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Chapter 36 : T-Lymphocyte Activation and Cell Signaling

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Abstract:

This chapter reviews signaling events associated with T-cell activation. Crystal structure analysis has confirmed that the molecular specificity of the T-cell receptor (TCR) is conferred by the solvent-exposed membrane-distal variable regions of the α and β chains. The earliest biochemical events elicited by T-cell activation are the phosphorylation of proteins in the TCR-CD3 complex and the activation and interaction of Syk and Src family protein tyrosine kinases (PTKs). Importantly, LAT is palmitoylated, which targets it (and the molecules it recruits) to glycolipid-enriched microdomains known as lipid rafts. Recruitment of the tyrosine-phosphorylated TCR to lipid rafts is a critical step in T-cell activation and presumably concentrates the downstream signaling machinery in close proximity, facilitating enzymatic activity and molecular scaffold formation. The reorientation and redistribution of the cytoskeleton constitute an important consequence of T-cell activation, since they are closely linked to functional outcome. Defects affecting the termination of T-cell activation and growth may result in systemic autoimmune disease. The sensitivity of the quantitative fluorescence is based on the use of the standard curve as described in the standardization section. The combined use of flow cytometry and evolving technologies, such as gene microarray systems and proteomics, will tremendously increase the understanding in the field of T-lymphocyte activation and signaling.

Citation: Currier J. 2006. T-Lymphocyte Activation and Cell Signaling, p 315-327. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch36

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Immune Receptors
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MHC Class I
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Human immunodeficiency virus 1
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Figures

Image of FIGURE 1
FIGURE 1

Three-color analysis of CD69 expression in CD3 CD4 lymphocytes in unlysed whole-blood preparations from an HIV-1-seronegative individual (top panels), an HIV-infected individual with a CD69 response in the normal range (middle panels), and an HIV-infected individual with a low CD69 response to stimulation with anti-CD2/2R MAbs (bottom panels). T cells were gated as 90° light scatter, and CD3 staining was carried out (left panels). CD69 expression was measured in CD3 CD4 lymphocytes cultured for 4 h in medium alone, with a stimulatory anti-CD2/2R MAb combination, or with PMA (right panels). PMA will spontaneously activate all cells within this time period and is considered the positive control.

Citation: Currier J. 2006. T-Lymphocyte Activation and Cell Signaling, p 315-327. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch36
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Image of FIGURE 2
FIGURE 2

Kinetics of CD69 expression on CD4 T cells. Whole blood from an HIV-1-seronegative individual (A) and an HIV-infected patient (B) was cultured without further stimulus (⊠) or stimulated with soluble anti-CD2 MAb (▀), bead-bound anti-CD3 MAb (▴), or PMA (•). At the indicated times, flow cytometric measurements were performed on cells stained with anti-CD3, anti-CD4, and anti-CD69 MAbs. The percentage of cells positive for CD69 was calculated for cells in the CD3 CD4 gate. Five uninfected controls and five HIV-infected patients were studied. Results shown are from a representative HIV-1-seronegative individual and an HIV-infected patient with a depressed CD69 response.

Citation: Currier J. 2006. T-Lymphocyte Activation and Cell Signaling, p 315-327. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch36
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Image of FIGURE 3
FIGURE 3

Representative histograms show the measurement of the CD38 intensity from CD3 CD8 T cells. (A) T-cell gate population (CD3 cells) on a side scatter-versus-CD3 histogram; (B) CD38 median measured on a CD38-versus-CD8 histogram; (C) calibration curve of antibody binding capacity values versus histogram channel number (scale, 0 to 255). Median channel number (relative linear channel) is converted to a 256-standard channel number and then read from the calibration curve (C) to determine the antibody binding capacity. In this example, a median channel measurement of 22 was determined to represent an antibody binding capacity of 21,272.

Citation: Currier J. 2006. T-Lymphocyte Activation and Cell Signaling, p 315-327. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch36
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Image of FIGURE 4
FIGURE 4

Representative calcium assay graphics showing the measurement of the v/b ratio versus time. The first cursor indicates the resting phase, and the second cursor represents the responding phase. (A) Baseline curve with no stimulation added; (B) INDO-1-labeled cells stimulated with ionomycin (2 μg); (C) INDO-1-labeled cells stimulated with avidin after pretreatment of cells with anti-CD3 (see control section for details); (D) INDO-1-labeled cells stimulated with SDF-1 (100 nM) after 7 days of cell activation.

Citation: Currier J. 2006. T-Lymphocyte Activation and Cell Signaling, p 315-327. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch36
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Tables

Generic image for table
TABLE 1

Culture medium reagents (Gibco BRL) required for the protocol

Citation: Currier J. 2006. T-Lymphocyte Activation and Cell Signaling, p 315-327. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch36
Generic image for table
TABLE 2

Experimental nanomolar equivalents for final concentrations of stimulants

Citation: Currier J. 2006. T-Lymphocyte Activation and Cell Signaling, p 315-327. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch36
Generic image for table
TABLE 3

Chemokine test layout

Citation: Currier J. 2006. T-Lymphocyte Activation and Cell Signaling, p 315-327. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch36
Generic image for table
TABLE 4

Problems and corrective actions

Citation: Currier J. 2006. T-Lymphocyte Activation and Cell Signaling, p 315-327. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch36

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