Live-Cell Dynamics and the Role of Costimulation in Immunological Synapse Formation

Wetzel, Scott A.; McKeithan, Timothy W.; Parker, David C.

doi:10.4049/jimmunol.169.11.6092

Cited by 85 publications

(85 citation statements)

References 60 publications

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“…The MCC:I-E k chimera used for the B7 lo line included the GyrB domains attached to the cytoplasmic tail, while that used for the B7 hi line included green fluorescent protein. These cytoplasmic domains do not affect presentation of the peptide complex on the surface of the APCs to T cells (27) and data not shown. Transfectants were selected with 500 g/ml G418 active drug, and drugresistant cells were screened for I-E k surface expression by flow cytometry and cloned by limiting dilution.…”

Section: Methodsmentioning

confidence: 82%

“…Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum (Hyclone) and supplemented with 1 mM L-glutamine, sodium pyruvate (100 mg/ml), 5 ϫ 10 Ϫ5 M ␤-mercaptoethanol, essential and non-essential amino acids (Invitrogen), 100 units/ml penicillin G, 100 units/ml streptomycin, and 50 g/ml gentamycin) at 37°C with 5% CO 2 . Fibroblasts were transfected with a plasmid encoding an MCC:I-E k chimera (27). The MCC:I-E k chimera used for the B7 lo line included the GyrB domains attached to the cytoplasmic tail, while that used for the B7 hi line included green fluorescent protein.…”

Section: Methodsmentioning

confidence: 99%

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Ectopic B-Raf Expression Enhances Extracellular Signal-regulated Kinase (ERK) Signaling in T Cells and Prevents Antigen-presenting Cell-induced Anergy

Dillon

Karpitski

Wetzel

et al. 2003

Journal of Biological Chemistry

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T cells that receive stimulation through the T cell receptor (TCR) in the absence of costimulation become anergic and are refractory to subsequent costimulation. This unresponsiveness is associated with the constitutive activation of the small G protein, Rap1, and the lack of Ras-dependent activation of ERK. Recent studies suggest that Rap1 can activate the MAP kinase kinase kinase B-Raf that is either endogenously or ectopically expressed. Peripheral T cells generally do not express B-Raf; therefore, to test the hypothesis that ectopic expression of B-Raf could permit Rap1 to activate ERK signaling, we generated transgenic mice expressing BRaf within peripheral T cells. This converted Rap1 into an activator of ERK, to enhance ERK activation and proliferation following TCR engagement in the absence of costimulation. When T cells were incubated with engineered APCs presenting antigen on I-E k and expressing low levels of B7, they became anergic, displayed constitutive activation of Rap1, and were deficient in Ras and ERK activation. However, when incubated with the same APCs, T cells expressing the B-Raf transgene proliferated upon restimulation and displayed elevated ERK activation. Thus B-Raf expression and enhanced ERK activation is sufficient to prevent anergy in a model of APC-induced T cell anergy. However, studies using anti-TCR antibody-induced anergy showed that the ability of ERKs to reverse T cell anergy is dependent on the anergic model utilized.The MAP 1 kinase cascade governs multiple cellular processes in T cell function, including proliferation and differentiation. Activation of the MAP kinase ERK (extracellular signalregulated kinase) is required for IL-2 gene transcription and T cell proliferation via engagement of the T cell receptor (TCR) (1). In addition, the magnitude of ERK activation may regulate the response of T cells to TCR engagement (2, 3). Therefore, it is expected that both the magnitude and duration of ERK activation are tightly regulated in T cells.One potential mechanism of ERK regulation is the modulation of Ras signaling by the small G protein, Rap1. Rap1, a member of the Ras super-family, was identified as an antagonist of Ras dependent transformation in fibroblasts (4). In many cell types, including T cells, Rap1 has been shown to inhibit ERK activation by blocking Ras-dependent activation of the MAP kinase kinase kinase, Raf-1 (5-8). Rap1 is activated upon TCR engagement in the absence of costimulation (5, 9, 10) and can limit TCR-induced signals to ERK (5, 11). Interestingly, Rap1 is inhibited by the engagement of the CD28 costimulatory receptor (10, 12), suggesting that one of the functions of Rap1 activation may be to limit ERK activation in the absence of costimulation (5). Indeed, in models of T cell anergy elicited by stimulation through the TCR alone, Rap1 is constitutively active and may limit signaling downstream of Ras (13).Recently other functions of Rap1 have been identified, particularly the enhancement of T cell adhesion (14, 15), raising the possibility that R...

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Section: Methodsmentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Ectopic B-Raf Expression Enhances Extracellular Signal-regulated Kinase (ERK) Signaling in T Cells and Prevents Antigen-presenting Cell-induced Anergy

Dillon

Karpitski

Wetzel

et al. 2003

Journal of Biological Chemistry

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“…Efficient T cell activation critically depends on integrinmediated adhesion. Whereas adhesion to the cell surface protein ICAM-1 is crucial for T cell/APC interactions (18,19), ␤1 integrin-mediated adhesion to extracellular matrix proteins such as fibronectin determines the capacity of T cells to migrate and infiltrate inflamed tissues (28,29). As shown in Figure 2a, teriflunomide caused a significant and concentrationdependent reduction of CD3-induced T cell adhesion to ICAM-1.…”

Section: Resultsmentioning

confidence: 99%

“…First, due to TCR-mediated signals, LFA-1 alters its adhesive state mainly by enhanced avidity, which is controlled by the actin cytoskeleton (15)(16)(17). This process of "insideout signaling" leads to enhanced binding of LFA-1 to its ligand intercellular adhesion molecule 1 (ICAM-1) and thus adhesion to APCs (18,19). Second, ligand binding of LFA-1 itself transduces critical signals ("outside-in signaling"), which augment TCR/CD3-induced T cell adhesion, surface molecule expression, and interleukin production, and then lead to Th1 differentiation (20)(21)(22).…”

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confidence: 99%

Disruption of the interaction of T cells with antigen‐presenting cells by the active leflunomide metabolite teriflunomide: Involvement of impaired integrin activation and immunologic synapse formation

Zeyda

Poglitsch

Geyeregger

et al. 2005

Arthritis & Rheumatism

105

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Objective. Leflunomide, a potent diseasemodifying antirheumatic drug of the isoxazole class, exhibits antiinflammatory, antiproliferative, and immunosuppressive effects by largely unknown mechanisms, although alterations of pyrimidine synthesis have been proposed. Successful immune responsiveness requires T cell activation by interaction with antigen-presenting cells (APCs), and integrin activation and formation of an immunologic synapse (IS). In this study, we evaluated the impact of the active leflunomide metabolite teriflunomide on T cell integrin activation, evolution of the IS, and antigen-specific formation of stable T cell/ APC conjugates.Methods. Effects of pharmacologic concentrations of teriflunomide on CD3/CD28-and lymphocyte function-associated antigen 1-induced signal transduction and activation of primary human T cells were investigated. Furthermore, T cells were stimulated with superantigen-and antigen-pulsed APCs to study relocalization of molecules to the IS and T cell/APC conjugate formation.Results. Teriflunomide inhibited T cell receptor (TCR)/CD3-mediated calcium mobilization, but other critical T cell signaling events, including activation of MAPK and NF-B, remained unaltered. In contrast, inhibition of TCR/CD3-triggered ␤1,2 integrin avidity and integrin-mediated costimulation (outside-in signaling) by teriflunomide revealed a striking interference with integrin function that was independent of altered pyrimidine synthesis. Moreover, teriflunomide abolished molecule relocalization to the IS and induction of T cell/APC conjugates.Conclusion. These data show that the active metabolite of leflunomide prevents the interaction of T cells with APCs to form an IS. Since IS formation is crucial for eliciting an immune response, this novel mechanism could underlie the beneficial effects of leflunomide in immune-mediated disorders such as rheumatoid arthritis.

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“…Live cell imaging. Live T-cell interactions were imaged as described previously (45). One day prior to the experiment, 2.5 ϫ 10 5 fibroblasts were seeded onto 0.17-mm Delta T-cell culture dishes (Bioptechs, Butler, PA) in 1 ml of complete Dulbecco's modified Eagle medium.…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Small GTPase Rap1 and Extracellular Signal-Regulated Kinases by the Costimulatory Molecule CTLA-4

Dillon

Carey

Wetzel

et al. 2005

Molecular and Cellular Biology

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The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) is activated following engagement of the T-cell receptor and is required for interleukin 2 (IL-2) production and T-cell proliferation. This activation is enhanced by stimulation of the coreceptor CD28 and inhibited by the coreceptor CTLA-4. We show that the small G protein Rap1 is regulated in the opposite manner; it is inhibited by CD28 and activated by CTLA-4. Together, CD3 and CTLA-4 activate Rap1 in a sustained manner. To delineate T-cell function in the absence of Rap1 activity, we generated transgenic mice expressing Rap1GAP1, a Rap1-specific GTPaseactivating protein. Transgenic mice showed lymphadenopathy, and transgenic T cells displayed increased ERK activation, proliferation, and IL-2 production. More significantly, the inhibitory effect of CTLA-4 on T-cell function in Rap1GAP1-transgenic T cells was reduced. We demonstrate that CTLA-4 activates Rap1, and we propose that intracellular signals from CTLA-4 antagonize CD28, at least in part, at the level of Rap1.The activation of T cells requires antigen recognition through the T-cell receptor (TCR) and costimulation via the CD28 coreceptor, which binds the family of B7 ligands (B7-1 and B7-2) expressed on antigen-presenting cells (APCs) (35). Additional complexity comes with another coreceptor, CTLA-4, that also binds B7 and can antagonize the ability of CD28 to stimulate T-cell activation and interleukin 2 (IL-2) accumulation (25,27,28,42,44). Recent studies have suggested that inhibition of extracellular signal-regulated kinases (ERKs) may contribute to this action (6, 9, 38). ERKs are activated following costimulation by TCR/CD28 and are required for AP-1-dependent transcription of IL-2, a cytokine that is essential for T-cell proliferation (32, 46). Cross-linking anti-CTLA-4 antibodies (that activate CTLA-4) inhibit both ERK activation and IL-2 production following costimulation by TCR/CD28 (6, 9). The mechanism by which CTLA-4 inhibits ERK activation is not known.The small G protein Ras mediates TCR activation of ERKs (7). TCR stimulation triggers the activation of Ras via the Ras guanine nucleotide exchange factors (GEF) Cal DAG-GEF II (also called Ras-GRP1) (17) and SOS (47). Ras is a positive activator of the mitogen-activated protein (MAP) kinase kinase kinase Raf-1 that can phosphorylate and activate the MAP kinase kinase MEK, which, in turn, can activate ERK. Potential negative regulatory molecules are also activated by TCR stimulation. One of these is the small G protein Rap1 that, like Ras, can be inhibited by specific GTPase-activating proteins (Rap1GAPs). Rap1 is activated following stimulation of the TCR (1, 8) and, like Ras, is rapidly recruited to the plasma membrane upon activation (3). During CD28 costimulation, Rap1 activation is inhibited (8,22,33) and ERK signaling is enhanced (8). Conversely, T lymphocytes lacking the Rap1GAP Spa-1 show constitutively elevated Rap1 activity, diminished ERK activation, and a decreased response to TCR stimulation (21). ...

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Live-Cell Dynamics and the Role of Costimulation in Immunological Synapse Formation

Cited by 85 publications

References 60 publications

Ectopic B-Raf Expression Enhances Extracellular Signal-regulated Kinase (ERK) Signaling in T Cells and Prevents Antigen-presenting Cell-induced Anergy

Ectopic B-Raf Expression Enhances Extracellular Signal-regulated Kinase (ERK) Signaling in T Cells and Prevents Antigen-presenting Cell-induced Anergy

Disruption of the interaction of T cells with antigen‐presenting cells by the active leflunomide metabolite teriflunomide: Involvement of impaired integrin activation and immunologic synapse formation

Regulation of the Small GTPase Rap1 and Extracellular Signal-Regulated Kinases by the Costimulatory Molecule CTLA-4

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