The Novel Murine CD4+CD8+ Thymocyte Cell Line Exhibits Lineage Commitment into Both CD4+ and CD8+ T Cells by Altering the Intensity and the Duration of Anti-CD3 Stimulation In Vitro

Nishida, Takashi; Matsuki, Yasunori; Ono, Takeshi; Oguma, Takemi; Tsujimoto, Kyoko; Satō, Masaki; Tadakuma, Takushi

doi:10.4049/jimmunol.172.11.6634

Cited by 8 publications

(6 citation statements)

References 36 publications

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“…1), we hypothesized that DP thymocytes were not efficiently transitioning from the DP to the CD4SP stage due to inefficient positive selection. Positive selection is associated with changes in the expression of surface molecules, including an increase of CD69 and a decrease of CD24 (27, 28). CD69 expression was lower and CD24 expression was higher on both n3.L2 + PKCθ -/- DP and n3.L2 + PKCθ -/- CD4SP thymocytes, compared to n3.L2 + WT DP and n3.L2 + WT CD4SP thymocytes (Fig.…”

Section: Resultsmentioning

confidence: 99%

Protein Kinase C-θ Is Required for Efficient Positive Selection

Morley

Weber

Kao

et al. 2008

The Journal of Immunology

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Protein kinase C-θ (PKCθ) is critical for TCR-initiated signaling in mature T cells, but initial reports found no requirement for PKCθ in thymocyte development. Thymocytes and peripheral T cells utilize many of the same signaling components and, given the significant role of PKCθ in peripheral T cells, it was surprising that it was not involved at all in TCR signaling in thymocytes. We decided to re-evaluate the role of PKCθ in thymocyte development using the well-characterized class II-restricted n3.L2 TCR-transgenic TCR model. Analysis of n3.L2 PKCθ−/− mice revealed a defect in thymocyte-positive selection, resulting in a 50% reduction in the generation of n3.L2 CD4 single-positive thymocytes and n3.L2 CD4 mature T cells. Competition between n3.L2 WT and n3.L2 PKCθ−/− thymocytes in bone marrow chimeras revealed a more dramatic defect, with a >80% reduction in generation of n3.L2 CD4 single-positive thymocytes derived from PKCθ−/− mice. Inefficient positive selection of n3.L2 PKCθ−/− CD4 single-positive cells resulted from “weaker” signaling through the TCR and correlated with diminished ERK activation. The defect in positive selection was not complete in the PKCθ−/− mice, most likely accounted for by compensation by other PKC isoforms not evident in peripheral cells. Similar decreased positive selection of both CD4 and CD8 single-positive thymocytes was also seen in nontransgenic PKCθ−/− mice. These findings now place PKCθ as a key signaling molecule in the positive selection of thymocytes as well as in the activation of mature T cells.

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Section: Resultsmentioning

confidence: 99%

Protein Kinase C-θ Is Required for Efficient Positive Selection

Morley

Weber

Kao

et al. 2008

The Journal of Immunology

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“…Flow cytometric analysis on the ␣␤ TCR-expressing DP (257-20-109) cell line (21), and the ␥␦ TCR expressing B9 cell line (22) indicates that 17A2 and 2C11 binding affinities are similar (Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

The αβ T Cell Receptor Is an Anisotropic Mechanosensor

Kim

Takeuchi

Sun

et al. 2009

Journal of Biological Chemistry

368

438

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Thymus-derived lymphocytes protect mammalian hosts against virus-or cancer-related cellular alterations through immune surveillance, eliminating diseased cells. In this process, T cell receptors (TCRs) mediate both recognition and T cell activation via their dimeric ␣␤, CD3⑀␥, CD3⑀␦, and CD3 subunits using an unknown structural mechanism. Here, site-specific binding topology of anti-CD3 monoclonal antibodies (mAbs) and dynamic TCR quaternary change provide key clues. Agonist mAbs footprint to the membrane distal CD3⑀ lobe that they approach diagonally, adjacent to the lever-like C␤ FG loop that facilitates antigen (pMHC)-triggered activation. In contrast, a non-agonist mAb binds to the cleft between CD3⑀ and CD3␥ in a perpendicular mode and is stimulatory only subsequent to an external tangential but not a normal force (ϳ50 piconewtons) applied via optical tweezers. Specific pMHC but not irrelevant pMHC activates a T cell upon application of a similar force. These findings suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into a biochemical signal upon specific pMHC ligation during immune surveillance. Activating anti-CD3 mAbs mimic this force via their intrinsic binding mode. A common TCR quaternary change rather than conformational alterations can better facilitate structural signal initiation, given the vast array of TCRs and their specific pMHC ligands. The T cell receptor (TCR)3 is a multimeric transmembrane complex composed of a disulfide-linked antigen binding clonotypic heterodimer (␣␤ or ␥␦) in non-covalent association with the signal-transducing CD3 subunits (CD3⑀␥, CD3⑀␦, and CD3) (reviewed in Ref. 1). TCR signaling via CD3 dimers evokes T cell lineage commitment and repertoire selection during development, maintains the peripheral T cell pool, and further differentiates naïve T cells into effector or memory cell populations upon immune stimulation (2-5). The interaction between an Fab-like ␣␤ TCR heterodimer and an antigenic peptide bound to a major histocompatibility complex molecule (pMHC) initiates a cascade of downstream signaling events via the immunoreceptor tyrosine-based activation motif elements in the cytoplasmic tails of the associated CD3 subunits (6 -9). The length of these CD3 cytoplasmic tails is substantial, relative to those of the TCR ␣ and ␤ chains (6, 7).How recognition of pMHC by a weakly interacting (ϳ1-100 M K d ) clonotypic heterodimer on the T cell surface evokes intracellular signaling via the adjacent CD3 components remains undefined (1). Solution structures of CD3⑀␥ and CD3⑀␦ heterodimers reveal a unique side-to-side hydrophobic interface with conjoined ␤-sheets involving the G-strands of the two Ig-like ectodomains of the pair (10, 11). The squat and rigid CD3 connecting segments contrast sharply with the long and flexible TCR ␣ and ␤ connecting peptides linking their respective constant domains to the transmembrane segments.To investigate the basis of signal transduction involving the ectodomain components within the TCR membrane complex,...

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“…The DP257–20-109 thymocyte-like cell line (16) was used as the basis for the stable expression of large amounts of full-length, transmembrane plexin-D1 and myristoylated cytoplasmic domain plexin-D1 (parental cell line provided by Dr. Takushi Tadakuma). The surface antigen phenotypic profile of DP257–20-109 is characteristic of a pre-selection DP thymocyte (fig.…”

Section: Methodsmentioning

confidence: 99%

Regulation of thymocyte trafficking by Tagap, a GAP domain protein linked to human autoimmunity

et al. 2018

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Multiple autoimmune pathologies are associated with single-nucleotide polymorphisms of the human gene , which encodes TAGAP, a guanosine triphosphatase (GTPase)-activating protein. We showed in mice that Tagap-mediated signaling by the sema3E/plexin-D1 ligand-receptor complex attenuates thymocytes' adhesion to the cortex through their β-containing integrins. By promoting thymocyte detachment within the cortex of the thymus, Tagap-mediated signaling enabled their translocation to the medulla, which is required for continued thymic selection. Tagap physically interacted with the cytoplasmic domain of plexin-D1 and directly stimulated the activity and signaling of the GTPase RhoA. In addition, Tagap indirectly mediated the activation of Cdc42 in response to the binding of sema3E to plexin-D1. Both RhoA and Cdc42 are key mediators of cytoskeletal and integrin dynamics in thymocytes. Knockdown of Tagap in mice suppressed the sema3E- and plexin-D1-mediated release of thymocytes that adhered within the cortex through β-containing integrins. This suppression led to the impaired translocation of thymocytes from the cortex to the medulla and resulted in the formation of ectopic medullary structures within the thymic cortex. Our results suggest that variation modulates the risk of autoimmunity by altering thymocyte migration during thymic selection.

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The Novel Murine CD4+CD8+ Thymocyte Cell Line Exhibits Lineage Commitment into Both CD4+ and CD8+ T Cells by Altering the Intensity and the Duration of Anti-CD3 Stimulation In Vitro

Cited by 8 publications

References 36 publications

Protein Kinase C-θ Is Required for Efficient Positive Selection

Protein Kinase C-θ Is Required for Efficient Positive Selection

The αβ T Cell Receptor Is an Anisotropic Mechanosensor

Regulation of thymocyte trafficking by Tagap, a GAP domain protein linked to human autoimmunity

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