Rapid <i>in vivo</i> analysis of mutant forms of the LAT adaptor using <i>Pax5‐Lat</i> double‐deficient pro‐B cells

Ardouin, Laurence; Rolink, Antonius; Mura, Anne-Marie; Gommeaux, Julien; Melchers, Fritz; Busslinger, Meinrad; Malissen, Bernard

doi:10.1002/eji.200425836

Cited by 3 publications

(3 citation statements)

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“…Previously, the Pax5‐deficient proB cell system has successfully been used to analyze mutant forms of the LAT adaptor in vivo 37. Herein, we have demonstrated that TCR‐β chains isolated from γδ T cells can very efficiently participate in αβ T‐cell development and can give rise to functional T cells.…”

Section: Discussionmentioning

confidence: 93%

TCR‐β chains derived from peripheral γδ T cells can take part in αβ T‐cell development

et al. 2008

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Between 10 and 20% of the peripheral cd T cells express cytoplasmic TCR-b proteins, but whether such TCR-b chains can partake in ab T-cell development has never been systematically investigated. Therefore, we reconstituted the T-cell compartment of CD3e-deficient mice with Pax5-TCR-b deficient proB cells expressing, via a retroviral vector, TCR-b chains from either peripheral cd or ab T cells. Recipient thymi reconstituted with proB cells containing empty vector were small (o15 Â 10 6 cells), contained few cd T but no ab T cells. In contrast, thymi from mice receiving proB cells containing cd or ab T-cellderived TCR-b chains contained 80-130 Â 10 6 cells, and showed a normal CD4, CD8 and ab TCR expression pattern. However, regardless of the source of TCR-b chain, reconstituted mice rapidly showed signs of autoimmunity dying 5-15 wk following reconstitution. Autoimmune disease induction could be prevented by co-transfer of Treg cells thereby allowing the functionality of the generated T cells to be assessed. Results obtained show that TCR-b chains from cd T cells can efficiently take part in ab T-cell development. The implications of these findings for cd T-cell development will be discussed.Key words: ab T cells . gd T cells . TCR-b chains . pre-TCR . Thymus Supporting Information available online Introduction T-cell development takes place in the thymus from progenitors of bone marrow origin. By now it is generally believed that a cell called a thymus settling progenitor (TSP), characterized by the expression of CD44, CD117, CD135 and the chemokine receptor CCR9 [1][2][3][4] is the bone marrow cell that enters the thymus. However, transplantation experiments in T-cell-deficient mice have indicated that other progenitors can enter the thymus and may take part in T-cell development [2,5,6].Early T-cell progenitors in the thymus lack CD4 and CD8 expression and are therefore called double negative (DN) cells. Based on the differential expression of CD44, CD25 and CD117, DN cells can be subdivided into DN1-4 subpopulations. Thus DN1 cells are CD44 + , CD25 À and CD117 High and within this population, TSP are included as a CD135 + subpopulation [2,4,7,8]. DN1 cells differentiate through a CD44 + , CD25 + , CD117 high DN2 stage to become CD44 À , CD25 + , CD117 À/low DN3 cells [8].Ã These authors contributed equally to this work. 3520Both DN1 and DN2 cells still possess multi-lineage developmental potential, including that for NK, dendritic and myeloid cells [9][10][11] whereas B-cell lineage potential seems to be restricted to the TSP population within the DN1 subset [1,2,4,7]. By largely unknown mechanisms, commitment to the T-cell lineage is acquired at the DN2 to DN3 cell transition [9,12]. However, cells at the DN3 stage still have the option to develop into either ab or gd T-cell lineage cells [13][14][15]. A relatively large number of studies have indicated that the expression of the pre-TCR plays a crucial role in the development of ab T cells [14][15][16]. Recent, elegant, single cell studies have indicated ...

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Section: Discussionmentioning

confidence: 93%

TCR‐β chains derived from peripheral γδ T cells can take part in αβ T‐cell development

et al. 2008

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“…Samelson and coworkers [12] showed that mutation of this single tyrosine affects none of the intracellular signals triggered upon TCR engagement. Moreover, using a system in which Rag-deficient mice were reconstituted with T cell precursors expressing the LAT Y235F mutant (in which tyrosine 235, the equivalent to tyrosine 226 in mice, was substituted by phenylalanine), this mutant form of LAT was capable of reconstituting normal T cell development [43]. Therefore, it is likely that the specific signaling phenotype observed in JCaM2.5 cells expressing mutant LAT 5S/A cannot be fully recapitulated by the expression of LAT molecules expressing a mutation of tyrosine 226, suggesting that the conserved serine motifs may have a direct role in recruiting some effectors or adaptors.…”

Section: Discussionmentioning

confidence: 98%

Serine residues in the LAT adaptor are essential for TCR-dependent signal transduction

Martínez-Florensa

García-Blesa

Yélamos

et al. 2010

Journal of Leukocyte Biology

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The adaptor protein LAT has a prominent role in the transduction of intracellular signals elicited by the TCR/CD3 complex. Upon TCR engagement, LAT becomes tyrosine-phosphorylated and thereby, recruits to the membrane several proteins implicated in the activation of downstream signaling pathways. However, little is known about the role of other conserved motifs present in the LAT sequence. Here, we report that the adaptor LAT contains several conserved serine-based motifs, which are essential for proper signal transduction through the TCR. Mutation of these serine motifs in the human T cell line Jurkat prevents proper calcium influx, MAPK activation, and IL-2 production in response to TCR/CD3 stimulation. Moreover, this mutant form of LAT has a reduced ability to bind to PLC-γ1 and SLP-76, although phosphorylation of tyrosine residues 132, 171, and 191 is not decreased, raising a possible role for the serine-based motifs of LAT for the binding of important partners. The functional role of LAT serine-based motifs in signal transduction could be mediated by an effect on tyrosine phosphorylation, as their mutation significantly diminishes the phosphorylation of tyrosine residue 226. In addition, these serine motifs seem to have a regulatory role, given that upon their mutation, ZAP-70 shows enhanced phosphorylation. Therefore, the LAT serine-based motifs likely regulate signaling pathways that are essential for T cell physiology.

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“…B. Malissen (Marseille, France) discussed the activation pathway that is initiated by TCR ligation. When TCR encounters an antigen presented by an MHC protein, the tyrosine kinase Zap70 is activated, the linker for activation of T cells (LAT)—a raft‐anchored molecule—is phosphorylated, and a platform for further molecular recruitment is created (Ardouin et al , 2005). Malissen likens LAT to “a protein fishing line”; flexible and extended in the cytosol to allow a large capture radius.…”

Section: Introductionmentioning

confidence: 99%

New frontiers in immunology

Beutler

Casanova

2005

EMBO Reports

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Rapid in vivo analysis of mutant forms of the LAT adaptor using Pax5‐Lat double‐deficient pro‐B cells

Cited by 3 publications

References 29 publications

TCR‐β chains derived from peripheral γδ T cells can take part in αβ T‐cell development

TCR‐β chains derived from peripheral γδ T cells can take part in αβ T‐cell development

Serine residues in the LAT adaptor are essential for TCR-dependent signal transduction

New frontiers in immunology

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