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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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Subunit composition of pre-TCR complexes expressed by  primary explanted thymocytes. (A)Digitonin extracts of biotin–labeled  TCR-α0 thymocytes were immunoprecipitated using Abs reactive with  the following subunits: TCR-β (H57-597), CD3-γ/ε (7D6), CD3-δ (R9)  or TCR-ζ (551). SDS eluates of the resultant immune complexes were  either resolved directly on SDS-PAGE gels under nonreducing conditions (Total) or after neutralization and immunoprecipitation with either  anti-pTα (pTα) or control rabbit IgG (rIgG). Control anti–TCR-α Ab  did not coprecipitate pTα–β heterodimers from TCR-α0 thymocytes  (data not shown). (B) CD3-γ/δ/ε and TCR-ζ are all associated with pTα–β  heterodimers within the same pre-TCR complex. Digitonin extracts of  biotin–labeled TCR-α0 thymocytes were either immunoprecipitated directly with anti–TCR-β or after the detergent extracts had been depleted  of CD3-δ, -γ/ε, or -ζ with Abs reactive with those proteins. Surface-labeled  proteins were visualized by HRP-Av and chemiluminescence.
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Figure 3: Subunit composition of pre-TCR complexes expressed by primary explanted thymocytes. (A)Digitonin extracts of biotin–labeled TCR-α0 thymocytes were immunoprecipitated using Abs reactive with the following subunits: TCR-β (H57-597), CD3-γ/ε (7D6), CD3-δ (R9) or TCR-ζ (551). SDS eluates of the resultant immune complexes were either resolved directly on SDS-PAGE gels under nonreducing conditions (Total) or after neutralization and immunoprecipitation with either anti-pTα (pTα) or control rabbit IgG (rIgG). Control anti–TCR-α Ab did not coprecipitate pTα–β heterodimers from TCR-α0 thymocytes (data not shown). (B) CD3-γ/δ/ε and TCR-ζ are all associated with pTα–β heterodimers within the same pre-TCR complex. Digitonin extracts of biotin–labeled TCR-α0 thymocytes were either immunoprecipitated directly with anti–TCR-β or after the detergent extracts had been depleted of CD3-δ, -γ/ε, or -ζ with Abs reactive with those proteins. Surface-labeled proteins were visualized by HRP-Av and chemiluminescence.

Mentions: Having established that the recapture assay was specific, we next wished to evaluate subunit composition of pre-TCR complexes expressed on the surface of primary thymocytes. Abs reactive with TCR-β, CD3-γ/ε, TCR-ζ, and CD3-δ coprecipitated pTα–β heterodimers from detergent extracts of surface-labeled thymocytes (Fig. 3 A). Thus, according to the above definition, our analysis demonstrates that the pre-TCR complexes expressed by primary thymocytes in vivo comprise pTα–β heterodimers associated with CD3-γ/δ/ε, and TCR-ζ.


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)

Subunit composition of pre-TCR complexes expressed by  primary explanted thymocytes. (A)Digitonin extracts of biotin–labeled  TCR-α0 thymocytes were immunoprecipitated using Abs reactive with  the following subunits: TCR-β (H57-597), CD3-γ/ε (7D6), CD3-δ (R9)  or TCR-ζ (551). SDS eluates of the resultant immune complexes were  either resolved directly on SDS-PAGE gels under nonreducing conditions (Total) or after neutralization and immunoprecipitation with either  anti-pTα (pTα) or control rabbit IgG (rIgG). Control anti–TCR-α Ab  did not coprecipitate pTα–β heterodimers from TCR-α0 thymocytes  (data not shown). (B) CD3-γ/δ/ε and TCR-ζ are all associated with pTα–β  heterodimers within the same pre-TCR complex. Digitonin extracts of  biotin–labeled TCR-α0 thymocytes were either immunoprecipitated directly with anti–TCR-β or after the detergent extracts had been depleted  of CD3-δ, -γ/ε, or -ζ with Abs reactive with those proteins. Surface-labeled  proteins were visualized by HRP-Av and chemiluminescence.
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Related In: Results  -  Collection

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Figure 3: Subunit composition of pre-TCR complexes expressed by primary explanted thymocytes. (A)Digitonin extracts of biotin–labeled TCR-α0 thymocytes were immunoprecipitated using Abs reactive with the following subunits: TCR-β (H57-597), CD3-γ/ε (7D6), CD3-δ (R9) or TCR-ζ (551). SDS eluates of the resultant immune complexes were either resolved directly on SDS-PAGE gels under nonreducing conditions (Total) or after neutralization and immunoprecipitation with either anti-pTα (pTα) or control rabbit IgG (rIgG). Control anti–TCR-α Ab did not coprecipitate pTα–β heterodimers from TCR-α0 thymocytes (data not shown). (B) CD3-γ/δ/ε and TCR-ζ are all associated with pTα–β heterodimers within the same pre-TCR complex. Digitonin extracts of biotin–labeled TCR-α0 thymocytes were either immunoprecipitated directly with anti–TCR-β or after the detergent extracts had been depleted of CD3-δ, -γ/ε, or -ζ with Abs reactive with those proteins. Surface-labeled proteins were visualized by HRP-Av and chemiluminescence.
Mentions: Having established that the recapture assay was specific, we next wished to evaluate subunit composition of pre-TCR complexes expressed on the surface of primary thymocytes. Abs reactive with TCR-β, CD3-γ/ε, TCR-ζ, and CD3-δ coprecipitated pTα–β heterodimers from detergent extracts of surface-labeled thymocytes (Fig. 3 A). Thus, according to the above definition, our analysis demonstrates that the pre-TCR complexes expressed by primary thymocytes in vivo comprise pTα–β heterodimers associated with CD3-γ/δ/ε, and TCR-ζ.

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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