Analysis and Expression of a Cloned Pre-T Cell Receptor Gene

Saint‐Ruf, Claude; Ungewiss, K; Groettrup, Marcus; Bruno, Ludovica; Fehling, HJ; Boehmer, Harald von

doi:10.1126/science.7973703

Cited by 372 publications

(268 citation statements)

References 38 publications

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“…After RT with oligo-dT and AMV RT, the resulting cDNA was subjected to PCR. For the pre-TCR-α, the primers were: 5′, 5′-CTGCAACTGGGTCATGCTTC-3′ and 3′, 5′-TCA-GACGGGTGGGTAAGATC-3′ (29). Each PCR cycle consisted of 95°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute.…”

Section: Methodsmentioning

confidence: 99%

Metabolites from apoptotic thymocytes inhibit thymopoiesis in adenosine deaminase–deficient fetal thymic organ cultures

Thompson¹,

Wiele²,

Laurent³

et al. 2000

J. Clin. Invest.

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Murine fetal thymic organ culture was used to investigate the mechanism by which adenosine deaminase (ADA) deficiency causes T-cell immunodeficiency. C57BL/6 fetal thymuses treated with the specific ADA inhibitor 2′-deoxycoformycin exhibited features of the human disease, including accumulation of dATP and inhibition of S-adenosylhomocysteine hydrolase enzyme activity. Although T-cell receptor (TCR) Vβ gene rearrangements and pre-TCR-α expression were normal in ADA-deficient cultures, the production of αβ TCR + thymocytes was inhibited by 95%, and differentiation was blocked beginning at the time of β selection. In contrast, the production of γδ TCR + thymocytes was unaffected. Similar results were obtained using fetal thymuses from ADA gene-targeted mice. Differentiation and proliferation were preserved by the introduction of a bcl-2 transgene or disruption of the gene encoding apoptotic protease activating factor-1. The pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone also significantly lessened the effects of ADA deficiency and prevented the accumulation of dATP. Thus, ADA substrates accumulate and disrupt thymocyte development in ADA deficiency. These substrates derive from thymocytes that undergo apoptosis as a consequence of failing to pass developmental checkpoints, such as β selection.

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Section: Methodsmentioning

confidence: 99%

Metabolites from apoptotic thymocytes inhibit thymopoiesis in adenosine deaminase–deficient fetal thymic organ cultures

Thompson¹,

Wiele²,

Laurent³

et al. 2000

J. Clin. Invest.

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“…17). In early thymic development, a surrogate ␣-chain, termed pT␣, is expressed in double-negative (DN) thymocytes along with the TCR ␤-chain to form the pre-TCR (18,19). The pre-TCR functions to terminate additional ␤-chain rearrangements and fosters the transition from DN3 to DN4 developmental stages (20 -22).…”

Section: Importance Of the Cd3␥ Ectodomain Terminal ␤-Strand And Membmentioning

confidence: 99%

Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

Touma

Sun

Clayton

et al. 2007

The Journal of Immunology

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CD3εγ and CD3εδ are noncovalent heterodimers; each consists of Ig-like extracellular domains associated side-to-side via paired terminal β-strands that are linked to individual subunit membrane proximal stalk segments. CD3ε, CD3γ, and CD3δ stalks contain the RxCxxCxE motif. To investigate the functional importance of a CD3 stalk and terminal β-strand, we created a CD3γ double mutant CD3γC82S/C85S and a CD3γ β-strand triple mutant CD3γQ76S/Y78A/Y79A for use in retroviral transduction of lymphoid progenitors for comparison with CD3γwt. Although both mutant CD3γ molecules reduced association with CD3ε in CD3εγ heterodimers, CD3γQ76S/Y78A/Y79A abrogated surface TCR expression whereas CD3γC82S/C85S did not. Furthermore, CD3γC82S/C85S rescued thymic development in CD3γ−/− fetal thymic organ culture. However, the numbers of double-positive and single-positive thymocytes after CD3γC82S/C85S transduction were significantly reduced despite surface pre-TCR and TCR expression comparable to that of CD3γ−/− thymocytes transduced in fetal thymic organ culture with a retrovirus harboring CD3γwt cDNA. Furthermore, double-negative thymocyte development was perturbed with attenuated double-negative 3/double-negative 4 maturation and altered surface-expressed CD3εγ, as evidenced by the loss of reactivity with CD3γ N terminus-specific antisera. Single histidine substitution of either CD3γ stalk cysteine failed to restore CD3εγ association and conformation in transient COS-7 cell transfection studies. Thus, CD3γC82 and CD3γC85 residues likely are either reduced or form a tight intrachain disulfide loop rather than contribute to a metal coordination site in conjunction with CD3εC80 and CD3εC83. The implications of these results for CD3εγ and TCR structure and signaling function are discussed.

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“…The recently identified pTa chain has been shown to be expressed as a heterodimer with TCR p as part of the CD3-associated pre-TCR complex (23,30). In order to determine at which stage during T cell development TCR p and pTa are first expressed, we performed Northern blot analysis of early thymus subsets As shown in Fig.…”

Section: Expression Of Pta and Tcr F S Mrna In Immature Thymocytesmentioning

confidence: 99%

“…In situ hybridization was performed on single cells FACS-sorted directly onto microscope slides (31). The probes used were as follows: TCR Cp2 and p-actin (31); CD3 e, a 1200 bp Xba\-Bam\-U insert in pBS.KSII; CD3 y, a 700 bp EcoRI-H/ndlll insert in pBS.KSII; CD3 8, a 800 bp Xho\ insert in pBS.KSII; CD3 £, a 1100 bp EcoRI insert in pBS.SK (these latter four probes were the kind gift of Bernard and Marie Malissen, Marseille-Luminy, France); and pTa which is a 660 bp insert in Bluescript KS + made by PCR amplification of cDNA from CD25 + DN thymocytes using the published primer sequences (30). All probes were labeled by RNA transcription with either [a-PJUTP (for Northern blot) or [a-^SJUTP (for in situ).…”

Section: Northern Blot Analysis and In Situ Hybridizationmentioning

confidence: 99%

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Expression of genes encoding the pre-TCR and CD3 complex during thymus development

MacDonald

Malissen

1995

International Immunology

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The mature TCR is composed of a clonotypic heterodimer (ap" or 76) associated with the Invariant CD3 components (y, 8, e and Q. There is now considerable evidence that more immature forms of the TCR-CD3 complex (consisting of either CD3 alone or CD3 associated with a heterodimer of TCR p and pre-Ta) can be expressed at the cell surface on early thymocytes. These pre-TCR complexes are believed to be necessary for the ordered progression of early T cell development. We have analyzed in detail the expression of both the pre-TCR and CD3 complex at various stages of adult thymus development. Our data indicate that all CD3 components are already expressed at the mRNA level by the earliest identifiable (CD4 to ) thymic precursor. In contrast, genes encoding the pre-TCR complex (pre-Ta and fully rearranged TCR fJ) are first expressed at the CD44'°CD25 + CD4~CD8~ stage. Detectable surface expression of both CD3 and TCR f) are delayed relative to expression of the corresponding genes, suggesting the existence of other (as yet unidentified) components of the pre-TCR complex.

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Analysis and Expression of a Cloned Pre-T Cell Receptor Gene

Cited by 372 publications

References 38 publications

Metabolites from apoptotic thymocytes inhibit thymopoiesis in adenosine deaminase–deficient fetal thymic organ cultures

Metabolites from apoptotic thymocytes inhibit thymopoiesis in adenosine deaminase–deficient fetal thymic organ cultures

Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

Expression of genes encoding the pre-TCR and CD3 complex during thymus development

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