Assembly of the TCR/CD3 complex: CD3ε/δ and CD3ε/γ dimers associate indistinctly with both TCRα and TCRβ chains. Evidence for a double TCR heterodimer model

Abstract

The TCR/CD3 complex is composed of six subunits which are expressed on the cell surface in a coordinate fashion after assembly in the endoplasmic reticulum (ER). The TCR/CD3 complex is assembled after a series of pairwise interactions involving the formation of dimers of CD3 4 with either CD3 + or CD3ˇ.CD3ˇCD3ˇ. These dimers assemble with TCR § and TCR g chains, and finally, the CD3´homodimerCD3´CD3´homodimer is added to allow export of the full complex from the ER. A model has been proposed suggesting that during assembly the CD3 4 /CD3 + dimer interacts exclusively with TCR g and the CD3 4 /CD3ˇdimerCD3ˇCD3ˇdimer with TCR § to form a complex with a single TCR § / g heterodimer. We show in this study, by immunoprecipitation and two-dimensional gel electrophoresis, that in the human T cell line Jurkat as well as in total human thymocytes, this preferential interaction does not occur and instead, the CD3 4 /CD3 + and CD3 4 /CD3ˇdimersCD3ˇCD3ˇdimers associate with both TCR chains simultaneously and indistinctly. These data are confirmed by the analysis of the TCR §-negative T cell line MOLT-4 in which TCR g is found separately associated with CD3 4 /CD3 + and with CD3 4 /CD3ˇdimersCD3ˇCD3ˇdimers. Indirectly, our results support a model of stoichiometry in which two TCR § / g heterodimers are present in a TCR/CD3 complex. Furthermore, immunoprecipitation with anti-CD3 + and anti-CD3ˇantiCD3ˇCD3ˇanti-bodies from 1 % NP40 and 1 % Brij96 cell lysates showed that these subunits form independent partial complexes which are cross-linked through the CD3´homodimerCD3´CD3´homodimer. This suggests that CD3´mediatesCD3´CD3´mediates the interaction between both TCR § / g heterodimers contained in the double TCR complex. Further proof for this hypothesis is obtained after analysis of a Jur-kat cell transfectant containing a point mutation in the transmembrane domain of TCR g that impairs the association of CD3´.CD3´CD3´. In this mutant cell line, unlike a control line with wild-type TCR g , the CD3 +-and CD3ˇ-CD3ˇCD3ˇ-containing complexes were found completely independent. Altogether, these results support a model of TCR/CD3 assembly and stoichiometry in which two TCR-§ / g heterodimers form two hemicomplexes containing either CD3 4 / + or CD3 4 / ˇ dimers which become associated via the CD3´homodimerCD3´CD3´homodimer.

Increased Sensitivity of Antigen-Experienced T Cells through the Enrichment of Oligomeric T Cell Receptor Complexes

“…IP, SDS-PAGE, and immunoblotting were performed in accordance to standard protocols. The anti-CD3z serum has been described (San José et al, 1998). The anti-CD3ε mAb M-20 was purchased from Santa Cruz.…”

Full Activation of the T Cell Receptor Requires Both Clustering and Conformational Changes at CD3

“…The rabbit anti-z antiserum 448 has been described (San Jose et al, 1998). The following antibodies were used: UCHT1 (anti-human CD3, P. Beverly, UK), 145-2C11 (anti-mouse CD3, J. Bluestone, USA), anti-sc (Ac146, M. Reth, Germany), 3D3 (anti-mouse Va/b, J.M.…”

The immunomodulation to diabetes control: New proposals for the reversion of this disease

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