway

Immunology_Janeway. CD40 is constitutively expressed on B cells and functions during B-cell activation in response to antigen recognition by the B-cell receptor. Additional TNF receptor superfamily members are also expressed on B cells, and each of them is important in B-cell survival at a particular stage of B-cell maturation, including B cells that have differentiated into antibody-secreting cells or memory cells. Similarly, TNF receptor superfamily members are expressed on T cells, many of which are upregulated following T-cell activation. These molecules, such as OX40, 4-1BB, CD30, and CD27, contribute important survival signals and function to enhance cellular metabolism at later stages of the T-cell response to infection (see Chapter 11). responses by interfering with co-stimulatory signaling pathways.


Immunology_Janeway

Immunology_Janeway. CD28 belongs to a family of structurally related receptors that are expressed by lymphocytes and bind B7-family ligands. Some, such as the receptor ICOS, which is discussed in Chapter 9, act as activating receptors, but others inhibit signaling by the antigen receptors, can stimulate apoptosis, and are important in regulating the immune response. Inhibitory receptors related to CD28 and expressed by T cells include CTLA-4 (CD152) and PD-1 (programmed death-1), while the B and T lymphocyte attenuator (BTLA) is expressed by both T cells and B cells. Of these, CTLA-4 seems to be the most important: mice lacking CTLA-4 die at a young age from an uncontrolled proliferation of T cells in multiple organs, whereas loss of PD-1 or BTLA causes less marked changes that alter the magnitude of responses following lymphocyte activation, rather than causing widespread spontaneous lymphoproliferation. Both CTLA-4 and PD-1 have been targeted for the development of protein-based therapeutics that


Immunology_Janeway

Immunology_Janeway. following lymphocyte activation, rather than causing widespread spontaneous lymphoproliferation. Both CTLA-4 and PD-1 have been targeted for the development of protein-based therapeutics that function to block the activities of these receptors. The goal of these therapeutics is to enhance T-cell responses by inhibiting these inhibitory receptors, a therapeutic strategy referred to as checkpoint blockade (see Chapter 16). Recent clinical trials demonstrate that both CTLA-4 and PD-1 blockade have remarkable efficacy in the treatment of cancer by enhancing the patient’s own antitumor T-cell responses.


Immunology_Janeway

Immunology_Janeway. CTLA-4 is induced on activated T cells and binds to the same co-stimulatory ligands (B7.1 and B7.2) as CD28, but CTLA-4 engagement is inhibitory for T-cell activation, rather than enhancing (Fig. 7.32). The function of CTLA-4 is controlled largely by regulation of its surface expression. Initially, CTLA-4 resides on intracellular membranes but moves to the cell surface after T-cell receptor signaling. The surface expression of CTLA-4 is controlled by phosphorylation of the tyrosine-based motif GVYVKM in its cytoplasmic tail. When this motif is not phosphorylated, it is able to bind to the clathrin adaptor molecule AP-2, which removes CTLA-4 from the surface. When it is phosphorylated, this motif cannot bind AP-2, and CTLA-4 remains in the membrane, where it can bind B7 molecules on antigen-presenting cells.


Immunology_Janeway

Immunology_Janeway. CTLA-4 has a higher affinity for its B7 ligands than does CD28, and, apparently of importance for its inhibitory function, it engages B7 molecules in a different orientation. CD28, CTLA-4, and B7.1 are all expressed as homodimers. A CD28 dimer engages one B7.1 dimer in a direct one-to-one correspondence, but a CTLA-4 dimer engages two different B7 dimers in a configuration that allows for extended cross-linkages that confer high avidity to the interaction (see Fig. 7.32). CTLA-4 was once presumed to act by recruiting inhibitory phosphatases, like some of the other inhibitory receptors described later, but this is no longer thought to be so. It is still not clear whether CTLA-4 directly activates inhibitory signaling pathways. Instead, its actions may result in part from blocking the binding of CD28 to B7, thereby reducing CD28-dependent co-stimulation.


Immunology_Janeway

Immunology_Janeway. CTLA-4-expressing T cells can also exert an inhibitory effect on the activation of other T cells. How they do this is not yet clear, but it might result from CTLA-4 binding to B7 molecules on antigen-presenting cells, in effect stealing the ligand for CD28 required by the other T cells. Direct actions of CTLA-4 on T cells have not been excluded, however. Notably, the regulatory T cells that are needed to suppress autoimmunity express high levels of CTLA-4 on their surface, and they require CTLA-4 to function normally. Regulatory cells are described in detail in Chapter 9. 7-25 Inhibitory receptors on lymphocytes downregulate immune responses by recruiting protein or lipid phosphatases.


Immunology_Janeway

Immunology_Janeway. Some other receptors that can inhibit lymphocyte activation possess motifs in their cytoplasmic regions that are known as the immunoreceptor tyrosine-based inhibitory motif (ITIM, consensus sequence [I/V]XYXX[L/I], where X is any amino acid) (Fig. 7.33) or the related immunoreceptor tyrosine-based switch motif (ITSM, consensus sequence TXYXX[V/I]). When the tyrosine in an ITIM or ITSM is phosphorylated, it can recruit either of two inhibitory phosphatases, called SHP (SH2-containing phosphatase) and SHIP (SH2containing inositol phosphatase), via their SH2 domains. SHP is a protein tyrosine phosphatase that removes phosphate groups added by tyrosine kinases to a variety of proteins. SHIP is an inositol phosphatase and removes the phosphate from PIP3 to generate PIP2, thus reversing the recruitment of proteins such as Tec kinases and Akt to the cell membrane and thereby inhibiting signaling.


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Immunology_Janeway. CD28 B7.1 One dimer of CD28 engages just one dimer of B7 A distinct binding orientation allows one dimer of CTLA-4 to bind two different B7 dimers, providing for high-avidity clustering CTLA-4


Immunology_Janeway

Immunology_Janeway. Fig. 7.32 CTLA-4 has a higher affinity than CD28 for B7 and engages it in a multivalent orientation. cd28 and cTLa-4 are both expressed as dimers on the cell surface and both bind to two ligands of B7.1, which is a dimer, and B7.2, which is not. however, the orientations of the B7 binding of cd28 and of cTLa-4 differ in a way that contributes to the inhibitory action of cTLa-4. one dimer of cd28 engages just one dimer of B7.1. But one dimer of cTLa-4 binds in such a way that two different dimers of B7.1 are engaged at once, allowing these molecules to cluster into complexes of high avidity. This, and the higher affinity of cTLa-4 for B7 molecules, may give it an advantage in competing for available B7 molecules on an antigen-presenting cell, providing one mechanism by which it could block the co-stimulation of T cells. B cells, T cells, and NK cells express receptors that contain immunoreceptor tyrosine-based inhibitory motifs PIR-B Fc˜RIIB-1 CD22 BTLA, KIR2DL KIR3DL PD-1


Immunology_Janeway

Immunology_Janeway. B cells, T cells, and NK cells express receptors that contain immunoreceptor tyrosine-based inhibitory motifs PIR-B Fc˜RIIB-1 CD22 BTLA, KIR2DL KIR3DL PD-1 Fig. 7.33 Some lymphocyte cell-surface receptors contain motifs involved in downregulating activation. Several receptors that transduce signals that inhibit lymphocyte or nK-cell activation contain one or more iTims (immunoreceptor tyrosine-based inhibitory motifs) in their cytoplasmic tails (red rectangles). iTims bind to various phosphatases that, when activated, inhibit signals derived from iTamcontaining receptors.


Immunology_Janeway

Immunology_Janeway. One ITIM-containing receptor is PD-1 (see Fig. 7.33), which is induced transiently on activated T cells, B cells, and myeloid cells. It can bind to the B7-family ligands PD-L1 (programmed death ligand-1, B7-H1) and PD-L2 (programmed death ligand-2, B7-DC). Despite their names, we now understand that these proteins function as ligands for the inhibitory receptor PD-1, rather than acting directly in cell death. PD-L1 is constitutively expressed by a wide variety of cells, whereas PD-L2 expression is induced on antigen-presenting cells during inflammation. Because PD-L1 is expressed constitutively, regulation of PD-1 expression could have a critical role in controlling T-cell responses. For example, signaling by pro-inflammatory cytokines can repress PD-1, thus enhancing the T-cell response. Mice lacking PD-1 gradually develop autoimmunity, presumably because of an inability to regulate T-cell activation. In chronic infections, the widespread expression of PD-1 reduces the effector


Immunology_Janeway

Immunology_Janeway. Mice lacking PD-1 gradually develop autoimmunity, presumably because of an inability to regulate T-cell activation. In chronic infections, the widespread expression of PD-1 reduces the effector activity of T cells; this helps to limit potential damage to bystander cells, but at the expense of pathogen clearance.


Immunology_Janeway

Immunology_Janeway. BTLA contains an ITIM and an ITSM and is expressed on activated T cells and B cells, as well as on some cells of the innate immune system. Unlike other CD28-family members, however, BTLA does not interact with B7 ligands but binds a member of the TNF receptor family; called the herpesvirus entry molecule (HVEM), this receptor is highly expressed on resting T cells and immature dendritic cells. When BTLA and HVEM are co-expressed on the same cell, BTLA utilizes a second mechanism that further inhibits lymphocyte activation. In this configuration, BLTA binds to HVEM and prevents HVEM from binding to alternative partners that would stimulate NFκB-dependent pro-survival signaling pathways downstream of HVEM. Alternatively, when BTLA and HVEM are expressed on different cells, the interaction of these two receptors functions to stimulate the positive pro-survival signal in the HVEM-expressing cell.


Immunology_Janeway

Immunology_Janeway. Other receptors on B cells and T cells also contain ITIMs and can inhibit cell activation when ligated along with the antigen receptors. One example is the receptor FcγRIIB-1 on B cells, which binds the Fc region of IgG antibodies. As a result, antigens present as immune complexes containing IgG antibodies are poor at activating naive B cells, due to the co-engagement of the B-cell receptor with this inhibitory Fc receptor. The ITIM in FcγRIIB-1 recruits SHIP into a complex with the B-cell receptor to block the actions of PI 3-kinase (Fig. 7.34). Another inhibitory receptor on B cells is CD22, a transmembrane protein that recognizes sialic acid-modified glycoproteins commonly found on mammalian cells but rarely on microbial pathogen surfaces. CD22 contains an ITIM that interacts with SHP, a phosphatase that can dephosphorylate adaptors such as SLP-65 that associate with CD22, thereby inhibiting signaling from the B-cell receptor.


Immunology_Janeway

Immunology_Janeway. The ITIM motif is also an important motif in signaling by receptors on NK cells that inhibit the killer activity of these cells (see Section 3-26). These inhibitory receptors recognize MHC class I molecules and transmit signals that inhibit the release of the NK cell’s cytotoxic granules when the NK cell recognizes a healthy uninfected cell. In NK cells, ITIM-containing receptors play an important role in setting the threshold for NK cell activation by balancing positive signals from ITAM-containing receptors. Summary.


Immunology_Janeway

Immunology_Janeway. Summary. Signaling through the antigen receptors on T cells and B cells is essential for the activation of these cells. However, for naive T and B cells, the signal through the T-cell receptor or B-cell receptor, respectively, is not sufficient to initiate a response. In addition to the antigen receptor signals, these cells require signals through accessory receptors that serve to monitor the environment of the cell to ensure the presence of an infection. An important secondary signaling


Immunology_Janeway

Immunology_Janeway. Fig. 7.34 The ITIM-containing Fc receptor inhibits B-cell receptor signaling by recruiting the inositol phosphatase SHIP. When the B-cell receptor binds an antigen that is already present in immune complexes with igG, the iTim-containing fc receptor fcγRiiB is engaged at the same time as the B-cell receptor. The Src-family kinase present at the B-cell receptor (BcR) phosphorylates the iTim motif of fcγRiiB, which then recruits the Sh2 domain-containing inositol phosphatase Ship. Ship dephosphorylates pip3 in the plasma membrane, generating pip2. ph domain-containing enzymes, such as vav, Btk, and pLc-γ, depend on their ph domain binding to pip3 for their stable recruitment to the activated B-cell receptor complex. The loss of pip3 terminates the recruitment of these enzymes and inhibits B-cell receptor signaling.


Immunology_Janeway

Immunology_Janeway. system in naive T cells is provided by the CD28 family of co-stimulatory proteins, which bind members of the B7 family of proteins. Activating members of the CD28 family provide co-stimulatory signals that amplify the signal from the T-cell receptor and are important in ensuring the activation of naive T cells by the appropriate target cell. In B cells, these secondary signals are provided by members of the TNF receptor superfamily, such as CD40. Inhibitory members of the CD28 and other receptor families function to attenuate or completely block signaling by activating receptors. The regulated expression of activating and inhibitory receptors and their ligands generates a sophisticated level of control of immune responses that is only beginning to be understood. Summary to Chapter 7.


Immunology_Janeway

Immunology_Janeway. Signaling by cell-surface receptors of many different sorts is crucial to the ability of the immune system to respond appropriately to foreign pathogens. The importance of these signaling pathways is demonstrated by the many diseases that are due to aberrant signaling, which include both immunodeficiency diseases and autoimmune diseases. Common features of many signaling pathways are the generation of second messengers such as calcium and phosphoinositides and the activation of both serine/threonine and tyrosine kinases. An important concept in the initiation of signaling pathways by receptor proteins is the recruitment of signaling proteins to the plasma membrane and the assembly of multiprotein signaling complexes. In many cases, signal transduction leads to the activation of transcription factors that lead directly or indirectly to the proliferation, differentiation, and effector function of activated lymphocytes. Other roles of signal transduction are to mediate changes in the


Immunology_Janeway

Immunology_Janeway. factors that lead directly or indirectly to the proliferation, differentiation, and effector function of activated lymphocytes. Other roles of signal transduction are to mediate changes in the cytoskeleton that are important for cell functions such as migration and shape changes. These steps of the T-cell and B-cell receptor signaling pathways are summarized in Fig. 7.28.


Immunology_Janeway

Immunology_Janeway. While we are beginning to understand the basic circuitry of signal transduction pathways, it is important to keep in mind that we do not yet understand why these pathways are so complex. One reason might be that the signaling pathways have roles in properties such as amplification, robustness, diversity, and efficiency of signaling responses. An important goal for the future will be to understand how the design of each signaling pathway contributes to the particular quality and sensitivity needed for specific signaling responses. Questions. 7.1 True or False: antigen receptors bear intrinsic kinase activity that allows for phosphorylation of cytoplasmic proteins and subsequent downstream signaling events. 7.2 Matching: indicate whether the following receptors are receptor tyrosine kinases (RTKs), are receptor serine/ threonine kinases (RSTKs), or have no intrinsic enzymatic activity (null). A. ___ Kit B. ___ B-cell receptor C. ___ fLT3


Immunology_Janeway

Immunology_Janeway. A. ___ Kit B. ___ B-cell receptor C. ___ fLT3 D. ___ TGf-β receptor 7.3 Short Answer: how can scaffolds and adaptors modulate signaling responses if they have no intrinsic enzymatic activity? 7.4 Multiple Choice: Which of the following alterations would result in increased activity of Ras (one or more may apply): A. a mutation in Ras that enhances its GTpase activity B. overexpression of Gefs C. depletion of GTp in the cytoplasm D. overexpression of Gaps


Immunology_Janeway

Immunology_Janeway. A. a mutation in Ras that enhances its GTpase activity B. overexpression of Gefs C. depletion of GTp in the cytoplasm D. overexpression of Gaps E. a mutation in Ras that renders it unsusceptible to the activities of Gaps 7.5 Matching: order (by numbering 1–5) the downstream signaling events that occur immediately after T-cell receptor engagement: ____ LaT and SLp-76, scaffold proteins linked by Gads, are phosphorylated ____ Zap-70, a tandem Sh2 domain containing kinase, binds to iTams ____ Recruitment and activation of Sh2, ph, and pX domain-containing proteins ____ pi 3-kinase is activated and produces pip3 ____ phosphorylation of iTams by Lck, a Src-family kinase 7.6 Fill-in-the-Blanks: for each of the following sentences, fill in the blanks with the best word selected from the list below. each word should be used only once.


Immunology_Janeway

Immunology_Janeway. LaT:Gads:SLp-76 akt antigen receptor signaling leads to many downstream events that branch out into many signaling pathways or modules. These can be activated by the scaffold complex ___________, the generation of pip3 from pip2 by the enzyme ____________, or both. phosphorylated tyrosine residues on the scaffold recruit proteins containing ___________ domains, while pip3 recruits proteins containing ___________ domains. These four modules are the activation of (1) ___________, which cleaves pip2 to produce daG and ip3, (2) ___________, which binds to pip3 and activates the mToR pathway by phosphorylating and inactivating the TSc complex, (3) ____________, an adaptor that recruits SKap55 and Riam, and (4) __________, a Gef that leads to activation of WaSp. These pathways ultimately lead to increased transcription of key genes, increased cellular metabolism, increased cellular adhesion, and actin polymerization, respectively.


Immunology_Janeway

Immunology_Janeway. 7.7 Matching: match the small G protein (GTpase) to its function. A. _____ Ras i. WaSp; actin polymerization B. _____ cdc42 ii. mToR; cellular metabolism (Rho family) C. _____ Rap1 iii. Lfa-1 aggregation; cellular adhesion D. _____ Rheb iv. mapK pathway; cellular proliferation 7.8 Multiple Choice: Which of the follow statements is false? A. K63 polyubiquitination leads to downstream cellular signaling. B. K48 polyubiquitination leads to degradation by the proteasome. C. out of the three families of enzymes involved in ubiquitination—e1 (ubiquitin-activating) enzymes, e2 (ubiquitin-conjugating) enzymes, and e3 enzymes (ubiquitin ligases)—cbl is an e3 enzyme that selects its target via its Sh2 domain. D. monoor di-ubiquitination of surface receptors leads to degradation by the proteasome. 7.9 Matching: match the human disease with the gene that is


Immunology_Janeway

Immunology_Janeway. D. monoor di-ubiquitination of surface receptors leads to degradation by the proteasome. 7.9 Matching: match the human disease with the gene that is D. ____ X-linked hypohidrotic iv. WaSp ectodermal dysplasia and immunodeficiency 7.10 Fill-in-the-Blanks: name the corresponding receptor or signaling component in its respective T/B cell counterpart: T cell B cell cd3ε:cd3δ:(cd3γ)2:(cd3ζ)2 A. __________ B. __________ cd21:cd19:cd81 cd28 C. __________ D. __________ fyn, Blk, Lyn E. __________ Syk LaT:Gads:SLp-76 F. __________ General references.


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Immunology_Janeway. D. __________ fyn, Blk, Lyn E. __________ Syk LaT:Gads:SLp-76 F. __________ General references. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P.: Molecular Biology of the Cell, 6th ed. New York: Garland Science, 2015. Gomperts, B., Kramer, I., and Tatham, P.: Signal Transduction. San Diego: Elsevier, 2002. Marks, F., Klingmler, U., and Mler-Decker, K.: Cellular Signal Processing. New York: Garland Science, 2009. Samelson, L., and Shaw, A. (eds.): Immunoreceptor Signaling. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2010. Section references. 7-1 Transmembrane receptors convert extracellular signals into intracellular biochemical events. Lin, J., and Weiss, A.: T cell receptor signalling. J. Cell Sci. 2001, 114:243–244. Smith-Garvin, J.E., Koretzky, G.A., and Jordan, M.S.: T cell activation. Annu. Rev. Immunol. 2009, 27:591–619.


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Immunology_Janeway. Lin, J., and Weiss, A.: T cell receptor signalling. J. Cell Sci. 2001, 114:243–244. Smith-Garvin, J.E., Koretzky, G.A., and Jordan, M.S.: T cell activation. Annu. Rev. Immunol. 2009, 27:591–619. 7.11 True or False: cTLa-4 and pd-1 are both iTim-containing inhibitory receptors that interfere with co-stimulatory signaling pathways by activating intracellular protein and/or lipid phosphatases. 7.12 Multiple Choice: intravenous administration of exogenous immunoglobulin is a widely used therapy for autoimmune disorders that involve the production of autoantibodies (antibodies against self antigens). Researchers have discovered that the presence of sialic acids on the infused immunoglobulins is critical for the inhibition of autoantibody production in the patient’s own B cells. Which of the following receptors could potentially be responsible for the inhibition of antibody production by B cells, given this finding? A. fcγRiiB B. cd22 C. pd-1 D. cd40


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Immunology_Janeway. A. fcγRiiB B. cd22 C. pd-1 D. cd40 E. BTLa 7-2 Intracellular signal propagation is mediated by large multiprotein signaling complexes. Balagopalan, L., Coussens, N.P., Sherman, E., Samelson, L.E., and Sommers, C.L.: The LAT story: a tale of cooperativity, coordination, and choreography. Cold Spring Harbor Perspect. Biol. 2010, 2:a005512. Jordan, M.S., and Koretzky, G.A.: Coordination of receptor signaling in multiple hematopoietic cell lineages by the adaptor protein SLP-76. Cold Spring Harbor Perspect. Biol. 2010, 2:a002501. Lim, W.A., and Pawson, T.: Phosphotyrosine signaling: evolving a new cellular communication system. Cell 2010, 142:661–667. Scott, J.D., and Pawson, T.: Cell signaling in space and time: where proteins come together and when they’re apart. Science 2009, 326:1220–1224. 7-3 Small G proteins act as molecular switches in many different signaling pathways. Etienne-Manneville, S., and Hall, A.: Rho GTPases in cell biology. Nature 2002, 420:629–635.


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Immunology_Janeway. 7-3 Small G proteins act as molecular switches in many different signaling pathways. Etienne-Manneville, S., and Hall, A.: Rho GTPases in cell biology. Nature 2002, 420:629–635. Mitin, N., Rossman, K.L., and Der, C.J.: Signaling interplay in Ras superfamily function. Curr. Biol. 2005, 15:R563–R574. 7-4 Signaling proteins are recruited to the membrane by a variety of mechanisms. Buday, L.: Membrane-targeting of signalling molecules by SH2/ SH3 domain-containing adaptor proteins. Biochim. Biophys. Acta 1999, 1422:187–204. Kanai, F., Liu, H., Field, S.J., Akbary, H., Matsuo, T., Brown, G.E., Cantley, L.C., and Yaffe, M.B.: The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat. Cell Biol. 2001, 3:675–678. Lemmon, M.A.: Membrane recognition by phospholipid-binding domains. Nat. Rev. Mol. Cell Biol. 2008, 9:99–111. 7-5 Post-translational modifications of proteins can both activate and inhibit signaling responses.


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Immunology_Janeway. 7-5 Post-translational modifications of proteins can both activate and inhibit signaling responses. Ciechanover, A.: Proteolysis: from the lysosome to ubiquitin and the proteasome. Nat. Rev. Mol. Cell Biol. 2005, 6:79–87. Hurley, J.H., Lee, S., and Prag, G.: Ubiquitin-binding domains. Biochem. J. 2006, 399:361–372. Liu, Y.C., Penninger, J., and Karin, M.: Immunity by ubiquitylation: a reversible process of modification. Nat. Rev. Immunol. 2005, 5:941–952. Wertz, I.E., and Dixit, V.M.: Signaling to NFκB: regulation by ubiquitination. Cold Spring Harbor Perspect. Biol. 2010, 2:a003350. 7-6 The activation of some receptors generates small-molecule second messengers. Kresge, N., Simoni, R.D., and Hill, R.L.: Earl W. Sutherland’s discovery of cyclic adenine monophosphate and the second messenger system. J. Biol. Chem. 2005, 280:39–40. Rall, T.W., and Sutherland, E.W.: Formation of a cyclic adenine ribonucleotide by tissue particles. J. Biol. Chem. 1958, 232:1065–1076.


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Immunology_Janeway. Rall, T.W., and Sutherland, E.W.: Formation of a cyclic adenine ribonucleotide by tissue particles. J. Biol. Chem. 1958, 232:1065–1076. 7-7 Antigen receptors consist of variable antigen-binding chains associated with invariant chains that carry out the signaling function of the receptor. Brenner, M.B., Trowbridge, I.S., and Strominger, J.L.: Cross-linking of human T cell receptor proteins: association between the T cell idiotype beta subunit and the T3 glycoprotein heavy subunit. Cell 1985, 40:183–190. Call, M.E., Pyrdol, J., Wiedmann, M., and Wucherpfennig, K.W.: The organizing principle in the formation of the T cell receptor-CD3 complex. Cell 2002, 111:967–979. Kuhns, M.S., and Davis, M.M.: TCR signaling emerges from the sum of many parts. Front. Immunol. 2012, 3:159. Samelson, L.E., Harford, J.B., and Klausner, R.D.: Identification of the components of the murine T cell antigen receptor complex. Cell 1985, 43:223–231.


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Immunology_Janeway. Samelson, L.E., Harford, J.B., and Klausner, R.D.: Identification of the components of the murine T cell antigen receptor complex. Cell 1985, 43:223–231. Tolar, P., Sohn, H.W., Liu, W., and Pierce, S.K.: The molecular assembly and organization of signaling active B-cell receptor oligomers. Immunol. Rev. 2009, 232:34–41. 7-8 Antigen recognition by the T-cell receptor and its co-receptors transduces a signal across the plasma membrane to initate signaling. Klausner, R.D., and Samelson, L.E.: T cell antigen receptor activation pathways: the tyrosine kinase connection. Cell 1991, 64:875–878. Weiss, A., and Littman, D.R.: Signal transduction by lymphocyte antigen receptors. Cell 1994, 76:263–274. 7-9 Antigen recognition by the T-cell receptor and its co-receptors leads to phosphorylation of ITAMs by Src-family kinases, generating the first intracellular signal in a signaling cascade.


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Immunology_Janeway. 7-9 Antigen recognition by the T-cell receptor and its co-receptors leads to phosphorylation of ITAMs by Src-family kinases, generating the first intracellular signal in a signaling cascade. Au-Yeung, B.B., Deindl, S., Hsu, L.-Y., Palacios, E.H., Levin, S.E., Kuriyan, J., and Weiss, A.: The structure, regulation, and function of ZAP-70. Immunol. Rev. 2009, 228:41–57. Bartelt, R.R., and Houtman, J.C.D.: The adaptor protein LAT serves as an integration node for signaling pathways that drive T cell activation. Wiley Interdiscip. Rev. Syst. Biol. Med. 2013, 5:101–110. Chan, A.C., Iwashima, M., Turck, C.W., and Weiss, A.: ZAP-70: a 70 Kd pro-tein-tyrosine kinase that associates with the TCR zeta chain. Cell 1992, 71:649–662. Iwashima, M., Irving, B.A., van Oers, N.S., Chan, A.C., and Weiss, A.: Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 1994, 263:1136–1139.


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Immunology_Janeway. Iwashima, M., Irving, B.A., van Oers, N.S., Chan, A.C., and Weiss, A.: Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 1994, 263:1136–1139. Okkenhaug, K., and Vanhaesebroeck, B.: PI3K in lymphocyte development, differentiation and activation. Nat. Rev. Immunol. 2003, 3:317–330. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R.P., and Samelson, L.E.: LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 1998, 92:83–92. 7-10 Phosphorylated ITAMs recruit and activate the tyrosine kinase ZAP-70. Yan, Q., Barros, T., Visperas, P.R., Deindl, S., Kadlecek, T.A., Weiss, A., and Kuriyan, J.: Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker. Mol. Cell. Biol. 2013, 33:2188–2201. 7-11 ITAMs are also found in other receptors on leukocytes that signal for cell activation.


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Immunology_Janeway. 7-11 ITAMs are also found in other receptors on leukocytes that signal for cell activation. Lanier, L.L.: Up on the tightrope: natural killer cell activation and inhibition. Nat. Immunol. 2008, 9:495–502. 7-12 Activated ZAP-70 phosphorylates scaffold proteins and promotes PI 3-kinase activation. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R.P., and Samelson, L.E.: LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 1998, 92:83–92. 7-13 Activated PLC-γ generates the second messengers diacylglycerol and inositol trisphosphate that lead to transcription factor activation. Berg, L.J., Finkelstein, L.D., Lucas, J.A., and Schwartzberg, P.L.: Tec family kinases in T lymphocyte development and function. Annu. Rev. Immunol. 2005, 23:549–600. Yang, Y.R., Choi, J.H., Chang, J.-S., Kwon, H.M., Jang, H.-J., Ryu, S.H., and Suh, P.-G.: Diverse cellular and physiological roles of phospholipase C-γ1. Adv. Biol. Regul. 2012, 52:138–151.


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Immunology_Janeway. Yang, Y.R., Choi, J.H., Chang, J.-S., Kwon, H.M., Jang, H.-J., Ryu, S.H., and Suh, P.-G.: Diverse cellular and physiological roles of phospholipase C-γ1. Adv. Biol. Regul. 2012, 52:138–151. 7-14 Ca2+ entry activates the transcription factor NFAT. Hogan, P.G., Chen, L., Nardone, J., and Rao, A.: Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 2003, 17:2205–2232. Hogan, P.G., Lewis, R.S., and Rao, A.: Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu. Rev. Immunol. 2010, 28:491–533. Picard, C., McCarl, C.A., Papolos, A., Khalil, S., Lhy, K., Hivroz, C., LeDeist, F., Rieux-Laucat, F., Rechavi, G., Rao, A., et al. STIM1 mutation associated with a syndrome of immunodeficiency and autoimmunity. N. Engl. J. Med. 2009, 360:1971–1980. Prakriya, M., Feske, S., Gwack, Y., Srikanth, S., Rao, A., and Hogan, P.G.: Orai1 is an essential pore subunit of the CRAC channel. Nature 2006, 443:230–233.


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Immunology_Janeway. Prakriya, M., Feske, S., Gwack, Y., Srikanth, S., Rao, A., and Hogan, P.G.: Orai1 is an essential pore subunit of the CRAC channel. Nature 2006, 443:230–233. 7-15 Ras activation stimulates the mitogen-activated protein kinase (MAPK) relay and induces expression of the transcription factor AP-1. Downward, J., Graves, J.D., Warne, P.H., Rayter, S., and Cantrell, D.A.: Stimulation of P21ras upon T-cell activation. Nature 1990, 346:719–723. Leevers, S.J., and Marshall, C.J.: Activation of extracellular signal-regulated kinase, ERK2, by P21ras oncoprotein. EMBO J. 1992, 11:569–574. References. Roskoski, R.: ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol. Res. 2012, 66:105–143. Shaw, A.S., and Filbert, E.L.: Scaffold proteins and immune-cell signalling. Nat. Rev. Immunol. 2009, 9:47–56. 7-16 Protein kinase C activates the transcription factors NFκB and AP-1.


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Immunology_Janeway. Shaw, A.S., and Filbert, E.L.: Scaffold proteins and immune-cell signalling. Nat. Rev. Immunol. 2009, 9:47–56. 7-16 Protein kinase C activates the transcription factors NFκB and AP-1. Blonska, M., and Lin, X.: CARMA1-mediated NFκB and JNK activation in lymphocytes. Immunol. Rev. 2009, 228:199–211. Matsumoto, R., Wang, D., Blonska, M., Li, H., Kobayashi, M., Pappu, B., Chen, Y., Wang, D., and Lin, X.: Phosphorylation of CARMA1 plays a critical role in T cell receptor-mediated NFκB activation. Immunity 2005, 23:575–585. Rueda, D., and Thome, M.: Phosphorylation of CARMA1: the link(Er) to NFκB activation. Immunity 2005, 23:551–553. Sommer, K., Guo, B., Pomerantz, J.L., Bandaranayake, A.D., Moreno-García, M.E., Ovechkina, Y.L., and Rawlings, D.J.: Phosphorylation of the CARMA1 linker controls NFκB activation. Immunity 2005, 23:561–574. Thome, M., and Weil, R.: Post-translational modifications regulate distinct functions of CARMA1 and BCL10. Trends Immunol. 2007, 28:281–288.


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Immunology_Janeway. Thome, M., and Weil, R.: Post-translational modifications regulate distinct functions of CARMA1 and BCL10. Trends Immunol. 2007, 28:281–288. 7-17 PI 3-kinase activation upregulates cellular metabolic pathways via the serine/threonine kinase Akt. Gamper, C.J., and Powell, J.D.: All PI3Kinase signaling is not mTOR: dissecting mTOR-dependent and independent signaling pathways in T cells. Front. Immunol. 2012, 3:312. Kane, L.P., and Weiss, A.: The PI-3 kinase/Akt pathway and T cell activation: pleiotropic pathways downstream of PIP3. Immunol. Rev. 2003, 192:7–20. Pearce, E.L.: Metabolism in T cell activation and differentiation. Curr. Opin. Immunol. 2010, 22:314–320. 7-18 T-cell receptor signaling leads to enhanced integrin-mediated cell adhesion. Bezman, N., and Koretzky, G.A.: Compartmentalization of ITAM and integrin signaling by adapter molecules. Immunol. Rev. 2007, 218:9–28.


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Immunology_Janeway. Bezman, N., and Koretzky, G.A.: Compartmentalization of ITAM and integrin signaling by adapter molecules. Immunol. Rev. 2007, 218:9–28. Mor, A., Dustin, M.L., and Philips, M.R.: Small GTPases and LFA-1 reciprocally modulate adhesion and signaling. Immunol. Rev. 2007, 218:114–125. 7-19 T-cell receptor signaling induces cytoskeletal reorganization by activating the small GTPase Cdc42. Burkhardt, J.K., Carrizosa, E., and Shaffer, M.H.: The actin cytoskeleton in T cell activation. Annu. Rev. Immunol. 2008, 26:233–259. Tybulewicz, V.L.J., and Henderson, R.B.: Rho family GTPases and their regulators in lymphocytes. Nat. Rev. Immunol. 2009, 9:630–644. 7-20 The logic of B-cell receptor signaling is similar to that of T-cell receptor signaling, but some of the signaling components are specific to B cells. Cambier, J.C., Pleiman, C.M., and Clark, M.R.: Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 1994, 12:457–486.