A commonly used assay to measure Ca 2+ influx in response to TCR stimulation inv

Question

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 the figure below, the characteristic pattern of Ca2+ influx is shown in the red line (wild-type; WT). In this you line you can see where TCR stimulation causes a sharp rise in cytoplasmic Ca2+, followed by a slow decline over hours. Also 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. (1.5 points)

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 a Western blot 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 the figure:

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.

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 points)

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

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. (0.5 point)

aCD3-biotin Streptavidin lonomycin T cells 1 minute 00o 8 minutes ??? Cytoplasmic 5 T calcium concentration4 Wild-type Mutant 200 600 Time 400 Streptavidin lonomycin

Explanation / Answer

Ans 1.

To activate acquired immune response, T lymphocytes plays an important role with specialized antigen-presenting cells (APCs). Peptide-specific T cell antigen receptor (TCR) and an APC bears peptides subsequently results in cell cycle progression and proliferation. T cell recognizes its target cell and polarizes towards the T cell–target cell interface, the immunological synapse (IS), secrets cytokines which eliminates the malignant or infected target cell.

TCR transduces its signals with the observation that TCR-deficient- T cells are stimulated with the combination of streptavidin and Ca2+ ionophores. This experiment tells that the TCR might stimulate the signals with the induced reagents. The source of the Ca2+ increase was shown to be a combination of Ca2+ released from an intracellular pool in response to streptavidin and biotin complex production and influx of Ca2+ from outside of the cell. It is observed phorbol esters are important because of their ability to activate protein kinase C (PKC, now known to be a family of enzymes), whose activity is regulated physiologically by diacylglycerol (DAG) and study tell that it generates both IP3 and DAG, to regulate the PLC activity by the function protein of TCR.

In mutant strains, T-cell proliferation activates the stimuli response and this observed by anti-TGF-? antibodies or exogenous interleukin-2 (IL-2). TGF-? signaling is required for the upregulation of growth factor production and normal T-cell proliferation which is followed by the receptor ligation. IL-2 and IL-4 levels can be reduced in response to anti-CD3? stimulation of mutant T cells, and transfected cells can be activated by an IL-2 reporter system in non-T cells.

Ans 2.

Protein extracts can be enzymatically digested to peptide and which can be seen in mutant cells CD3 proteins. As, LAT, ITK, SLP-76 and PLC - ? could not be quantified beacause the peptide sequence is identical among the kinases enzyme. Phosphorylation of Tyr appeared to be constitutive and did not change response to any of stimulation.

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