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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.

Show MeSH
A model accounting  for the role of the pre-TCR/ CD3 complex in the establishment of allelic exclusion at the  TCR-β locus. Upon entering  the CD44−/low CD25+ compartment, the TCR Vβ gene segment cluster [(V)n] become  accessible to the V(D)J recombinase (dashed lines sandwiching the  TCR-β alleles a and b). At that  stage of development, the pTα  and CD3 components of the  pre-TCR are already available  and it is the TCR-β polypeptides that constitute the rate limiting factor in the assembly of the  pre-TCR/CD3 complex. From a  cohort of nine CD44−/low CD25+  triple negative thymocytes, three  are expected to produce a functional Vβ gene (VDJ+) as a result  of their first attempt of rearrangement (step 1, see reference 29).  The resulting TCR-β polypeptide participates in the assembly  of a pre-TCR complex (step 2). As soon as assembled, this complex triggers (step 3) the transition beyond the CD44−/low CD25+ stage and activates a  negative feedback loop that will close the accessibility of the second, partially rearranged, allele (allele b) to the V(D)J recombinase (continuous lines sandwiching the TCR-β alleles), thereby restricting such a T cell to the expression of only a single TCR-β chain allele. The p56lck kinase (lck) constitutes one  of the effector operating downstream of the pre-TCR/CD3 complex since the overexpression of a catalytically active form of p56lck inhibits endogenous  Vβ to DβJβ rearrangements while inducing coincidently the transition to the DP stage (30). As proposed previously (29), time delay along this negative  feedback loop, and/or the existence of a few cells in which Vβ to DβJβ rearrangements can be attempted quasisimultaneously on both β alleles, may explain the presence of rare cells with two productively rearranged TCR-β alleles (52, 53). Based on the comparison of TCR-β transgenic, p56lck−/−, and  TCR-β transgenic, CD3-ζ/η−/− mice (see Discussion), it is tempting to speculate that TCR-β gene allelic exclusion is brought about via two contingent pathways. One of which (step 4b), by inducing cell proliferation and DNA replication, enables the reprogrammation of the chromatin structure at  the TCR-β locus, and thereby permits factor(s) induced by the second pathway (step 5) to act and render the TCR-β locus inaccessible to further V(D)J  recombination. Note that the degradation of RAG-2, which results from cyclin-dependent kinase phosphorylation (loop 4a; reference 38) and occurs  during the burst of divisions associated with the transition from the DN to the DP stage, appears to constitute a fail-safe mechanism not essential for the  execution of TCR-β allelic exclusion (39).
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Figure 9: A model accounting for the role of the pre-TCR/ CD3 complex in the establishment of allelic exclusion at the TCR-β locus. Upon entering the CD44−/low CD25+ compartment, the TCR Vβ gene segment cluster [(V)n] become accessible to the V(D)J recombinase (dashed lines sandwiching the TCR-β alleles a and b). At that stage of development, the pTα and CD3 components of the pre-TCR are already available and it is the TCR-β polypeptides that constitute the rate limiting factor in the assembly of the pre-TCR/CD3 complex. From a cohort of nine CD44−/low CD25+ triple negative thymocytes, three are expected to produce a functional Vβ gene (VDJ+) as a result of their first attempt of rearrangement (step 1, see reference 29). The resulting TCR-β polypeptide participates in the assembly of a pre-TCR complex (step 2). As soon as assembled, this complex triggers (step 3) the transition beyond the CD44−/low CD25+ stage and activates a negative feedback loop that will close the accessibility of the second, partially rearranged, allele (allele b) to the V(D)J recombinase (continuous lines sandwiching the TCR-β alleles), thereby restricting such a T cell to the expression of only a single TCR-β chain allele. The p56lck kinase (lck) constitutes one of the effector operating downstream of the pre-TCR/CD3 complex since the overexpression of a catalytically active form of p56lck inhibits endogenous Vβ to DβJβ rearrangements while inducing coincidently the transition to the DP stage (30). As proposed previously (29), time delay along this negative feedback loop, and/or the existence of a few cells in which Vβ to DβJβ rearrangements can be attempted quasisimultaneously on both β alleles, may explain the presence of rare cells with two productively rearranged TCR-β alleles (52, 53). Based on the comparison of TCR-β transgenic, p56lck−/−, and TCR-β transgenic, CD3-ζ/η−/− mice (see Discussion), it is tempting to speculate that TCR-β gene allelic exclusion is brought about via two contingent pathways. One of which (step 4b), by inducing cell proliferation and DNA replication, enables the reprogrammation of the chromatin structure at the TCR-β locus, and thereby permits factor(s) induced by the second pathway (step 5) to act and render the TCR-β locus inaccessible to further V(D)J recombination. Note that the degradation of RAG-2, which results from cyclin-dependent kinase phosphorylation (loop 4a; reference 38) and occurs during the burst of divisions associated with the transition from the DN to the DP stage, appears to constitute a fail-safe mechanism not essential for the execution of TCR-β allelic exclusion (39).

Mentions: Our data also bear on the causal relationships between pre-TCR–induced cell proliferation and the establishment of TCR-β allelic exclusion. It has been suggested that preTCR–induced cell cycle progression is essential for the establishment of allelic exclusion at the TCR-β locus (36–39; see also references 40 and 41 in the case of B cell development). As outlined in Fig. 9, one or more rounds of DNA replication are speculated to enable the reprogrammation of the chromatin structure of the TCR-β loci and make them inaccessible to the V(D)J recombinase. According to that model, the lack of TCR-β allelic exclusion observed in the CD3-εΔ5/Δ5 thymuses would be fully accounted for by the fact that their TCR-β pTα heterodimers are prevented from inducing cell cycle entry. Mice carrying a mutation in the lck gene display a pronounced thymic atrophy associated with a dramatic reduction in the number of DP cells (42). In these mutant mice, TCR-β gene allelic exclusion is not severely compromised as the presence of a productively rearranged TCR-β transgene resulted in an almost complete inhibition of endogenous TCR-β gene rearrangements (43). Considering that TCR-β transgenic, CD3-ζ/η−/− thymuses display the same composition and cellularity as TCR-β transgenic, lck−/− thymuses (compare our data with those of Wallace et al., reference 43), it came as a surprise to find that there was in the former a clear dissociation between the transition to the DP stage and the establishment of TCR-β gene allelic exclusion. Thus, in the absence of CD3-ζ/η subunit, TCR-β selection may have led to differentiation rather than proliferation and, consistent with the above model, resulted in the lack of TCR-β gene allelic exclusion. However, the frequency of dividing early DP cells is only slightly smaller in CD3-ζ/η−/− mice than in wild-type littermates, indicating that CD3-ζ/η–less pre-TCR complexes are still capable of triggering cell cycle entry (9). Collectively, these observations suggest that burst of cell divisions induced by the pre-TCR may be enabling rather than inductive for the establishment of TCR-β gene allelic exclusion, and that the pre-TCR is likely to contribute additional signals to effect TCR-β gene allelic exclusion. According to that view and under physiological conditions, the signals emanating from both the lck– and CD3-ζ/η–less pre-TCR complexes suffice to trigger cell cycle entry and CD4/CD8 expression, whereas only those emanating from the former can reach the higher threshold plausibly required to activate the regulatory loop required for mediating allelic exclusion (denoted as 5 in Fig. 9). However, it should be noted that upon massive and artefactual cross-linking, even the partial pre-TCR complexes expressed at the surface of CD3-ζ/η−/− DN thymocytes are capable of inducing both maturation to the DP stage and TCR-β gene allelic exclusion (as suggested by the finding that most of the CD3-ζ/η−/− DP cells that develop after injection of anti–CD3-ε antibodies do not contain intracellular TCR-β chains; reference 44). Therefore, our results are reminiscent of those obtained with the TCR complexes expressed on mature T cells (e.g., reference 45) in that they suggest that different pre-TCR–mediated responses display distinct activation thresholds.


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)

A model accounting  for the role of the pre-TCR/ CD3 complex in the establishment of allelic exclusion at the  TCR-β locus. Upon entering  the CD44−/low CD25+ compartment, the TCR Vβ gene segment cluster [(V)n] become  accessible to the V(D)J recombinase (dashed lines sandwiching the  TCR-β alleles a and b). At that  stage of development, the pTα  and CD3 components of the  pre-TCR are already available  and it is the TCR-β polypeptides that constitute the rate limiting factor in the assembly of the  pre-TCR/CD3 complex. From a  cohort of nine CD44−/low CD25+  triple negative thymocytes, three  are expected to produce a functional Vβ gene (VDJ+) as a result  of their first attempt of rearrangement (step 1, see reference 29).  The resulting TCR-β polypeptide participates in the assembly  of a pre-TCR complex (step 2). As soon as assembled, this complex triggers (step 3) the transition beyond the CD44−/low CD25+ stage and activates a  negative feedback loop that will close the accessibility of the second, partially rearranged, allele (allele b) to the V(D)J recombinase (continuous lines sandwiching the TCR-β alleles), thereby restricting such a T cell to the expression of only a single TCR-β chain allele. The p56lck kinase (lck) constitutes one  of the effector operating downstream of the pre-TCR/CD3 complex since the overexpression of a catalytically active form of p56lck inhibits endogenous  Vβ to DβJβ rearrangements while inducing coincidently the transition to the DP stage (30). As proposed previously (29), time delay along this negative  feedback loop, and/or the existence of a few cells in which Vβ to DβJβ rearrangements can be attempted quasisimultaneously on both β alleles, may explain the presence of rare cells with two productively rearranged TCR-β alleles (52, 53). Based on the comparison of TCR-β transgenic, p56lck−/−, and  TCR-β transgenic, CD3-ζ/η−/− mice (see Discussion), it is tempting to speculate that TCR-β gene allelic exclusion is brought about via two contingent pathways. One of which (step 4b), by inducing cell proliferation and DNA replication, enables the reprogrammation of the chromatin structure at  the TCR-β locus, and thereby permits factor(s) induced by the second pathway (step 5) to act and render the TCR-β locus inaccessible to further V(D)J  recombination. Note that the degradation of RAG-2, which results from cyclin-dependent kinase phosphorylation (loop 4a; reference 38) and occurs  during the burst of divisions associated with the transition from the DN to the DP stage, appears to constitute a fail-safe mechanism not essential for the  execution of TCR-β allelic exclusion (39).
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Figure 9: A model accounting for the role of the pre-TCR/ CD3 complex in the establishment of allelic exclusion at the TCR-β locus. Upon entering the CD44−/low CD25+ compartment, the TCR Vβ gene segment cluster [(V)n] become accessible to the V(D)J recombinase (dashed lines sandwiching the TCR-β alleles a and b). At that stage of development, the pTα and CD3 components of the pre-TCR are already available and it is the TCR-β polypeptides that constitute the rate limiting factor in the assembly of the pre-TCR/CD3 complex. From a cohort of nine CD44−/low CD25+ triple negative thymocytes, three are expected to produce a functional Vβ gene (VDJ+) as a result of their first attempt of rearrangement (step 1, see reference 29). The resulting TCR-β polypeptide participates in the assembly of a pre-TCR complex (step 2). As soon as assembled, this complex triggers (step 3) the transition beyond the CD44−/low CD25+ stage and activates a negative feedback loop that will close the accessibility of the second, partially rearranged, allele (allele b) to the V(D)J recombinase (continuous lines sandwiching the TCR-β alleles), thereby restricting such a T cell to the expression of only a single TCR-β chain allele. The p56lck kinase (lck) constitutes one of the effector operating downstream of the pre-TCR/CD3 complex since the overexpression of a catalytically active form of p56lck inhibits endogenous Vβ to DβJβ rearrangements while inducing coincidently the transition to the DP stage (30). As proposed previously (29), time delay along this negative feedback loop, and/or the existence of a few cells in which Vβ to DβJβ rearrangements can be attempted quasisimultaneously on both β alleles, may explain the presence of rare cells with two productively rearranged TCR-β alleles (52, 53). Based on the comparison of TCR-β transgenic, p56lck−/−, and TCR-β transgenic, CD3-ζ/η−/− mice (see Discussion), it is tempting to speculate that TCR-β gene allelic exclusion is brought about via two contingent pathways. One of which (step 4b), by inducing cell proliferation and DNA replication, enables the reprogrammation of the chromatin structure at the TCR-β locus, and thereby permits factor(s) induced by the second pathway (step 5) to act and render the TCR-β locus inaccessible to further V(D)J recombination. Note that the degradation of RAG-2, which results from cyclin-dependent kinase phosphorylation (loop 4a; reference 38) and occurs during the burst of divisions associated with the transition from the DN to the DP stage, appears to constitute a fail-safe mechanism not essential for the execution of TCR-β allelic exclusion (39).
Mentions: Our data also bear on the causal relationships between pre-TCR–induced cell proliferation and the establishment of TCR-β allelic exclusion. It has been suggested that preTCR–induced cell cycle progression is essential for the establishment of allelic exclusion at the TCR-β locus (36–39; see also references 40 and 41 in the case of B cell development). As outlined in Fig. 9, one or more rounds of DNA replication are speculated to enable the reprogrammation of the chromatin structure of the TCR-β loci and make them inaccessible to the V(D)J recombinase. According to that model, the lack of TCR-β allelic exclusion observed in the CD3-εΔ5/Δ5 thymuses would be fully accounted for by the fact that their TCR-β pTα heterodimers are prevented from inducing cell cycle entry. Mice carrying a mutation in the lck gene display a pronounced thymic atrophy associated with a dramatic reduction in the number of DP cells (42). In these mutant mice, TCR-β gene allelic exclusion is not severely compromised as the presence of a productively rearranged TCR-β transgene resulted in an almost complete inhibition of endogenous TCR-β gene rearrangements (43). Considering that TCR-β transgenic, CD3-ζ/η−/− thymuses display the same composition and cellularity as TCR-β transgenic, lck−/− thymuses (compare our data with those of Wallace et al., reference 43), it came as a surprise to find that there was in the former a clear dissociation between the transition to the DP stage and the establishment of TCR-β gene allelic exclusion. Thus, in the absence of CD3-ζ/η subunit, TCR-β selection may have led to differentiation rather than proliferation and, consistent with the above model, resulted in the lack of TCR-β gene allelic exclusion. However, the frequency of dividing early DP cells is only slightly smaller in CD3-ζ/η−/− mice than in wild-type littermates, indicating that CD3-ζ/η–less pre-TCR complexes are still capable of triggering cell cycle entry (9). Collectively, these observations suggest that burst of cell divisions induced by the pre-TCR may be enabling rather than inductive for the establishment of TCR-β gene allelic exclusion, and that the pre-TCR is likely to contribute additional signals to effect TCR-β gene allelic exclusion. According to that view and under physiological conditions, the signals emanating from both the lck– and CD3-ζ/η–less pre-TCR complexes suffice to trigger cell cycle entry and CD4/CD8 expression, whereas only those emanating from the former can reach the higher threshold plausibly required to activate the regulatory loop required for mediating allelic exclusion (denoted as 5 in Fig. 9). However, it should be noted that upon massive and artefactual cross-linking, even the partial pre-TCR complexes expressed at the surface of CD3-ζ/η−/− DN thymocytes are capable of inducing both maturation to the DP stage and TCR-β gene allelic exclusion (as suggested by the finding that most of the CD3-ζ/η−/− DP cells that develop after injection of anti–CD3-ε antibodies do not contain intracellular TCR-β chains; reference 44). Therefore, our results are reminiscent of those obtained with the TCR complexes expressed on mature T cells (e.g., reference 45) in that they suggest that different pre-TCR–mediated responses display distinct activation thresholds.

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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