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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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pTα–β heterodimers  are specifically recaptured by Abs  reactive with the cytoplasmic domain of pTα. (A) Recapture assay.  Recapture assay is schematized in  the top panel. (Bottom) Brij 96  detergent extracts of surface-labeled VL3-3M2 and SL-12β.12  cells were immunoprecipitated  with anti–TCR-β. After elution  by boiling in SDS, the anti– TCR-β immune complexes  were quenched with NP-40 and  split into three equal parts which  were either: (a) held on ice; (b)  immunoprecipitated with anti-pTα Ab; or (c) immunoprecipitated with control rabbit IgG.  The samples were then resolved on 2D NR×R SDS-PAGE gels, after  which the surface-labeled proteins were visualized with HRP-Av and  chemiluminescence. (B) Anti-pTα Ab specifically recaptured pTα–β  heterodimers from detergent extracts of surface-labeled pre-TCR+ (SL-12β.12) but not from α/β-TCR+ (VL3-3M2) cells. A recapture assay was  performed as above except that the samples were resolved on one-dimensional SDS-PAGE under nonreducing conditions.
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Figure 2: pTα–β heterodimers are specifically recaptured by Abs reactive with the cytoplasmic domain of pTα. (A) Recapture assay. Recapture assay is schematized in the top panel. (Bottom) Brij 96 detergent extracts of surface-labeled VL3-3M2 and SL-12β.12 cells were immunoprecipitated with anti–TCR-β. After elution by boiling in SDS, the anti– TCR-β immune complexes were quenched with NP-40 and split into three equal parts which were either: (a) held on ice; (b) immunoprecipitated with anti-pTα Ab; or (c) immunoprecipitated with control rabbit IgG. The samples were then resolved on 2D NR×R SDS-PAGE gels, after which the surface-labeled proteins were visualized with HRP-Av and chemiluminescence. (B) Anti-pTα Ab specifically recaptured pTα–β heterodimers from detergent extracts of surface-labeled pre-TCR+ (SL-12β.12) but not from α/β-TCR+ (VL3-3M2) cells. A recapture assay was performed as above except that the samples were resolved on one-dimensional SDS-PAGE under nonreducing conditions.

Mentions: The apparent absence of CD3-δ and TCR-ζ from pre-TCR complexes does not exclude them as potential pre-TCR components, as subunit visibility in this assay is dependent upon accessibility during surface labeling. Accordingly, proteins that did not label efficiently would be missed, providing an inaccurate view of pre-TCR subunit composition. In particular, surface-labeled TCR-ζ molecules were conspicuously absent from α/β-TCR complexes expressed by the VL3-3M2 thymic lymphoma (Fig. 2 A), despite the fact that these α/β-TCR complexes do contain TCR-ζ (data not shown). Likewise, our inability to identify labeled CD3-δ subunits in the pre-TCRs expressed by primary thymocytes may result from inefficient biotin-labeling of CD3-δ and/or from comigration with a contaminating protein that obscures CD3-δ.


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)

pTα–β heterodimers  are specifically recaptured by Abs  reactive with the cytoplasmic domain of pTα. (A) Recapture assay.  Recapture assay is schematized in  the top panel. (Bottom) Brij 96  detergent extracts of surface-labeled VL3-3M2 and SL-12β.12  cells were immunoprecipitated  with anti–TCR-β. After elution  by boiling in SDS, the anti– TCR-β immune complexes  were quenched with NP-40 and  split into three equal parts which  were either: (a) held on ice; (b)  immunoprecipitated with anti-pTα Ab; or (c) immunoprecipitated with control rabbit IgG.  The samples were then resolved on 2D NR×R SDS-PAGE gels, after  which the surface-labeled proteins were visualized with HRP-Av and  chemiluminescence. (B) Anti-pTα Ab specifically recaptured pTα–β  heterodimers from detergent extracts of surface-labeled pre-TCR+ (SL-12β.12) but not from α/β-TCR+ (VL3-3M2) cells. A recapture assay was  performed as above except that the samples were resolved on one-dimensional SDS-PAGE under nonreducing conditions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199111&req=5

Figure 2: pTα–β heterodimers are specifically recaptured by Abs reactive with the cytoplasmic domain of pTα. (A) Recapture assay. Recapture assay is schematized in the top panel. (Bottom) Brij 96 detergent extracts of surface-labeled VL3-3M2 and SL-12β.12 cells were immunoprecipitated with anti–TCR-β. After elution by boiling in SDS, the anti– TCR-β immune complexes were quenched with NP-40 and split into three equal parts which were either: (a) held on ice; (b) immunoprecipitated with anti-pTα Ab; or (c) immunoprecipitated with control rabbit IgG. The samples were then resolved on 2D NR×R SDS-PAGE gels, after which the surface-labeled proteins were visualized with HRP-Av and chemiluminescence. (B) Anti-pTα Ab specifically recaptured pTα–β heterodimers from detergent extracts of surface-labeled pre-TCR+ (SL-12β.12) but not from α/β-TCR+ (VL3-3M2) cells. A recapture assay was performed as above except that the samples were resolved on one-dimensional SDS-PAGE under nonreducing conditions.
Mentions: The apparent absence of CD3-δ and TCR-ζ from pre-TCR complexes does not exclude them as potential pre-TCR components, as subunit visibility in this assay is dependent upon accessibility during surface labeling. Accordingly, proteins that did not label efficiently would be missed, providing an inaccurate view of pre-TCR subunit composition. In particular, surface-labeled TCR-ζ molecules were conspicuously absent from α/β-TCR complexes expressed by the VL3-3M2 thymic lymphoma (Fig. 2 A), despite the fact that these α/β-TCR complexes do contain TCR-ζ (data not shown). Likewise, our inability to identify labeled CD3-δ subunits in the pre-TCRs expressed by primary thymocytes may result from inefficient biotin-labeling of CD3-δ and/or from comigration with a contaminating protein that obscures CD3-δ.

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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