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Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 delta is physically associated but not functionally required.

Berger MA, Davé V, Rhodes MR, Bosma GC, Bosma MJ, Kappes DJ, Wiest DL - J. Exp. Med. (1997)

Bottom Line: However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear.Interestingly, while CD3-delta is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-delta-deficient mice.Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (gamma, delta, epsilon, and zeta), possibly because pT alpha itself possesses signaling capabilities.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Immunobiology Working Group, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. ma_berger@fccc.edu

ABSTRACT
Maturation of immature CD4-CD8- (DN) thymocytes to the CD4+CD8+ (DP) stage of development is driven by signals transduced through a pre-T cell receptor (TCR) complex, whose hallmark is a novel subunit termed pre-T alpha (pT alpha). However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear. Moreover, progress in understanding pre-TCR function has been hampered thus far because previous attempts to demonstrate expression of pT alpha-containing pre-TCRs on the surface of normal thymocytes have been unsuccessful. In this report, we demonstrate for the first time that pT alpha-containing pre-TCR complexes are expressed at low levels on the surface of primary thymocytes and that these pre-TCR complexes comprise a disulfide-linked pT alpha-TCR-beta heterodimer associated not only with CD3-gamma and -epsilon, as previously reported, but also with zeta and delta. Interestingly, while CD3-delta is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-delta-deficient mice. The fact that any of the signaling components of the pre-TCR are dispensable for pre-TCR function is indeed surprising, given that few pre-TCR complexes are actually expressed on the surface of primary thymocytes in vivo. Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (gamma, delta, epsilon, and zeta), possibly because pT alpha itself possesses signaling capabilities.

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Schematic of the role of the pre-TCR in thymocyte development. After β gene rearrangement, immature thymocytes express surface pre-TCR complexes comprising pTα–β heterodimers associated  with CD3-γ/δ/ε and TCR-ζ signaling subunits. Pre-TCR complexes  are thought to regulate development of thymocytes from the DN to the  DP stage by evaluating the status of the TCR-β protein product after β  gene rearrangement. The molecular interactions underlying this evaluation process are currently unclear; however, development is blocked by  elimination of pTα, TCR-ζ or CD3-γ/δ/ε, but not CD3-δ. Curiously,  elimination of CD3-δ does impair the ability of the α/β-TCR to signal  maturation of DP thymocytes to the CD4+ or CD8+ stage. Thus, while  both pre-TCR and TCR complexes possess the same array of signaling subunits, they exhibit differential dependence on the individual components.
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Figure 5: Schematic of the role of the pre-TCR in thymocyte development. After β gene rearrangement, immature thymocytes express surface pre-TCR complexes comprising pTα–β heterodimers associated with CD3-γ/δ/ε and TCR-ζ signaling subunits. Pre-TCR complexes are thought to regulate development of thymocytes from the DN to the DP stage by evaluating the status of the TCR-β protein product after β gene rearrangement. The molecular interactions underlying this evaluation process are currently unclear; however, development is blocked by elimination of pTα, TCR-ζ or CD3-γ/δ/ε, but not CD3-δ. Curiously, elimination of CD3-δ does impair the ability of the α/β-TCR to signal maturation of DP thymocytes to the CD4+ or CD8+ stage. Thus, while both pre-TCR and TCR complexes possess the same array of signaling subunits, they exhibit differential dependence on the individual components.

Mentions: The ability of TCR and pre-TCR complexes to transduce signals resides in their invariant CD3-γ/δ/ε and TCR-ζ subunits. While both receptors carry the same array (γ, δ, ε, and ζ), the requirements of these receptors for individual signaling subunits differ (Fig. 5), raising the fundamental question of whether the different subunits subserve redundant or unique roles in receptor function. If CD3-γ/δ/ε and TCR-ζ subunits are redundant and function to amplify signals, then it is surprising that the pre-TCR complex can tolerate the loss of CD3-δ, given that surface expression levels of the pre-TCR complex are so low (Figs. 1 and 4). In that regard, the pre-TCR may be able to tolerate loss of CD3-δ because pre-TCR signals need not be as quantitatively intense or because pre-TCRs have a lower signaling threshold (than α/β-TCR complexes). In support of the latter possibility, pre-TCR complexes function before expression of surface molecules that can be inhibitory, such as CD4, which we have shown can decrease signaling competence of the α/β-TCR on DP thymocytes by sequestering p56lck protein tyrosine kinase (22). It is also possible that the individual CD3-γ/δ/ε and TCR-ζ signaling subunits perform unique functions. Consistent with this hypothesis, it has been shown that immunoreceptor tyrosine-based activation motifs of different signaling subunits are able to interact with different cytoplasmic signaling effector molecules and induce phosphorylation of different substrates (23). Finally, it is possible that pre-TCR complexes can tolerate loss of CD3-δ because in the absence of CD3-δ the pTα subunit itself is also able to function as a signaling subunit. Murine pTα has a cytoplasmic tail of ∼30 amino acids which contains consensus motifs for phosphorylation by protein kinase C and for docking of SH3 domain containing proteins (12); however, the functional importance of these motifs is unclear as there is little sequence conservation between the cytoplasmic tails of the murine and human pTα homologues (5). Recently, the role of the cytoplasmic tail of pTα was tested by reconstitution of pTα-deficient mice with a pTα transgene lacking the cytoplasmic tail (24). While tailless pTα compensated for pTα deficiency, it did so only partially, leaving open the possibility that the cytoplasmic tail of pTα does function as a signaling domain within the pre-TCR complex and might underlie the pre-TCR's ability to tolerate loss of the CD3-δ subunit. In that regard it would be informative to analyze the function of pre-TCR complexes in CD3-δ0 thymocytes expressing tailless pTα molecules.


Subunit composition of pre-T cell receptor complexes expressed by primary thymocytes: CD3 delta is physically associated but not functionally required.

Berger MA, Davé V, Rhodes MR, Bosma GC, Bosma MJ, Kappes DJ, Wiest DL - J. Exp. Med. (1997)

Schematic of the role of the pre-TCR in thymocyte development. After β gene rearrangement, immature thymocytes express surface pre-TCR complexes comprising pTα–β heterodimers associated  with CD3-γ/δ/ε and TCR-ζ signaling subunits. Pre-TCR complexes  are thought to regulate development of thymocytes from the DN to the  DP stage by evaluating the status of the TCR-β protein product after β  gene rearrangement. The molecular interactions underlying this evaluation process are currently unclear; however, development is blocked by  elimination of pTα, TCR-ζ or CD3-γ/δ/ε, but not CD3-δ. Curiously,  elimination of CD3-δ does impair the ability of the α/β-TCR to signal  maturation of DP thymocytes to the CD4+ or CD8+ stage. Thus, while  both pre-TCR and TCR complexes possess the same array of signaling subunits, they exhibit differential dependence on the individual components.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199111&req=5

Figure 5: Schematic of the role of the pre-TCR in thymocyte development. After β gene rearrangement, immature thymocytes express surface pre-TCR complexes comprising pTα–β heterodimers associated with CD3-γ/δ/ε and TCR-ζ signaling subunits. Pre-TCR complexes are thought to regulate development of thymocytes from the DN to the DP stage by evaluating the status of the TCR-β protein product after β gene rearrangement. The molecular interactions underlying this evaluation process are currently unclear; however, development is blocked by elimination of pTα, TCR-ζ or CD3-γ/δ/ε, but not CD3-δ. Curiously, elimination of CD3-δ does impair the ability of the α/β-TCR to signal maturation of DP thymocytes to the CD4+ or CD8+ stage. Thus, while both pre-TCR and TCR complexes possess the same array of signaling subunits, they exhibit differential dependence on the individual components.
Mentions: The ability of TCR and pre-TCR complexes to transduce signals resides in their invariant CD3-γ/δ/ε and TCR-ζ subunits. While both receptors carry the same array (γ, δ, ε, and ζ), the requirements of these receptors for individual signaling subunits differ (Fig. 5), raising the fundamental question of whether the different subunits subserve redundant or unique roles in receptor function. If CD3-γ/δ/ε and TCR-ζ subunits are redundant and function to amplify signals, then it is surprising that the pre-TCR complex can tolerate the loss of CD3-δ, given that surface expression levels of the pre-TCR complex are so low (Figs. 1 and 4). In that regard, the pre-TCR may be able to tolerate loss of CD3-δ because pre-TCR signals need not be as quantitatively intense or because pre-TCRs have a lower signaling threshold (than α/β-TCR complexes). In support of the latter possibility, pre-TCR complexes function before expression of surface molecules that can be inhibitory, such as CD4, which we have shown can decrease signaling competence of the α/β-TCR on DP thymocytes by sequestering p56lck protein tyrosine kinase (22). It is also possible that the individual CD3-γ/δ/ε and TCR-ζ signaling subunits perform unique functions. Consistent with this hypothesis, it has been shown that immunoreceptor tyrosine-based activation motifs of different signaling subunits are able to interact with different cytoplasmic signaling effector molecules and induce phosphorylation of different substrates (23). Finally, it is possible that pre-TCR complexes can tolerate loss of CD3-δ because in the absence of CD3-δ the pTα subunit itself is also able to function as a signaling subunit. Murine pTα has a cytoplasmic tail of ∼30 amino acids which contains consensus motifs for phosphorylation by protein kinase C and for docking of SH3 domain containing proteins (12); however, the functional importance of these motifs is unclear as there is little sequence conservation between the cytoplasmic tails of the murine and human pTα homologues (5). Recently, the role of the cytoplasmic tail of pTα was tested by reconstitution of pTα-deficient mice with a pTα transgene lacking the cytoplasmic tail (24). While tailless pTα compensated for pTα deficiency, it did so only partially, leaving open the possibility that the cytoplasmic tail of pTα does function as a signaling domain within the pre-TCR complex and might underlie the pre-TCR's ability to tolerate loss of the CD3-δ subunit. In that regard it would be informative to analyze the function of pre-TCR complexes in CD3-δ0 thymocytes expressing tailless pTα molecules.

Bottom Line: However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear.Interestingly, while CD3-delta is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-delta-deficient mice.Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (gamma, delta, epsilon, and zeta), possibly because pT alpha itself possesses signaling capabilities.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Immunobiology Working Group, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. ma_berger@fccc.edu

ABSTRACT
Maturation of immature CD4-CD8- (DN) thymocytes to the CD4+CD8+ (DP) stage of development is driven by signals transduced through a pre-T cell receptor (TCR) complex, whose hallmark is a novel subunit termed pre-T alpha (pT alpha). However, the precise role of pre-TCRs in mediating the DN to DP transition remains unclear. Moreover, progress in understanding pre-TCR function has been hampered thus far because previous attempts to demonstrate expression of pT alpha-containing pre-TCRs on the surface of normal thymocytes have been unsuccessful. In this report, we demonstrate for the first time that pT alpha-containing pre-TCR complexes are expressed at low levels on the surface of primary thymocytes and that these pre-TCR complexes comprise a disulfide-linked pT alpha-TCR-beta heterodimer associated not only with CD3-gamma and -epsilon, as previously reported, but also with zeta and delta. Interestingly, while CD3-delta is associated with the pre-TCR complex, it is not required for pre-TCR function, as evidenced by the generation of normal numbers of DP thymocytes in CD3-delta-deficient mice. The fact that any of the signaling components of the pre-TCR are dispensable for pre-TCR function is indeed surprising, given that few pre-TCR complexes are actually expressed on the surface of primary thymocytes in vivo. Thus, pre-TCRs do not require the full array of TCR-associated signaling subunits (gamma, delta, epsilon, and zeta), possibly because pT alpha itself possesses signaling capabilities.

Show MeSH
Related in: MedlinePlus