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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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Expression of pTα-containing pre-TCR complexes on the  surface of primary thymocytes. Surface biotin–labeled thymocytes from  TCR-α0 mice were solubilized in Brij 96, immunoprecipitated with  anti–TCR-β Ab (H57-597; right), or control hamster IgG (left) and resolved on 2D NR×R SDS-PAGE gels. Surface-labeled proteins were  visualized by HRP-Av and chemiluminescence. Migration positions of  individual subunits are indicated.
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Figure 1: Expression of pTα-containing pre-TCR complexes on the surface of primary thymocytes. Surface biotin–labeled thymocytes from TCR-α0 mice were solubilized in Brij 96, immunoprecipitated with anti–TCR-β Ab (H57-597; right), or control hamster IgG (left) and resolved on 2D NR×R SDS-PAGE gels. Surface-labeled proteins were visualized by HRP-Av and chemiluminescence. Migration positions of individual subunits are indicated.

Mentions: It is well established that immature thymocytes express surface TCR complexes containing TCR-β without TCR-α; however, it remains unclear whether these complexes comprise TCR-β homodimers or, alternatively, heterodimers of TCR-β and pTα, the hallmark of the pre-TCR complex (4, 12, 19). This issue remains unresolved because previous attempts to demonstrate expression of pTα-containing pre-TCR complexes on the surface of primary thymocytes have been unsuccessful (4, 12, 19). Consequently, we wished to determine if pTα-containing pre-TCR complexes were actually expressed on the surface of primary thymocytes. Thymocytes from TCR-α0 mice were used because they lack α/β-TCR complexes which might otherwise complicate analysis of pre-TCR structure, yet they still exhibit normal pre-TCR function (2, 14). Detergent extracts of surface biotin–labeled thymocytes were immunoprecipitated either with control hamster IgG or with an anti–TCR-β mAb after which the resultant immune complexes were resolved by two-dimensional nonreducing × reducing (2D NR×R) SDS-PAGE (Fig. 1). The anti–TCR-β mAb immunoprecipitation contained associated CD3-γ/ε heterodimers as well as disulfide-linked pTα–β heterodimers. Curiously, neither surface-labeled TCR-ζ nor CD3-δ was visible. Thus, primary thymocytes do indeed express surface pre-TCR complexes in which pTα–β and CD3-γ/ε heterodimers are evident.


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)

Expression of pTα-containing pre-TCR complexes on the  surface of primary thymocytes. Surface biotin–labeled thymocytes from  TCR-α0 mice were solubilized in Brij 96, immunoprecipitated with  anti–TCR-β Ab (H57-597; right), or control hamster IgG (left) and resolved on 2D NR×R SDS-PAGE gels. Surface-labeled proteins were  visualized by HRP-Av and chemiluminescence. Migration positions of  individual subunits are indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199111&req=5

Figure 1: Expression of pTα-containing pre-TCR complexes on the surface of primary thymocytes. Surface biotin–labeled thymocytes from TCR-α0 mice were solubilized in Brij 96, immunoprecipitated with anti–TCR-β Ab (H57-597; right), or control hamster IgG (left) and resolved on 2D NR×R SDS-PAGE gels. Surface-labeled proteins were visualized by HRP-Av and chemiluminescence. Migration positions of individual subunits are indicated.
Mentions: It is well established that immature thymocytes express surface TCR complexes containing TCR-β without TCR-α; however, it remains unclear whether these complexes comprise TCR-β homodimers or, alternatively, heterodimers of TCR-β and pTα, the hallmark of the pre-TCR complex (4, 12, 19). This issue remains unresolved because previous attempts to demonstrate expression of pTα-containing pre-TCR complexes on the surface of primary thymocytes have been unsuccessful (4, 12, 19). Consequently, we wished to determine if pTα-containing pre-TCR complexes were actually expressed on the surface of primary thymocytes. Thymocytes from TCR-α0 mice were used because they lack α/β-TCR complexes which might otherwise complicate analysis of pre-TCR structure, yet they still exhibit normal pre-TCR function (2, 14). Detergent extracts of surface biotin–labeled thymocytes were immunoprecipitated either with control hamster IgG or with an anti–TCR-β mAb after which the resultant immune complexes were resolved by two-dimensional nonreducing × reducing (2D NR×R) SDS-PAGE (Fig. 1). The anti–TCR-β mAb immunoprecipitation contained associated CD3-γ/ε heterodimers as well as disulfide-linked pTα–β heterodimers. Curiously, neither surface-labeled TCR-ζ nor CD3-δ was visible. Thus, primary thymocytes do indeed express surface pre-TCR complexes in which pTα–β and CD3-γ/ε heterodimers are evident.

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