Flow Cytometric Analysis of Kinase Signaling Cascades

Perez, Omar D.; Krutzik, Peter O.; Nolan, Garry P.

doi:10.1385/1-59259-773-4:067

Cited by 64 publications

(82 citation statements)

References 16 publications

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“…The T cell clone JKF6, specific for the melanoma differentiation Ag MART-1 [27][28][29][30][31][32][33][34][35] , was derived from tumor-infiltrating lymphocytes, as previously described (14). These T cells were expanded and activated by using 30 ng/ ml mAb OKT-3, irradiated PBMCs from three donors, and 500 IU/ml recombinant human IL-2.…”

Section: Cellsmentioning

confidence: 99%

“…1). Briefly, a specific TCR-like clonotypic mAb that recognizes the MART-1 [27][28][29][30][31][32][33][34][35] peptide, only when in complex with the HLA-A2 molecule, was used (16,17). JY cells were pulsed with different peptide concentrations, ranging from 500 mM down to 100 pM.…”

Section: Ag Quantitationmentioning

confidence: 99%

“…JY cells were stained with 1 mM PKH26 (Sigma) and pulsed with 500 mM, 100 nM, or 100 pM MART-1 [27][28][29][30][31][32][33][34][35] peptide for 1.5 h. CTLs were stained with 1 mM CFSE and 1 mM Indo-1 for 45 min in CTL culture media at 37˚C. All cells were washed thoroughly with media, resuspended in HBSS (Biological Industries), and incubated at 37˚C.…”

Section: Calcium Flux Measurementmentioning

confidence: 99%

“…To this end, CTLs were stained with both the calcium indicator Indo-1 and CFSE. CTLs were cocultured with PKH26-stained target cells pulsed with MART-1 [27][28][29][30][31][32][33][34][35] peptide at concentrations yielding presentation of 0, 20, 100, or 1200 pMHC-I complexes/cell. Conjugates of CTLs with APCs were analyzed based on their double-positive CFSE + PKH26 + -staining properties, and intracellular calcium was determined ( Fig.…”

Section: Ag-induced Hyporesponsiveness In Ctls Is Not Caused By Impaimentioning

confidence: 99%

“…To evaluate the role of PD-1 in Ag-induced hyporesponsiveness, JKF6 T cells were cocultured with target cells pulsed with MART-1 [27][28][29][30][31][32][33][34][35] peptide in a dose-dependent manner. This was followed by measuring cytotoxicity, as well as surface expression of PD-1.…”

Section: Ag-induced Hyporesponsiveness In Ctls Is Not Caused By Impaimentioning

confidence: 99%

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Antigen-Dependent Integration of Opposing Proximal TCR-Signaling Cascades Determines the Functional Fate of T Lymphocytes

Wolchinsky

Hod-Marco

Oved

et al. 2014

The Journal of Immunology

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T cell anergy is a key tolerance mechanism to mitigate unwanted T cell activation against self by rendering lymphocytes functionally inactive following Ag encounter. Ag plays an important role in anergy induction where high supraoptimal doses lead to the unresponsive phenotype. How T cells “measure” Ag dose and how this determines functional output to a given antigenic dose remain unclear. Using multiparametric phospho-flow and mass cytometry, we measured the intracellular phosphorylation-dependent signaling events at a single-cell resolution and studied the phosphorylation levels of key proximal human TCR activation- and inhibition-signaling molecules. We show that the intracellular balance and signal integration between these opposing signaling cascades serve as the molecular switch gauging Ag dose. An Ag density of 100 peptide–MHC complexes/cell was found to be the transition point between dominant activation and inhibition cascades, whereas higher Ag doses induced an anergic functional state. Finally, the neutralization of key inhibitory molecules reversed T cell unresponsiveness and enabled maximal T cell functions, even in the presence of very high Ag doses. This mechanism permits T cells to make integrated “measurements” of Ag dose that determine subsequent functional outcomes.

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Section: Cellsmentioning

confidence: 99%

Section: Ag Quantitationmentioning

confidence: 99%

Section: Calcium Flux Measurementmentioning

confidence: 99%

Section: Ag-induced Hyporesponsiveness In Ctls Is Not Caused By Impaimentioning

confidence: 99%

Section: Ag-induced Hyporesponsiveness In Ctls Is Not Caused By Impaimentioning

confidence: 99%

See 3 more Smart Citations

Antigen-Dependent Integration of Opposing Proximal TCR-Signaling Cascades Determines the Functional Fate of T Lymphocytes

Wolchinsky

Hod-Marco

Oved

et al. 2014

The Journal of Immunology

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Single‐Cell Phospho‐Protein Analysis by Flow Cytometry

et al. 2007

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This protocol describes methods for monitoring intracellular phosphorylation-dependent signaling events on a single-cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho-specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagnostic or drug efficacy information can be retrieved by discovering how a disease affects the ability of cells to respond to growth factors. Basic scientists can use this technique to analyze signaling events in cell lines and human or murine primary cells, including rare populations, like B1 cells or stem cells. This technique has broad applications to take standard biochemical analysis into primary cells to garner valuable information about signaling events in physiologic settings.

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Multiparameter Analysis of Intracellular Phosphoepitopes in Immunophenotyped Cell Populations by Flow Cytometry

et al. 2005

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This unit presents protocols for measuring intracellular phosphoepitopes by flow cytometry for biochemical investigations in both human and murine primary cells as well as in cell lines. Conventional methods that require cellular lysis cannot discriminate between proteins from different cellular subsets. Intracellular detection of phosphorylated and nonphosphorylated signaling molecules, on the other hand, has recently exposed the heterogeneity that can be observed upon signal transduction. Although staining methodologies for cytokines and cell cycle antigens have been appreciated for years, detection of phosphorylated molecules presents several new challenges, including generation of reagents and details of the staining technique. As these techniques are adapted to new applications, the protocols continue to be refined. This unit describes signal amplification of intracellular signals upon detergent‐based permeabilizations, staining protocol for adherent cells, methodology for whole‐blood staining, and multiparameter staining procedures for surface and intracellular antigens.

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Flow Cytometric Analysis of Kinase Signaling Cascades

Cited by 64 publications

References 16 publications

Antigen-Dependent Integration of Opposing Proximal TCR-Signaling Cascades Determines the Functional Fate of T Lymphocytes

Antigen-Dependent Integration of Opposing Proximal TCR-Signaling Cascades Determines the Functional Fate of T Lymphocytes

Single‐Cell Phospho‐Protein Analysis by Flow Cytometry

Multiparameter Analysis of Intracellular Phosphoepitopes in Immunophenotyped Cell Populations by Flow Cytometry

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