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The CD3-gammadeltaepsilon and CD3-zeta/eta modules are each essential for allelic exclusion at the T cell receptor beta locus but are both dispensable for the initiation of V to (D)J recombination at the T cell receptor-beta, -gamma, and -delta loci.

Ardouin L, Ismaili J, Malissen B, Malissen M - J. Exp. Med. (1998)

Bottom Line: The pre-T cell receptor (TCR) associates with CD3-transducing subunits and triggers the selective expansion and maturation of T cell precursors expressing a TCR-beta chain.Furthermore, using mutant mice lacking both the CD3-epsilon and CD3-zeta/eta genes, we established that the CD3 gene products are dispensable for the onset of V to (D)J recombination (V, variable; D, diversity; J, joining) at the TCR-beta, TCR-gamma, and TCR-delta loci.Thus, the CD3 components are differentially involved in the sequential events that make the TCR-beta locus first accessible to, and later insulated from, the action of the V(D)J recombinase.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France.

ABSTRACT
The pre-T cell receptor (TCR) associates with CD3-transducing subunits and triggers the selective expansion and maturation of T cell precursors expressing a TCR-beta chain. Recent experiments in pre-Talpha chain-deficient mice have suggested that the pre-TCR may not be required for signaling allelic exclusion at the TCR-beta locus. Using CD3-epsilon- and CD3-zeta/eta-deficient mice harboring a productively rearranged TCR-beta transgene, we showed that the CD3-gammadeltaepsilon and CD3-zeta/eta modules, and by inference the pre-TCR/CD3 complex, are each essential for the establishment of allelic exclusion at the endogenous TCR-beta locus. Furthermore, using mutant mice lacking both the CD3-epsilon and CD3-zeta/eta genes, we established that the CD3 gene products are dispensable for the onset of V to (D)J recombination (V, variable; D, diversity; J, joining) at the TCR-beta, TCR-gamma, and TCR-delta loci. Thus, the CD3 components are differentially involved in the sequential events that make the TCR-beta locus first accessible to, and later insulated from, the action of the V(D)J recombinase.

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Assessment of TCR-β  transgene expression by RNA-PCR and intracytoplasmic staining. (A) CD25+ cells sorted from  TCR-β transgenic wild-type  thymuses (CD3-ε+/+ TCR-β)  and total thymocytes from  TCR-β transgenic CD3-εΔ5/Δ5  mice (CD3-εΔ5/Δ5 TCR-β) were  analyzed for the presence of transcripts originating from the P14  TCR-β transgene using the  RNA-PCR strategy depicted in  the bottom diagram. The P14  TCR-β cDNA is expressed under the control of the H-2Kb  promotor and IgH chain intronic  enhancer (EH). The 3′ end of the  P14 TCR-β cDNA is linked to  a genomic fragment of the human β globin gene that provides  both an intron and a polyadenylation sequence (24). Owing to  the presence of this intron, primers for PCR amplification can be chosen to distinguish amplification  products corresponding to transgene transcription (expected size: 0.8 kb)  from those resulting from adventitious DNA contamination (expected  size: 1.6 kb). Accordingly, an antisense primer specific for the 3′ untranslated region of the human β globin gene (primer 4) was used in combination with a sense primer (primer 3) straddling the sequence corresponding  to the third complementarity region of the P14 TCR-β gene. RNA extracted from nontransgenic CD3-εΔ5/Δ5 thymocytes was also included as a  negative control. A second pair of primers (denoted 1 and 2) was used in  parallel to detect both endogenous and transgenic transcripts containing  the TCR Cβ2 exon. The products resulting from amplification with primer  pairs 1 + 2 (TCR Cβ2) and 3 + 4 (TCR-β Tg) were gel fractionated,  blotted, and hybridized with a Cβ2-specific probe (p). The location of  specific primers are indicated by arrowheads and the transcription start  site of the TCR-β transgene by an arrow. Control PCR were set up in  parallel using a pair of primers specific for the actin gene to control for  the quantity and quality of RNA in each sample, run on agarose gel, and  revealed by ethidium bromide staining (Actin). (B) The presence of the  transgenic P14 TCR-β chain within the CD25+ subset present in CD3-εΔ5Δ5 transgenic β thymocytes was revealed by intracellular staining with  an antibody (F23.1) specific for the Vβ8 gene segment used by the P14  TCR β chain. RAG-1−/− thymocytes were also included as negative  controls. Cytoplasmic staining of the CD25+ compartment from nontransgenic CD3-εΔ5/Δ5 mice revealed <1% F23.1+ cells.
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Figure 3: Assessment of TCR-β transgene expression by RNA-PCR and intracytoplasmic staining. (A) CD25+ cells sorted from TCR-β transgenic wild-type thymuses (CD3-ε+/+ TCR-β) and total thymocytes from TCR-β transgenic CD3-εΔ5/Δ5 mice (CD3-εΔ5/Δ5 TCR-β) were analyzed for the presence of transcripts originating from the P14 TCR-β transgene using the RNA-PCR strategy depicted in the bottom diagram. The P14 TCR-β cDNA is expressed under the control of the H-2Kb promotor and IgH chain intronic enhancer (EH). The 3′ end of the P14 TCR-β cDNA is linked to a genomic fragment of the human β globin gene that provides both an intron and a polyadenylation sequence (24). Owing to the presence of this intron, primers for PCR amplification can be chosen to distinguish amplification products corresponding to transgene transcription (expected size: 0.8 kb) from those resulting from adventitious DNA contamination (expected size: 1.6 kb). Accordingly, an antisense primer specific for the 3′ untranslated region of the human β globin gene (primer 4) was used in combination with a sense primer (primer 3) straddling the sequence corresponding to the third complementarity region of the P14 TCR-β gene. RNA extracted from nontransgenic CD3-εΔ5/Δ5 thymocytes was also included as a negative control. A second pair of primers (denoted 1 and 2) was used in parallel to detect both endogenous and transgenic transcripts containing the TCR Cβ2 exon. The products resulting from amplification with primer pairs 1 + 2 (TCR Cβ2) and 3 + 4 (TCR-β Tg) were gel fractionated, blotted, and hybridized with a Cβ2-specific probe (p). The location of specific primers are indicated by arrowheads and the transcription start site of the TCR-β transgene by an arrow. Control PCR were set up in parallel using a pair of primers specific for the actin gene to control for the quantity and quality of RNA in each sample, run on agarose gel, and revealed by ethidium bromide staining (Actin). (B) The presence of the transgenic P14 TCR-β chain within the CD25+ subset present in CD3-εΔ5Δ5 transgenic β thymocytes was revealed by intracellular staining with an antibody (F23.1) specific for the Vβ8 gene segment used by the P14 TCR β chain. RAG-1−/− thymocytes were also included as negative controls. Cytoplasmic staining of the CD25+ compartment from nontransgenic CD3-εΔ5/Δ5 mice revealed <1% F23.1+ cells.

Mentions: RNA samples were extracted from total (CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β samples) or CD25high (sorted from the CD3-ε+/+ TCR-β sample) thymocytes using TRIzolTM (GIBCO BRL, Cergy Pontoise, France) as recommended by the manufacturer. Before conversion to cDNA, RNA samples were treated with DNAseI–RNAse free (Pharmacia). Conversion to cDNA was done on 1 μg of total RNA using the Ready-to-GoTM T-primed first strand kit (Pharmacia). 1/15 of each reaction was used for PCR amplification. The pair of Cβ2 primers used to detect transcripts incorporating the TCR Cβ2 exon was as described in reference 25. They are denoted as primers 1 and 2 in Fig. 3. The sequences of the other PCR primers used in these experiments were: CDR3 P14β: 5′-GTGATGCCGGGGGGCGGAACAC-3′; and β-globin 3′UT: 5′-GGGCATTAGCCACACCAGCCACCA-3′, and denoted in Fig. 3 as primers 3 and 4, respectively. The amplified products were analyzed on 1.5% agarose gel, transferred to nylon membrane (Gene Screen Plus; NEN Life Science Products, LeBlanc Mesnil, France), and hybridized using a 5′-kinased oligonucleotide (Cβ2A; reference 26).


The CD3-gammadeltaepsilon and CD3-zeta/eta modules are each essential for allelic exclusion at the T cell receptor beta locus but are both dispensable for the initiation of V to (D)J recombination at the T cell receptor-beta, -gamma, and -delta loci.

Ardouin L, Ismaili J, Malissen B, Malissen M - J. Exp. Med. (1998)

Assessment of TCR-β  transgene expression by RNA-PCR and intracytoplasmic staining. (A) CD25+ cells sorted from  TCR-β transgenic wild-type  thymuses (CD3-ε+/+ TCR-β)  and total thymocytes from  TCR-β transgenic CD3-εΔ5/Δ5  mice (CD3-εΔ5/Δ5 TCR-β) were  analyzed for the presence of transcripts originating from the P14  TCR-β transgene using the  RNA-PCR strategy depicted in  the bottom diagram. The P14  TCR-β cDNA is expressed under the control of the H-2Kb  promotor and IgH chain intronic  enhancer (EH). The 3′ end of the  P14 TCR-β cDNA is linked to  a genomic fragment of the human β globin gene that provides  both an intron and a polyadenylation sequence (24). Owing to  the presence of this intron, primers for PCR amplification can be chosen to distinguish amplification  products corresponding to transgene transcription (expected size: 0.8 kb)  from those resulting from adventitious DNA contamination (expected  size: 1.6 kb). Accordingly, an antisense primer specific for the 3′ untranslated region of the human β globin gene (primer 4) was used in combination with a sense primer (primer 3) straddling the sequence corresponding  to the third complementarity region of the P14 TCR-β gene. RNA extracted from nontransgenic CD3-εΔ5/Δ5 thymocytes was also included as a  negative control. A second pair of primers (denoted 1 and 2) was used in  parallel to detect both endogenous and transgenic transcripts containing  the TCR Cβ2 exon. The products resulting from amplification with primer  pairs 1 + 2 (TCR Cβ2) and 3 + 4 (TCR-β Tg) were gel fractionated,  blotted, and hybridized with a Cβ2-specific probe (p). The location of  specific primers are indicated by arrowheads and the transcription start  site of the TCR-β transgene by an arrow. Control PCR were set up in  parallel using a pair of primers specific for the actin gene to control for  the quantity and quality of RNA in each sample, run on agarose gel, and  revealed by ethidium bromide staining (Actin). (B) The presence of the  transgenic P14 TCR-β chain within the CD25+ subset present in CD3-εΔ5Δ5 transgenic β thymocytes was revealed by intracellular staining with  an antibody (F23.1) specific for the Vβ8 gene segment used by the P14  TCR β chain. RAG-1−/− thymocytes were also included as negative  controls. Cytoplasmic staining of the CD25+ compartment from nontransgenic CD3-εΔ5/Δ5 mice revealed <1% F23.1+ cells.
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Figure 3: Assessment of TCR-β transgene expression by RNA-PCR and intracytoplasmic staining. (A) CD25+ cells sorted from TCR-β transgenic wild-type thymuses (CD3-ε+/+ TCR-β) and total thymocytes from TCR-β transgenic CD3-εΔ5/Δ5 mice (CD3-εΔ5/Δ5 TCR-β) were analyzed for the presence of transcripts originating from the P14 TCR-β transgene using the RNA-PCR strategy depicted in the bottom diagram. The P14 TCR-β cDNA is expressed under the control of the H-2Kb promotor and IgH chain intronic enhancer (EH). The 3′ end of the P14 TCR-β cDNA is linked to a genomic fragment of the human β globin gene that provides both an intron and a polyadenylation sequence (24). Owing to the presence of this intron, primers for PCR amplification can be chosen to distinguish amplification products corresponding to transgene transcription (expected size: 0.8 kb) from those resulting from adventitious DNA contamination (expected size: 1.6 kb). Accordingly, an antisense primer specific for the 3′ untranslated region of the human β globin gene (primer 4) was used in combination with a sense primer (primer 3) straddling the sequence corresponding to the third complementarity region of the P14 TCR-β gene. RNA extracted from nontransgenic CD3-εΔ5/Δ5 thymocytes was also included as a negative control. A second pair of primers (denoted 1 and 2) was used in parallel to detect both endogenous and transgenic transcripts containing the TCR Cβ2 exon. The products resulting from amplification with primer pairs 1 + 2 (TCR Cβ2) and 3 + 4 (TCR-β Tg) were gel fractionated, blotted, and hybridized with a Cβ2-specific probe (p). The location of specific primers are indicated by arrowheads and the transcription start site of the TCR-β transgene by an arrow. Control PCR were set up in parallel using a pair of primers specific for the actin gene to control for the quantity and quality of RNA in each sample, run on agarose gel, and revealed by ethidium bromide staining (Actin). (B) The presence of the transgenic P14 TCR-β chain within the CD25+ subset present in CD3-εΔ5Δ5 transgenic β thymocytes was revealed by intracellular staining with an antibody (F23.1) specific for the Vβ8 gene segment used by the P14 TCR β chain. RAG-1−/− thymocytes were also included as negative controls. Cytoplasmic staining of the CD25+ compartment from nontransgenic CD3-εΔ5/Δ5 mice revealed <1% F23.1+ cells.
Mentions: RNA samples were extracted from total (CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β samples) or CD25high (sorted from the CD3-ε+/+ TCR-β sample) thymocytes using TRIzolTM (GIBCO BRL, Cergy Pontoise, France) as recommended by the manufacturer. Before conversion to cDNA, RNA samples were treated with DNAseI–RNAse free (Pharmacia). Conversion to cDNA was done on 1 μg of total RNA using the Ready-to-GoTM T-primed first strand kit (Pharmacia). 1/15 of each reaction was used for PCR amplification. The pair of Cβ2 primers used to detect transcripts incorporating the TCR Cβ2 exon was as described in reference 25. They are denoted as primers 1 and 2 in Fig. 3. The sequences of the other PCR primers used in these experiments were: CDR3 P14β: 5′-GTGATGCCGGGGGGCGGAACAC-3′; and β-globin 3′UT: 5′-GGGCATTAGCCACACCAGCCACCA-3′, and denoted in Fig. 3 as primers 3 and 4, respectively. The amplified products were analyzed on 1.5% agarose gel, transferred to nylon membrane (Gene Screen Plus; NEN Life Science Products, LeBlanc Mesnil, France), and hybridized using a 5′-kinased oligonucleotide (Cβ2A; reference 26).

Bottom Line: The pre-T cell receptor (TCR) associates with CD3-transducing subunits and triggers the selective expansion and maturation of T cell precursors expressing a TCR-beta chain.Furthermore, using mutant mice lacking both the CD3-epsilon and CD3-zeta/eta genes, we established that the CD3 gene products are dispensable for the onset of V to (D)J recombination (V, variable; D, diversity; J, joining) at the TCR-beta, TCR-gamma, and TCR-delta loci.Thus, the CD3 components are differentially involved in the sequential events that make the TCR-beta locus first accessible to, and later insulated from, the action of the V(D)J recombinase.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France.

ABSTRACT
The pre-T cell receptor (TCR) associates with CD3-transducing subunits and triggers the selective expansion and maturation of T cell precursors expressing a TCR-beta chain. Recent experiments in pre-Talpha chain-deficient mice have suggested that the pre-TCR may not be required for signaling allelic exclusion at the TCR-beta locus. Using CD3-epsilon- and CD3-zeta/eta-deficient mice harboring a productively rearranged TCR-beta transgene, we showed that the CD3-gammadeltaepsilon and CD3-zeta/eta modules, and by inference the pre-TCR/CD3 complex, are each essential for the establishment of allelic exclusion at the endogenous TCR-beta locus. Furthermore, using mutant mice lacking both the CD3-epsilon and CD3-zeta/eta genes, we established that the CD3 gene products are dispensable for the onset of V to (D)J recombination (V, variable; D, diversity; J, joining) at the TCR-beta, TCR-gamma, and TCR-delta loci. Thus, the CD3 components are differentially involved in the sequential events that make the TCR-beta locus first accessible to, and later insulated from, the action of the V(D)J recombinase.

Show MeSH