Janeway Immunobiology 9th Edition By Kenneth Murphy Test Bank

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Janeway Immunobiology 9th Edition By Kenneth Murphy Test Bank

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WITH ANSWERS

 

Janeways Immunobiology 9th Edition By Kenneth Murphy  Test Bank

 

Janeways Immunobiology, 9th Edition

Chapter 7: Lymphocyte Receptor Signaling

 

7-1  Transmembrane receptors convert extracellular signals into intracellular biochemical events

 

7.1       Short answer: Many receptors of the immune system activate protein kinases as a mechanism of initiating signaling. For antigen receptors on lymphocytes, ligand binding induces receptor clustering, and the enzymes activated are protein tyrosine kinases. Based on this mechanism, predict the outcome of expressing a mutant form of the receptor-associated tyrosine kinase in cells that still express the wild-type version of this enzyme, and explain your reasoning. This mutant is unable to bind ATP and therefore is catalytically inactive; assume the mutant and wild-type forms of the kinase are expressed in equimolar amounts.

 

7-2  Intracellular signal propagation is mediated by large multiprotein signaling complexes

 

7.2       True/False: All of the modular protein domains used for signaling protein interactions bind to ligands that are transiently generated following receptor stimulation.

 

7-3  Small G proteins act as molecular switches in many different signaling pathways

 

7.3       Multiple choice: Small GTPases, such as Ras, Rho, and cdc42, are activated when they exchange their bound GDP for GTP. In the GTP-bound state, these proteins contribute to signaling by:

  1. Hydrolyzing the bound GTP back to GDP
  2. Interacting with GTPase-activating proteins (GAPs)
  3. Interacting with target proteins and altering their activity
  4. Diffusing from the membrane and entering the nucleus
  5. Inducing calcium release from the endoplasmic reticulum

 

7-4  Signaling proteins are recruited to the membrane by a variety of mechanisms

 

7.4       Short answer: Antigen receptors use multiple mechanisms to recruit signaling proteins to the plasma membrane, where they can propagate downstream signals. In some cases, recruitment of proteins to the membrane is induced following antigen receptor stimulation, whereas other proteins are constitutively associated with the membrane.

 

Name one mechanism that is induced by antigen receptor stimulation, and one that is constitutive, and give an example a protein recruited by each mechanism.

 

7-5  Post-translational modifications of proteins can both activate and inhibit signaling responses

 

7.5       Multiple choice: Scaffold proteins are often phosphorylated at multiple sites, allowing the recruitment of several different signaling proteins. In antigen receptor signaling pathways, this mechanism is used to bring enzymes in close proximity to their substrates. Termination of this signaling mechanism would be most efficiently accomplished by:

  1. Ubiquitination of the scaffold protein, leading to its degradation
  2. Binding of the enzyme to a GTPase activating protein (GAP)
  3. Depletion of the substrate due to enzyme catalysis
  4. Dephosphorylation of the scaffold by a phosphatase
  5. Ubiquitination of the enzyme by K63-linkage of polyubiquitin

 

7-6  The activation of some receptors generates small-molecule second messengers

 

7.6       Multiple choice: Second messengers, such as calcium ions (Ca2+), are chemical mediators commonly used in intracellular signaling pathways. Despite its common usage in many different cell types in the body, Ca2+ has specific effects in lymphocytes following antigen receptor stimulation. The specific responses of lymphocytes to increased concentrations of intracellular Ca2+ are determined by:

  1. The expression of a specific subset of Ca2+-responsive enzymes in lymphocytes compared to other cell types
  2. The increased expression of calmodulin in lymphocytes compared to other cell types
  3. The presence of enzymes that bind calmodulin in lymphocytes but not other cell types
  4. The high levels of Ca2+ in the endoplasmic reticulum of lymphocytes compared to other cell types
  5. The ability of Ca2+ to amplify signaling pathways in lymphocytes but not other cell types

 

7-7  Antigen receptors consist of variable antigen-binding chains associated with invariant chains that carry out the signaling function of the receptor

 

7.7       Multiple choice: The TCR and BCR are multi-subunit receptor complexes. Experiments examining the synthesis and transport of these receptors to the lymphocyte cell surface have shown that the signaling subunits of each receptor complex are required for transport of the ligand-binding receptor subunits to the cell surface. One possible reason for this stringent control on cell surface expression is:

  1. To ensure that very few complete TCRs or BCRs are expressed on the lymphocyte surface
  2. To ensure that each lymphocyte expresses only a single specificity of antigen receptor
  3. To prevent surface expression of receptors that will bind ligand but fail to induce signals
  4. To prevent lymphocytes from triggering antigen receptor signaling pathways from intracellular forms of the receptors
  5. To ensure that equimolar amounts of all antigen receptor signaling subunits are produced

 

7-8  Antigen recognition by the T-cell receptor and its co-receptors transduces a signal across the plasma membrane to initiate signaling

 

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

 

7.8       Short answer: Antigen receptor signaling pathways are regulated by a balanced equilibrium between tyrosine kinases and tyrosine phosphatases. In general, activation of signaling proceeds when the kinase activities leading to auto-phosphorylation of Lck, to phosphorylation of ZAP-70, and to phosphorylation of downstream adapters and scaffolds exceeds the activity of phosphatases acting on these substrates. Therefore, it came as a surprise when T cells lacking the membrane tyrosine phosphatase, CD45, were first generated, and were found to be unable to be activated by TCR stimulation. Name one important function of CD45 in T cells that explains the requirement for this phosphatase in TCR signaling.

 

7.9       Multiple choice: Antigen receptor signaling pathways are initiated by the action of a Src-family kinase. In T cells, the predominant Src-kinase is Lck. In resting T cells, Lck is maintained in an inactive state by allosteric interactions involving multiple domains of the enzyme. When T cells are treated with a small molecule inhibitor of the tyrosine kinase Csk, TCR signaling is initiated even in the absence of a ligand to stimulate the TCR. This occurs because:

  1. Csk phosphorylates Lck in its kinase domain, leading to Lck activation.
  2. Csk phosphorylates ZAP-70, maintaining ZAP-70 in an auto-inhibited state.
  3. Csk phosphorylates the ITAM motifs in the TCR z chain, leading to ZAP-70 recruitment.
  4. Csk phosphorylates and activates the membrane tyrosine phosphatase CD45.
  5. Csk phosphorylates the C-terminal negative regulatory tyrosine in Lck.

 

7-10  Phosphorylated ITAMs recruit and activate the tyrosine kinase ZAP-70

 

7.10     Multiple choice: Immunoreceptor signaling proteins, such as the TCR z chain and CD3 subunits, have conserved ITAM motifs in their cytoplasmic tails. When fully phosphorylated, the ITAM recruits a tyrosine kinase with a tandem SH2 domain structure at the amino-terminal end of the protein. Tandem SH2 domain-containing kinases do not bind to sequences in other proteins, even if they contain a phosphorylated tyrosine because:

  1. The amino acid sequence adjacent to the phosphorylated tyrosines in the ITAM motif is unique, and not found in any other proteins.
  2. The affinity of a single SH2 domain within these kinases for a tyrosine phosphorylated sequence is too low for efficient binding.
  3. The amino-terminal SH2 domain of the kinase has very high affinity for both of the phosphorylated tyrosines in the ITAM motif, so will not bind to other proteins.
  4. The amino-terminal SH2 domain of the kinase is in an autoinhibited conformation and can only bind to a phosphorylated ITAM.
  5. The tandem SH2 domain-containing kinase phosphorylates the tyrosines in the ITAM itself, so can only bind to these sequences.

 

7-11  ITAMs are also found in other receptors on leukocytes that signal for cell activation

 

7.11     Short answer: The TCR and BCR are each composed of two modules, an antigen-binding module and a signaling module; furthermore, in each case, the two functional modules are encoded by distinct polypeptides. In addition, the tyrosine kinases that initiate antigen receptor signaling are also separate proteins from those of each receptor. This is a different strategy for receptor signaling than the case of receptor tyrosine kinases, where the enzyme is an intrinsic component of the ligand-binding receptor protein. Name one advantage of this organization of the TCR and BCR that accounts for the expression of ZAP-70 and Syk, as well as ITAM-containing immunoreceptors, in many different subsets of immune cells.

 

7-12  Activated ZAP-70 phosphorylates scaffold proteins and promotes PI 3-kinase activation

 

7.12     True/False: Following TCR or BCR signaling, the most important events downstream of the activation of ZAP-70 or SYK, respectively, are the activation of transcription factors leading to new gene expression.

 

7.13     True/False: The LAT:Gads:SLP-76 complex that assembles following TCR stimulation provides the scaffold for initiating multiple downstream signaling modules, leading to actin polymerization, integrin activation, and gene expression.

 

7-13  Activated PLC-g generates the second messengers diacylglycerol and inositol trisphosphate that lead to transcription factor activation

 

7.14     Multiple choice: The TCR signaling module leading to transcription factor activation is dependent on the enzyme phospholipase-C-g (PLC-g). The mechanism by which PLC-g activates multiple transcription factors is by:

  1. Generating two small second messengers that act on multiple target proteins in the T cell
  2. Directly cleaving inhibitory subunits of multiple transcription factors, thereby releasing the active transcription factors
  3. Generating two small second messengers that diffuse to the nucleus and activate transcription factors present there
  4. Generating two small second messengers that act as chaperones to promote nuclear localization of transcription factors
  5. Directly cleaving the lipid binding domain from membrane-tethered transcription factors, allowing them to migrate to the nucleus

 

7-14  Ca2+ entry activates the transcription factor NFAT

 

7.15     Multiple choice: Using an antibody that recognizes the phosphorylated, but not the non-phosphorylated form of the transcription factor, NFAT, T cells are permeabilized, stained with this antibody, and analyzed by flow cytometry. Which of the data in Figure Q7.15 represent the expected pattern of staining from wild-type T cells before and after TCR stimulation.

Figure Q7.15

 

7.16     Multiple choice: Human patients with genetic defects that result in a failure to produce the calcium channel protein ORAI1, or the ER calcium sensor protein STIM1, have severe immunodeficiency diseases. An immunosuppressive drug that would most closely mimic these primary immunodeficiencies is:

  1. Rituximab, a drug that depletes B cells
  2. Cyclosporin A, a calcineurin inhibitor
  3. Rapamycin, an mTOR inhibitor
  4. Tysabri, an inhibitor of integrin binding
  5. Enbrel or Humira that inhibit TNF

 

7.17     Multiple choice: A new strain of immunodeficient mice has been discovered, and found to have T cells that are unresponsive to TCR stimulation. The T cells from these mice have normal levels of the TCR complex on their surface, but when this TCR is stimulated, the cells fail to secrete IL-2. As a first step in determining the signaling defect responsible for this immunodeficiency, the T cells are stimulated with a phorbol ester (PMA) and Ionomycin. It is found that this treatment elicits IL-2 production by the immunodeficient T cells. Based on this information, candidate genes that could be mutated in these T cells include all of the following EXCEPT:

  1. ZAP-70
  2. PLC-g
  3. SLP-76
  4. ITK
  5. Calcineurin

 

7-15  Ras activation stimulates the mitogen-activated protein kinase (MAPK) relay and induces expression of the transcription factor AP-1

 

7.18     Multiple choice: Following TCR stimulation, the small GTPase Ras is activated. Ras activation is induced by the Ras GTP-exchange factor (GEF), RasGRP. Both Ras and RasGRP are constitutively expressed in resting T cells. The reason Ras activation is only induced following TCR stimulation is:

  1. RasGRP undergoes a Ca2+-dependent conformational change required for its activity.
  2. RasGRP requires tyrosine phosphorylation for its activity.
  3. RasGRP is ubiquitinated and degraded in the absence of TCR stimulation.
  4. RasGRP recruitment to the plasma membrane requires TCR stimulation.
  5. Ras is only recruited to the activated TCR following assembly of the LAT:Gads:SLP-76 complex.

 

7.19     True/False: Diacyl-glycerol (DAG) is one of the two products generated when PLC-g cleaves the membrane phospholipid, PIP2. This small lipid mediator remains associated with the plasma membrane and functions to inhibit tyrosine phosphatases that remove activating phosphate groups from ZAP-70 and the Tec-family kinase, ITK.

 

7.20     True/False: Several small GTPases play critical roles in antigen receptor signaling pathways. When activated by binding to GTP, these mediators induce changes in cytoskeletal organization, adhesion, and metabolism, but have no role in transcription factor activation.

 

7.21     Multiple choice: T cells with defective TCR signaling are discovered, and found to have an inactivating mutation in a key TCR signaling protein. Using an antibody that recognizes the phosphorylated (activated) form of the Erk Map-kinase, stimulated T cells are permeabilized, stained with this antibody, and analyzed on the flow cytometer. These data are shown in Figure Q7.21.

Figure Q7.21

Additional experiments examining Ca2+ influx into T cells following TCR stimulation show a normal response in the mutant T cells. One likely candidate gene that could be mutated in the defective cells is:

  1. PLC-g
  2. ITK
  3. RasGRP
  4. Calcineurin
  5. WASp

 

7-16  Protein kinase C activates the transcription factors NFkB and AP-1

 

7.22     Multiple choice: An important transcription factor activated by antigen receptor signaling in lymphocytes is an NFkB heterodimer of the two subunits, p50 and p65Rel. Defects in the IkB-kinase complex (NEMO) or mutations in IkB that prevent its phosphorylation interfere with NFkB activation and result in severe immunodeficiency diseases. This is due to the important function of:

  1. NEMO in targeting p50:p65Rel for ubiquitination and degradation
  2. NEMO in ubiquitinating IkB causing its release from NFkB
  3. IkB in blocking the DNA binding activity of NFkB
  4. IkB as a chaperone to promote NFkB nuclear localization
  5. NEMO in phosphorylating IkB inducing its degradation, thereby releasing NFkB

 

7-17  PI 3-kinase activation up-regulates cellular metabolic pathways via the serine/threonine kinase Akt

 

7.23     Multiple choice: Lymphocyte activation leads to robust proliferation and effector cell differentiation. The metabolic demands of these processes are met, in part, by up-regulation of glycolytic enzymes and nutrient transporters on the activated cell membrane. A key intermediate in the signaling pathway leading to enhanced glucose metabolism following antigen receptor stimulation is:

  1. The lipid mediator diacyl-glycerol (DAG)
  2. The phosphoinositide, PIP3
  3. Increases in cytoplasmic Ca2+
  4. Cleavage of the membrane phospholipid, PIP2
  5. The mitochondrial protein, Bcl-2

 

7.24     Multiple choice: The immunosuppressive drug rapamycin acts by inhibiting mTOR. When activated T cells are treated with rapamycin in a cell culture assay, they show greatly diminished proliferation, and accumulate to much lower numbers than control-treated cells. This is because:

  1. Rapamycin inhibits cells from increasing their synthesis of lipids and proteins.
  2. Rapamycin inhibits cells from activating the pro-survival protein, Bcl-2.
  3. Rapamycin inhibits DNA synthesis in activated T cells.
  4. Rapamycin inhibits cell cycle progression in activated T cells.
  5. Rapamycin inhibits the T cells production of the growth factor, IL-2.

 

7.25     True/False: Phosphorylation of signaling proteins can have activating or inhibitory effects on protein function. In many cases, such as the activation of mTOR, the phosphorylation of an inhibitory protein leads to inactivation of the inhibitor, resulting in downstream signaling.

 

7-18  T-cell receptor signaling leads to enhanced integrin-mediated cell adhesion

 

7.26     Multiple choice: The integrin LFA-1 is constitutively expressed on the surface of resting T cells. Yet, integrin-dependent T cell adhesion to antigen-presenting cells increases substantially following TCR stimulation. This increased integrin-dependent adhesion is mediated in part by:

  1. Increased synthesis of the LFA-1 protein
  2. Increased transport of intracellular pools of LFA-1 to the cell surface
  3. LFA-1 conversion to a high affinity binding state
  4. Increased phosphorylation of the LFA-1 cytoplasmic tail
  5. Activation of cdc42 and WASp

 

7.27     Multiple choice: Humans with defective expression of the integrin LFA-1 have an immunodeficiency disease characterized by the failure of lymphocytes and granulocytes to migrate to tissues at sites of infection or inflammation. A similar immunodeficiency would be expected if individuals had mutations disrupting the gene for:

  1. CD3z
  2. The complement receptor, CD21
  3. WASp
  4. Rap1
  5. SLP-76

 

7.28     Short answer: TCR stimulation was shown to affect ICAM-1 (integrin ligand) binding to LFA-1 (integrin) on T cells. To demonstrate this, varying concentrations of purified ICAM-1 were added to unstimulated or TCR-stimulated T cells, and the amount of ICAM-1 binding was measured. The data from such an experiment are displayed on Figure Q7.28. Assign the red or blue lines correctly to unstimulated or TCR-stimulated T cells, and explain the reasoning for your answer.

Figure Q7.28

 

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

 

7.29     Multiple choice: WiskottAldrich syndrome is an immunodeficiency disease due to mutations in the gene encoding WASp. Individuals with this disease make poor antibody responses to protein antigens, due to impaired T cell help for B cells. WASp-deficient T cells are likely impaired in providing adequate help to B cells due to:

  1. Defects in up-regulating expression of genes encoding cytokines required by B cells
  2. Defects in up-regulating metabolic pathways for T cell macromolecular synthesis
  3. Defects in up-regulating expression of genes needed for T cell survival
  4. Defects in cytoskeletal reorganization needed for directed T cell cytokine secretion
  5. Defects in up-regulating T cell integrin adhesion for stable interactions with B cells

 

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

 

7.30     True/False: Unlike TCR signaling, B cell receptor (BCR) signaling is not initiated by a Src-family kinase phosphorylating tyrosine resides in ITAM motifs of BCR signaling subunits.

 

7.31     Matching: BCR stimulation and TCR stimulation generally activate similar downstream signaling modules, but do so using related, but not identical, signaling proteins. From the list below, match each B cell protein to its T cell counterpart.

a. CD3z

b. SLP-76

c. PLC-g

d. ZAP-70

e. CD28

f. LAT

g. ITK

h. Vav

i. PI-3-kinase

j. Lck

1. Syk

2. Lyn, Blk or Fyn

3. SLP-65

4. CD19/CD21/CD81

5. BTK

6. Ig-a or Ig-b

 

 

7.32     Multiple choice: A mutant B cell line is examined by confocal microscopy after incubation with a microbial pathogen recognized by the BCR on these B cells. The B cells have been stained with antibodies to visualize the localization of polymerized actin and microtubules. As a control, wild-type B cells are examined. The results are shown in Figure Q7.32, with the numbers indicating the proportion of cells examined that show each pattern of staining.

Figure Q7.32

To identify the specific signaling defect in these mutant B cells, a reasonable biochemical assay would be to:

  1. Determine if BCR stimulation of mutant B cells produces enhanced binding of the B cell to the microbe
  2. Determine whether the mutant B cells have reduced levels of the enzyme Protein kinase C-q
  3. Determine whether the mutant B cells are overexpressing the enzyme Vav
  4. Determine whether BCR stimulation of mutant B cells promotes exchange of GDP for GTP on cdc42
  5. Determine whether BCR stimulation of mutant B cells produces increased levels of DAG

 

7.33     Multiple choice: The B cell co-receptor, composed of CD19/CD21/CD81, is a receptor that binds to complement fragments such as C3dg. When an antigen bound by the BCR on a B cell has also been tagged with C3dg, the B cell co-receptor is stimulated together with the BCR. Signaling through the co-receptor:

  1. Inhibits BCR signaling by leading to ITAM dephosphorylation
  2. Inhibits BCR signaling by leading to PIP3 dephosphorylation
  3. Enhances BCR signaling by recruiting and activating PI 3-kinase
  4. Enhances BCR signaling by bringing the Src-kinase together with Ig-a and Ig-b.
  5. Inhibits BCR signaling by sequestering the antigen away from the BCR.

 

7-21  The cell-surface protein CD28 is a required co-stimulatory signaling receptor for naive T cell activation

 

7-22  Maximal activation of PLC-g, which is important for transcription factor activation, requires a co-stimulatory signal induced by CD28

 

7.34     True/False: The only mechanism by which CD28 co-stimulation enhances T cell activation is by recruiting and activating PI 3-kinase, leading to Akt activation.

 

7.35     Multiple choice: TCR and CD28 signaling together lead to maximal production of IL-2 by the activated T cell. Experiments investigating the mechanism underlying the CD28 co-stimulation-mediated increase in IL-2 production show that T cells stimulated through the TCR plus CD28 have increased levels of IL-2 mRNA compared to cells stimulated through the TCR alone. One important component contributing to increased IL-2 mRNA levels is:

  1. Increased protein synthesis due to increased production of ribosomes
  2. Increased glucose metabolism due to increased production of glycolytic enzymes
  3. Increased mRNA stability after transcription and splicing
  4. Enhanced mRNA transport from the nucleus to the cytoplasm
  5. Increased levels of splicing enzymes that increase IL-2 mRNA splicing efficiency

 

7-23  TNF receptor superfamily members augment T-cell and B-cell activation

 

7.36     Multiple choice: In patients with CD40 ligand deficiency, T cell-dependent B cell activation is impaired, leading to poor antibody responses to protein antigens. The signaling pathway missing in these patients B cells is important for:

  1. Inducing integrin activation to promote adhesion
  2. Inducing NFkB activation by the noncanonical pathway
  3. Inducing WASp activation and actin polymerization
  4. Inducing Ca2+ influx leading to NFAT activation
  5. Inducing Ras activation and Erk Map-kinase signaling

 

7.37     Multiple choice: TNF-receptor signaling commonly includes several steps that are regulated by ubiquitination. One important step following TNF-receptor stimulation is the:

  1. K48-linked ubiquitination and degradation of a TRAF protein, itself a ubiquitin-ligase
  2. K48-linked ubiquitination of the TNF-receptor cytoplasmic tail, leading to its degradation
  3. K63-linked ubiquitination of the TNF-receptor, providing a docking site for TRAF protein binding
  4. K48-linked ubiquitination of NIK, the NFkB-inducing kinase
  5. K63-linked ubiquitination of cIAP, leading to its binding to NIK, the NFkB-inducing kinase

 

7-24  Inhibitory receptors on lymphocytes down-regulate immune responses by interfering with co-stimulatory signaling pathways

 

7.38     True/False: The mechanism by which CTLA-4 inhibits T cell activation is by recruiting inhibitory phosphatases.

 

7.39     Multiple choice: Checkpoint blockade is a therapeutic strategy based on enhancing T cell responses by inhibiting the function of inhibitory receptors, such as CTLA-4, and PD-1. Patients being treated with these protein-based therapeutics would likely be suffering from:

  1. An autoimmune disease
  2. An immunodeficiency disease
  3. Cancer
  4. Inflammatory bowel disease
  5. A neurodegenerative disease

 

7-25  Inhibitory receptors on lymphocytes down-regulate immune responses by recruiting protein or lipid phosphatases

 

7.40     Multiple choice: BCR signaling on B cells is initiated by antigen binding, leading to mTOR activation. This occurs, for instance, when the antigen is a live microbe that binds to the BCR on the B cells. Which one of the forms of antigen shown below the graph would correctly account for the data shown in Figure Q7.40.

Figure Q7.40

 

7.41     Synthesis question: Antigen receptor signaling and lymphocyte activation.

A commonly used assay to measure Ca2+ influx in response to TCR stimulation involves loading T cells with a Ca2+sensitive dye, and then stimulating the TCR using anti-CD3 antibody coupled to biotin, followed by cross-linking with Streptavidin (S-Av). As the antibody and then S-Av are added, the cells are run on the flow cytometer to examine the fluorescence of the Ca2+-sensitive dye. After several minutes of analysis, the cells are stimulated with ionomycin (Iono), to induce Ca2+ influx; this is used as a positive control to ensure that the cells are loaded with the dye. In Figure Q7.41A, the characteristic pattern of Ca2+ influx is shown in the red line (wild-type; WT), where TCR stimulation causes a sharp rise in cytoplasmic Ca2+, followed by a slow decline over hours. As shown below, cytoplasmic Ca2+ concentrations do not normally return to baseline for the timecourse of this experiment. A mutant mouse is identified with a defect in T cell activation in response to TCR stimulation. The calcium response of T cells from the mutant mouse is shown in the blue line.

Figure Q7.41A

 

  1. a) Given these data, name three T cell signaling proteins that could be defective in the mutant T cells. Then name three T cell signaling proteins that could not be responsible for this defect, even if mutated.

 

Additional experiments are performed to analyze protein tyrosine phosphorylation in response to TCR stimulation. For these experiments, T cells are stimulated with anti-CD3 antibody, and then lysates are prepared and run on a protein (SDS-PAGE) gel to separate the proteins by molecular weight. The proteins are transferred from the gel to a membrane for immunoblotting using an antibody that binds to all phosphorylated tyrosine residues in any protein; this antibody is called anti-phospho-tyrosine antibody, and is abbreviated as anti-P-Y. The results are shown in Figure Q7.41B.

Figure Q7.41B

 

You confirm that the mutant T cells express normal levels of all the proteins detected in the WT cells, including PLC-g, SLP-76, ITK, ZAP-70, LCK, LAT, and the CD3 and TCRz proteins.

 

  1. b) Based on these additional data, which of the candidate proteins in your answer to part (a) are ruled out? Briefly explain your answer.

 

  1. c) What protein is most likely defective in the mutant cells and why?

 

  1. d) For the protein you named in your answer to part (c), which amino acids or domain of the protein could be mutated to account for all the data.

 

7.42     Synthesis question: Co-stimulatory and inhibitory receptors modulate antigen receptor signaling in T and B lymphocytes. A new receptor is discovered, expressed on the surface of T cells, and called X. An antibody to X is generated, and used in T cell stimulation experiments. In these experiments, antibodies to the TCR complex (anti-CD3) and to CD28 (anti-CD28) are known to stimulate signaling through those receptors, as does the antibody to X. The data from an experiment measuring IL-2 secretion by the T cells stimulated with different combinations of antibodies are shown in Figure Q7.42.

Figure Q7.42

 

  1. a) Does stimulation of receptor X alone induce IL-2 production by T cells? Does it enhance or inhibit TCR signaling? Indicate the evidence supporting your answers.

 

  1. b) If you examined the amino acid sequence of the receptor X cytoplasmic tail, what motif would you expect to find?

 

Biochemical studies show that when receptor X is stimulated, a tyrosine residue in the cytoplasmic tail becomes phosphorylated.

 

  1. c) From these data, what are the two most likely signaling proteins that might be recruited to receptor X when it is stimulated? Does the T cell stimulation data shown in the graph rule in or out either of your candidate proteins? Why or why not?

 

  1. d) Describe a biochemical experiment (analysis of proteins) that would indicate which enzyme is recruited to receptor X when it is stimulated.

 

 

ANSWERS

 

7.1: Following stimulation of the antigen receptor, downstream signaling would be greatly diminished. This would be visible as reduced auto-phosphorylation of the kinase and as reduced phosphorylation of downstream substrates of the pathway. In cells expressing equimolar amounts of wild-type and inactive forms of the kinase, the majority of the receptors would be associated with two inactive proteins, or one active plus one inactive; few receptors would have two active kinases associated with them. Therefore, after receptor clustering, kinase activation that normally occurs by the two associated kinases phosphorylating each other would fail to occur in the majority of receptor complexes. In this situation, the inactive form of the kinase is known as a dominant-negative mutant, referring to its ability to poison signaling even in the presence of the wild-type kinase.

 

7.2: False.

SH3 domains, which bind proline (PXXP) motifs, and PDZ domains, which bind C termini of proteins, are modular domains that bind constitutive ligands present even in the absence of receptor stimulation.

 

7.3: C.

Small GTPases undergo a conformational change after releasing GDP and binding GTP. In the GTP-bound state, they bind to target proteins, and change the activity or function of these target proteins.

 

7.4:

Recruitment induced by antigen receptor stimulation:

  • binding to a membrane associated scaffold protein that is phosphorylated in response to antigen receptor stimulation (examples: Grb2, Gads, SLP-76)
  • binding to the membrane phospholipid PIP3 that is generated by phosphorylation of PIP2 in response to antigen receptor stimulation (examples: Akt, Itk, PLC-g)

 

Recruitment that is constitutive:

  • lipid modified proteins that associate constitutively with the plasma membrane (examples: small GTPases such as Ras, Rap1)

 

7.5: D.

Multiple mechanisms contribute to the termination of signaling. Ubiquitination of a target protein can lead to that proteins degradation. However, for a phosphorylated scaffold protein, the most immediate mechanism to terminate its signaling function is by dephosphorylation, thereby eliminating the binding sites for recruited proteins.

 

7.6: A.

The Ca2+-calmodulin complex binds to many different enzymes in many cell types. Its specific signaling effects will depend on the exact identify of which Ca2+-responsive enzymes are expressed in each cell type.

 

7.7: C.

The TCR and BCR are complex signaling receptors that require all of their subunits for proper ligand binding and optimal signaling. The surface expression of antigen receptors lacking one or more of their domains would interfere with T or B cell activation. This would be particularly detrimental in the case of cells expressing the ligand-binding domains of an antigen receptor without the signaling subunits; such a receptor would bind the ligand, but fail to signal. If antigen levels are low, this might result in a failure to mount a T or B cell response altogether.

 

7.8: CD45 is the phosphatase that de-phosphorylates the C-terminal negative regulatory tyrosine of Lck. When this negative regulatory tyrosine is phosphorylated and binds to the Lck SH2 domain, Lck is held in an inactive conformation. TCR signaling initiated by Lck cannot occur without CD45 to dephosphorylate this site.

 

7.9: E.

Csk is a tyrosine kinase that phosphorylates the C-terminal negative regulatory tyrosine in Lck, maintaining Lck in an inactive state. In the absence of Csk, tyrosine phosphatases in the T cell will dephosphorylate this reside, leading to Lck being in a primed state. Full Lck activation will occur when Lck autophosphorylates on its activation loop tyrosine in the kinase domain, a process that takes place at a low level, even in the absence of TCR stimulation. This low basal autophosphorylation activity of Lck is normally prevented in non-stimulated T cells by the phosphorylation of the C-terminal negative regulatory tyrosine, which locks Lck in an inactive state.

 

7.10: B.

Tandem SH2-containing tyrosine kinases, such as ZAP-70 and Syk, have SH2 domains that individually have very low affinity for their tyrosine-phosphorylated binding sites. Therefore, these kinases will not bind to a tyrosine-phosphorylated sequence that contains a binding site for only one of their SH2 domains. It is only when both tyrosines in an ITAM are phosphorylated, recruiting both SH2 domains together, that these kinases can stably bind to ITAM-containing signaling subunits.

 

7.11: Since the ITAM-containing signaling subunits and the enzymes are each present as separate polypeptides, these proteins can be re-used by other immune cells to mediate signaling via distinct ligand-binding receptors (i.e., mix and match the receptors, signaling subunits and tyrosine kinases). For instance, Fc receptors on NK cells can use the TCR z chain as a signaling subunit, and can use ZAP-70 as an enzyme. As another example, neutrophils use ITAM-containing subunits DAP-12 or Fcr-g to activate Syk downstream of the PSGL-1 (P-selectin binding) receptor.

 

7.12: False.

Explanation: Four important signaling modules are activated downstream of the TCR or BCR. In addition to transcription factor activation, the three additional modules lead to increased cellular metabolic activity, actin polymerization and cytoskeletal reorganization, and increased integrin adhesiveness and clustering.

 

7.13: True.

 

7.14: A.

PLC-g cleaves the membrane phospholipid PIP2 into two second messengers, DAG and IP3. DAG remains tethered to the plasma membrane, and leads to activation of Ras and Protein kinase C. IP3 diffuses to the endoplasmic reticulum (ER), and binds to and activates the IP3 receptor, ultimately leading to calcium influx into the cell. Collectively, signaling pathways arising from these two second messengers activate multiple transcription factors, including NFAT, NFkB, and AP-1.

 

7.15: B.

In resting T cells, NFAT is heavily phosphorylated on serine/threonine residues. Following TCR stimulation, calcineurin is activated and dephosphorylates NFAT.

 

7.16: B.

Patients with defects in ORAI1 or STIM1 have impaired TCR-induced Ca2+ influx, leading to defective activation of calcineurin and its downstream target NFAT. The immunosuppressive drug cyclosporin A inhibits calcineurin and causes a similar defect in NFAT activation.

 

7.17: E.

Since PMA plus Ionomycin restore normal IL-2 production in response to TCR stimulation, the defect must lie in PLC-g activation or a step upstream of this. Possible candidates would be ZAP-70, PLC-g, SLP-76 or ITK. Calcineurin would not be a possible candidate since cells with defective calcineurin would not show normal responses to PMA plus Ionomycin. This is because the Ca2+ influx induced by Ionomycin would fail to activate NFAT in the absence of calcineurin.

 

7.18: D.

Although Ras is constitutively present at the plasma membrane, it requires interaction with a GEF such as RasGRP for activation. While RasGRP is also constitutively expressed in non-stimulated T cells, it is not present at the membrane, and therefore cannot interact with Ras. TCR stimulation generates DAG, a second messenger that remains associated with the plasma membrane. DAG recruits Ras GRP, which can activate Ras.

 

7.19: False.

DAG remains associated with the plasma membrane and recruits RasGRP and the serine/threonine kinase, Protein kinase C (PKC)-q. RasGRP activates Ras, leading to the formation of the active transcription factor, AP-1, and PKC-q leads to the activation of a second transcription factor, NKkB.

 

7.20: False.

 

7.21: C.

The defect in these cells is failure to activate the Ras/Map-kinase pathway. Since Ca2+ influx in response to TCR stimulation is normal, this indicates that activation of PLC-g is normal. This information rules out PLC-g and ITK as candidates. Calcineurin is not required for Erk Map-kinase activation, so it is not a correct answer. WASp is also not required for Erk Map-kinase activation. The correct answer is RasGRP. When DAG is produced following PLC-g activation, RasGRP is required to bind DAG and activate Ras, leading to Erk Map-kinase activation.

 

7.22: E.

In non-stimulated T cells, the p50/p65Rel NFkB heterodimer is inactive due to retention in the cytoplasm by binding to the inhibitory subunit, IkB. Following TCR stimulation, NEMO is activated and phosphorylates IkB. This leads to IkB ubiquitination and degradation, thereby releasing NFkB. The NFkB heterodimer then enters the nucleus where it activates transcription of target genes.

 

7.23: B.

Increased glucose metabolism is induced by activation of the serine/threonine kinase Akt following TCR stimulation. Akt is activated by PDK1. Both Akt and PDK1 are recruited to the plasma membrane by binding to PIP3, as both kinases have PH domains. Binding to PIP3 activates PDK1 to phosphorylate and activate Akt.

 

7.24: A.

mTOR activation leads to increases in several macromolecular synthesis pathways in T cells. They include increases in lipid metabolism, protein synthesis, and RNA transcription. Without these increases, activated T cells are unable to produce the components needed for cells to undergo the robust proliferation seen normally. mTOR is not required to activate Bcl2, nor is it required for DNA synthesis, cell cycle progression or IL-2 synthesis.

 

7.25: True.

In response to antigen receptor stimulation, mTOR is activated by the small GTPase Rheb. In non-stimulated cells, Rheb is held in an inactive conformation by the protein complex TSC1/2. Activation of the serine/threonine kinase Akt leads to phosphorylation of TSC1/2, causing release of Rheb and activation of mTOR.

 

7.26: C.

TCR stimulation induces increased binding of the integrin LFA-1 to its ligand, ICAM-1, expressed on antigen-presenting-cells. In large part, increased LFA-1 binding is mediated by a conformational change that converts LFA-1 from a low-affinity to a high-affinity binding state. TCR stimulation also induces LFA-1 clustering on the plasma membrane, which also contributes to increased integrin-dependent adhesion.

 

7.27: D.

Integrin activation is induced by activation of the small GTPase Rap1 following antigen receptor stimulation. Therefore a defect in Rap1 would mimic a defect in LFA-1 itself. Neither CD21 nor WASp are required for integrin activation. Defects in CD3z or SLP-76 would lead to T cell defects that are much more severe than failure of cells to migrate to tissues.

 

7.28: Solid line = TCR-stimulated; dashed line = unstimulated.

When the TCR is stimulated, T cells bind the integrin ligand with higher affinity; hence, lower concentrations of ligand are required for maximal binding to activated T cells.

 

7.29: D.

WASp is required for actin polymerization and formation of the Immune Synapse. These cytoskeletal changes are required for directed secretion of cytokines from the T cell to the B cell during the TB interactions that are essential for T cell-dependent antibody responses. WASp is not required for cytokine or survival gene expression, up-regulation of metabolic pathways, or for integrin-mediated adhesion.

 

7.30: False.

BCR signaling, like TCR signaling, is initiated by a Src-family kinase phosphorylating ITAM tyrosines on BCR signaling subunit proteins, Ig-a and Ig-b.

 

7.31:

  1. d
  2. j
  3. b
  4. e
  5. g
  6. a

 

7.32: D.

These mutant B cells are defective in cytoskeletal reorganization following BCR stimulation. In this case, BCR stimulation is induced by binding the antigen, which is a microbe. In wild-type cells, BCR stimulation will induce polarization of the cytoskeletal elements, the microtubule organizing center and the polymerized actin, to the immune synapse where the activated BCR is localized. BCR-induced actin polymerization and cytoskeletal reorganization is activated by the Vav GTP-exchange factor (GEF) activating the small GTPase, cdc42, a reasonable experiment would be to determine whether BCR stimulation is functioning to activate cdc42. Cdc42 activation is dependent on release of GDP and binding of GTP, so the experiment would require assessing whether this nucleotide exchange process is occurring following BCR stimulation.

 

7.33: C.

 

7.34: False.

CD28 co-stimulation does lead to activation of PI 3-kinase, and to production of PIP3 at the plasma membrane. In addition to Akt activation, PIP3 recruits PLC-g, ITK, and Vav. These enzymes contribute to T cell activation by enhancing transcription factor activation and actin polymerization and cytoskeletal reorganization, pathways that are not dependent on Akt.

 

7.35: C.

TCR plus CD28 stimulation together lead to enhanced activation of transcription factors compared to TCR stimulation alone. This leads to increased mRNA synthesis of the IL-2 gene. In addition, CD28 co-stimulation leads to activation of Akt. Akt phosphorylates the RNA binding protein, NF-90. When phosphorylated, NF-90 translocates from the nucleus to the cytoplasm, where it binds to and stabilizes the IL-2 mRNA.

 

7.36: B.

CD40 ligand deficiency results in a failure to stimulate CD40 on B cells during TB interactions for antibody responses to protein antigens. CD40 is a TNF-receptor family member. Stimulation of CD40 leads to NFkB activation by the non-canonical pathway.

 

7.37: A.

TNF-receptors stimulate NFkB activation by the noncanonical pathway. This pathways includes several steps regulated by ubiquitination. These include K63-linked ubiquitination of cIAP, inducing cIAP to ubiquitinate TRAF3 with K-48-linked poly-ubiquitin. This causes degradation of TRAF3 and release of NIK. NIK then activates IkB-kinase-a, which phosphorylates the NFkB precursor protein, p100. Phosphorylation of p100 leads to its K48-linked ubiquitination, inducing cleavage of p100 to generate p52. P52 binds to relB to form the active NFkB heterodimer.

 

7.38: False.

Unlike the ITIM- and ITSM-containing receptors, CTLA-4 is no longer believed to recruit inhibitor phosphatases as a mechanism of inhibitory signaling. Instead, a major mechanism of CLTA-4 action is by competing with CD28 for binding to the shared B7 ligands.

 

7.39: C.

Checkpoint blockade aims to enhance T cell activation, by inhibiting the activation of inhibitory receptors. This would be beneficial in patients where the goal is to enhance or increase their T cell activation and function. This strategy is currently being investigated for use in cancer patients, to enhance the ability of their immune system to destroy their tumor cells.

 

7.40: A.

When the microbe is already coated with antibody proteins prior to incubation with the B cells, the inhibitory Fc receptor (FcgRIIb) on the B cell is stimulated along with the BCR. The co-engagement of FcgRIIb recruits the lipid phosphatase, SHIP, leading to dephosphorylation of PIP3. Loss of PIP3 reduces Akt activation, leading to reduced activation of mTOR. As a result, much higher concentrations of antigen are required to generate the same level of mTOR activation as would be seen with the live microbe alone. When the microbe is coated with C3dg, BCR signaling would be enhanced, and this form of antigen would stimulate mTOR activation at lower doses than unmodified antigen. The heat-killed microbe and membrane fragments would stimulate at similar antigen doses to live microbes.

 

7.41:

  1. a) Any TCR signaling protein required for PLC-g activation could be responsible. This would include Lck, ZAP-70, SLP-76, LAT, ITK, or PLC-g Additional candidates could be PI 3-kinase, ORAI1, or STIM1. Proteins that could not be responsible for the calcium signaling defect include: WASp, cdc42, Vav, Akt, mTOR, Rheb, Protein kinase C-q, Rap1, Nck, Ras, RasGRP, Erk-MAP-kinase or calcineurin. This is not a complete list, but the most common answers.
  2. b) LCK is normal, as CD3 and TCRz phosphorylation are normal. Also, increased LCK phosphorylation in response to TCR stimulation (autophosphorylation) is also normal. In addition, ZAP-70 phosphorylation is normal, indicating normal LCK activity. Since there is no phosphorylation of LAT or SLP-76, there is likely a defect in ZAP-70 kinase activity. These data cannot directly rule out a defect in ITK or PLC-g, but given the lack of phosphorylation of SLP-76 and LAT, a more likely explanation is a defect in ZAP-70.
  3. c) ZAP-70 for reasons explained in (b).
  4. d) ZAP-70 could have a mutation in its kinase domain that prevents kinase activity. This could be a mutation that prevents ATP binding, or one that prevents phosphorylation on the activation loop tyrosine. Alternatively, ZAP-70 could have a mutation in the linker region between the SH2 domains and the kinase domain that prevents phosphorylation of this linker region. In the absence of phosphorylation of this linker region, ZAP-70 remains in an auto-inhibited conformation, and would not phosphorylate its downstream substrates, ZAP-70 and LAT.

 

7.42:

  1. a) Stimulation of X does not induce IL-2 production by itself. Stimulation of X inhibits TCR signaling because anti-CD3 + anti-X stimulation leads to reduced IL-2 secretion compared to stimulation with anti-CD3 alone.
  2. b) An ITIM or ITSM motif.
  3. c) Inhibitory receptors that contain ITIM or ITSM motifs often recruit a phosphatase. This could be a protein tyrosine phosphatase, such as SHP or a lipid phosphatase, SHIP. The T cell stimulation data are not sufficient to identify which type of phosphatase is recruited. It could be SHP2, which would dephosphorylate signaling proteins such as ZAP-70, SLP-76 or ITK; dephosphorylation of any of these (or other) TCR signaling proteins would lead to reduced IL-2 production. Alternatively, it could be SHIP, which would dephosphorylate PIP3; this would prevent recruitment of ITK and PLC-g, either of which would lead to reduced IL-2 production.
  4. d) Possible answers:
  5. Stimulate T cells with anti-X antibody and then perform an immunoprecipitation of receptor X. Analyze the proteins in the immunoprecipitation for SHP or SHIP by Western blotting.

 

  1. Stimulate T cells with anti-CD3 + anti-CD28 or with anti-CD3 + anti-CD28 + anti-X. Lyse the T cells and examine candidate proteins for reduced tyrosine phosphorylation when stimulation of receptor X is included compared to stimulation of CD3 + CD28 alone. If X recruits SHP, cells stimulated with anti-X plus the other antibodies should show reduced tyrosine phosphorylation compared to cells stimulated with anti-CD3 + anti-CD28 alone.

 

In parallel, prepare lipids from T cells stimulated with anti-CD3 + anti-CD28 versus anti-CD3 + anti-CD28 + anti-X, and analyze for amounts of PIP3. If X recruits SHIP, then cells stimulated with all three antibodies should have reduced levels of PIP3 compared to cells stimulated with anti-CD3 + anti-CD28 alone.

 

 

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