Question 1

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, with the numbers indicating the proportion of cells examined that show each pattern of staining.  
 To identify the specific signaling defect in these mutant B cells, a reasonable biochemical assay would be to:

Accepted Answer

The staining patterns in the mutant B cells suggest a defect in actin and microtubule organization, both of which are regulated by the small GTPase cdc42. Therefore, a biochemical assay that tests whether BCR stimulation of mutant B cells promotes exchange of GDP for GTP on cdc42 would help identify the specific signaling defect in these cells. Choices A, B, C, and E are not directly related to the observed phenotype, or are not specific enough to identify the defect in these mutant B cells.

Question 2

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.

Accepted Answer

BCR signaling is initiated by a Src-family kinase phosphorylating tyrosine residues in ITAM motifs of BCR signaling subunits.

Question 3

Antigen receptor signaling and lymphocyte activation.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 Q41)A, 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. 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 . You confirm that the mutant T cells express normal levels of all the proteins detected in the WT cells, including PLC-$\gamma$, SLP-76, ITK, ZAP-70, LCK, LAT, and the CD3 and TCR$\zeta$ proteins. b) Based on these additional data, which of the candidate proteins in your answer to part (a) are ruled out? Briefly explain your answer. c) What protein is most likely defective in the mutant cells and why? 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.

Accepted Answer

The answer of Antigen receptor signaling and lymphocyte activation.antibody coupled...

Question 4

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 Q21). Figure Q21) 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:

Accepted Answer

The Erk Map-kinase is downstream of RasGRP in the TCR signaling pathway. Therefore, a mutation in RasGRP would prevent Erk phosphorylation and activation, as seen in the defective T cells. PLC-γ, ITK, Calcineurin, and WASp are not directly involved in the RasGRP/Erk signaling pathway. The normal response of Ca2+ influx also suggests that the defect lies downstream of PLC-γ activation.

Question 5

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. T-cell receptor signaling leads to enhanced integrin-mediated cell adhesion

Accepted Answer

This statement is true. T-cell receptor signaling can lead to enhanced integrin-mediated cell adhesion, which is important for the activation and movement of T cells.

Question 6

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:

Accepted Answer

Rap1 is involved in the inside-out signaling that activates integrins like LFA-1, so a mutation disrupting Rap1 would impair integrin activation and cell migration, leading to a similar immunodeficiency.

Question 7

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:

Accepted Answer

Signaling through the B cell co-receptor enhances BCR signaling by recruiting and activating PI 3-kinase, leading to downstream signaling events that promote B cell activation, differentiation, and antibody production. The other options either inhibit BCR signaling or are not related to the function of the co-receptor.

Question 8

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

Accepted Answer

While recruitment and activation of PI3-kinase and Akt signaling is an important mechanism by which CD28 co-stimulation enhances T cell activation, it is not the only mechanism. CD28 co-stimulation can also upregulate cytokine production, increase survival and proliferation signals, and enhance metabolic activity of T cells.

Question 9

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:

Accepted Answer

The question specifically mentions increased IL-2 mRNA levels, and mRNA stability after transcription and splicing is the most likely explanation for this increase. Increased protein synthesis (A) and increased glucose metabolism (B) would not directly affect mRNA levels. Enhanced mRNA transport (D) and increased splicing efficiency (E) could both indirectly affect mRNA levels, but the question does not provide any specific information to suggest that these mechanisms are involved. Therefore, C is the best choice.

Question 10

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:

Accepted Answer

T cell activation through TCR stimulation induces LFA-1 conversion from a low to high affinity binding state, allowing for increased binding to its ligands on antigen-presenting cells. There is no evidence that increased synthesis or transport of LFA-1 accounts for the observed increase in adhesion. While LFA-1 phosphorylation and activation of cdc42 and WASp may play a role in integrin-dependent adhesion, they are not the primary mechanisms for the observed increase in adhesion following TCR stimulation.

Question 11

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. Assign the red or blue lines correctly to 'unstimulated' or 'TCR-stimulated' T cells, and explain the reasoning for your answer.

Accepted Answer

The answer of TCR stimulation was shown to affect ICAM-1...

Question 12

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:

Accepted Answer

PIP₃ is a key intermediate in the signaling pathway that enhances glucose metabolism following antigen receptor stimulation, as it is involved in the activation of the Akt pathway, which promotes glucose uptake and glycolysis.

Question 13

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.

Accepted Answer

The answer of BCR signaling on B cells is initiated...

Question 14

The mechanism by which CTLA-4 inhibits T cell activation is by recruiting inhibitory phosphatases.

Accepted Answer

The answer of The mechanism by which CTLA-4 inhibits T...

Question 15

'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:

Accepted Answer

Checkpoint blockade therapy is used to activate T cells and enhance their responses against cancer cells by blocking inhibitory receptors such as CTLA-4 and PD-1. Therefore, patients being treated with this protein-based therapy are most likely suffering from cancer. It is not typically used for autoimmune, immunodeficiency, inflammatory bowel, or neurodegenerative diseases.

Question 16

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:

Accepted Answer

The answer of In patients with 'CD40 ligand deficiency', T...

Question 17

Wiskott-Aldrich 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:

Accepted Answer

WASp is important for cytoskeletal reorganization, which allows for directed cytokine secretion by T cells. Without WASp, T cells may not be able to properly interact with B cells and provide the necessary cytokine support for B cell activation and antibody production.

Question 18

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

Accepted Answer

K48-linked ubiquitination and degradation of a TRAF protein, itself a ubiquitin-ligase, is an important step following TNF-receptor stimulation.

Question 19

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

Accepted Answer

NEMO is part of the IKK complex that phosphorylates IκB, leading to its degradation and the release of NF-κB, allowing NF-κB to translocate to the nucleus and activate gene transcription.

Question 20

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:

Accepted Answer

The answer of The immunosuppressive drug rapamycin acts by inhibiting...

Quiz 7: Lymphocyte Receptor Signaling