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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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The CD3-δ deficiency  does not attenuate the DN to the DP  transition. (A) The absence of CD3-δ  does not affect pre-TCR expression.  Digitonin extracts of surface biotin– labeled TCR-α0CD3-δ0 thymocytes  were immunoprecipitated with anti– CD3-γ/ε or anti–CD3-δ Abs after  which the resultant immune complexes  were analyzed by recapture assay. Immune complexes were resolved by  one-dimensional SDS-PAGE under  nonreducing conditions. The absence  of any recaptured pTα–β heterodimers in the anti–CD3-δ immunoprecipitations demonstrates the specificity of the anti–CD3-δ Ab. (B)  Thymocytes from both TCR-α0CD3-δ+ and TCR-α0CD3-δ0 mice have the same percentage of CD4+CD8+ cells (top) and express similar levels of  TCR-β on the cell surface (bottom; thick line), as measured by flow cytometry with fluorochrome conjugated mAbs. The thin line represents control staining  with anti–human CD3-ε Ab. The mean number of cells per thymus for each strain is indicated at the bottom of the figure.
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Figure 4: The CD3-δ deficiency does not attenuate the DN to the DP transition. (A) The absence of CD3-δ does not affect pre-TCR expression. Digitonin extracts of surface biotin– labeled TCR-α0CD3-δ0 thymocytes were immunoprecipitated with anti– CD3-γ/ε or anti–CD3-δ Abs after which the resultant immune complexes were analyzed by recapture assay. Immune complexes were resolved by one-dimensional SDS-PAGE under nonreducing conditions. The absence of any recaptured pTα–β heterodimers in the anti–CD3-δ immunoprecipitations demonstrates the specificity of the anti–CD3-δ Ab. (B) Thymocytes from both TCR-α0CD3-δ+ and TCR-α0CD3-δ0 mice have the same percentage of CD4+CD8+ cells (top) and express similar levels of TCR-β on the cell surface (bottom; thick line), as measured by flow cytometry with fluorochrome conjugated mAbs. The thin line represents control staining with anti–human CD3-ε Ab. The mean number of cells per thymus for each strain is indicated at the bottom of the figure.

Mentions: Since we had demonstrated CD3-δ was a component of the pre-TCR complex, it was important to determine if δ were critical to pre-TCR function. Thus, we analyzed TCR-α0CD3-δ0 mice to determine if the loss of the CD3-δ signaling component affected pre-TCR expression as well as two manifestations of pre-TCR function, thymic cellularity and maturation of DN thymocytes to the DP stage (5, 6). Thymocytes from TCR-α0CD3-δ0 mice expressed surface pre-TCRs comprising pTα–β heterodimers associated with CD3-γ/ε and TCR-ζ, indicating that CD3-δ deficiency does not prevent assembly and surface expression of the remaining pre-TCR subunits (Fig. 4, A and B, and data not shown). Likewise, δ deficiency did not attenuate pre-TCR function. Flow cytometric analysis of thymocytes from TCR-α0CD3-δ+ and TCR-α0CD3-δ0 mice revealed that each contained 96% DP thymocytes (Fig. 4 B). Furthermore, total thymic cellularity in TCR-α0CD3-δ0 mice was equivalent to that in TCR-α0CD3-δ+ mice (2.80 × 108 versus 3.31 × 108, respectively; P <0.01). Thus, the absence of CD3-δ from the pre-TCR complex does not adversely affect the progression of immature thymocytes from the DN to the DP stage of development, demonstrating that CD3-δ is not necessary for pre-TCR function.


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)

The CD3-δ deficiency  does not attenuate the DN to the DP  transition. (A) The absence of CD3-δ  does not affect pre-TCR expression.  Digitonin extracts of surface biotin– labeled TCR-α0CD3-δ0 thymocytes  were immunoprecipitated with anti– CD3-γ/ε or anti–CD3-δ Abs after  which the resultant immune complexes  were analyzed by recapture assay. Immune complexes were resolved by  one-dimensional SDS-PAGE under  nonreducing conditions. The absence  of any recaptured pTα–β heterodimers in the anti–CD3-δ immunoprecipitations demonstrates the specificity of the anti–CD3-δ Ab. (B)  Thymocytes from both TCR-α0CD3-δ+ and TCR-α0CD3-δ0 mice have the same percentage of CD4+CD8+ cells (top) and express similar levels of  TCR-β on the cell surface (bottom; thick line), as measured by flow cytometry with fluorochrome conjugated mAbs. The thin line represents control staining  with anti–human CD3-ε Ab. The mean number of cells per thymus for each strain is indicated at the bottom of the figure.
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Figure 4: The CD3-δ deficiency does not attenuate the DN to the DP transition. (A) The absence of CD3-δ does not affect pre-TCR expression. Digitonin extracts of surface biotin– labeled TCR-α0CD3-δ0 thymocytes were immunoprecipitated with anti– CD3-γ/ε or anti–CD3-δ Abs after which the resultant immune complexes were analyzed by recapture assay. Immune complexes were resolved by one-dimensional SDS-PAGE under nonreducing conditions. The absence of any recaptured pTα–β heterodimers in the anti–CD3-δ immunoprecipitations demonstrates the specificity of the anti–CD3-δ Ab. (B) Thymocytes from both TCR-α0CD3-δ+ and TCR-α0CD3-δ0 mice have the same percentage of CD4+CD8+ cells (top) and express similar levels of TCR-β on the cell surface (bottom; thick line), as measured by flow cytometry with fluorochrome conjugated mAbs. The thin line represents control staining with anti–human CD3-ε Ab. The mean number of cells per thymus for each strain is indicated at the bottom of the figure.
Mentions: Since we had demonstrated CD3-δ was a component of the pre-TCR complex, it was important to determine if δ were critical to pre-TCR function. Thus, we analyzed TCR-α0CD3-δ0 mice to determine if the loss of the CD3-δ signaling component affected pre-TCR expression as well as two manifestations of pre-TCR function, thymic cellularity and maturation of DN thymocytes to the DP stage (5, 6). Thymocytes from TCR-α0CD3-δ0 mice expressed surface pre-TCRs comprising pTα–β heterodimers associated with CD3-γ/ε and TCR-ζ, indicating that CD3-δ deficiency does not prevent assembly and surface expression of the remaining pre-TCR subunits (Fig. 4, A and B, and data not shown). Likewise, δ deficiency did not attenuate pre-TCR function. Flow cytometric analysis of thymocytes from TCR-α0CD3-δ+ and TCR-α0CD3-δ0 mice revealed that each contained 96% DP thymocytes (Fig. 4 B). Furthermore, total thymic cellularity in TCR-α0CD3-δ0 mice was equivalent to that in TCR-α0CD3-δ+ mice (2.80 × 108 versus 3.31 × 108, respectively; P <0.01). Thus, the absence of CD3-δ from the pre-TCR complex does not adversely affect the progression of immature thymocytes from the DN to the DP stage of development, demonstrating that CD3-δ is not necessary for pre-TCR function.

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