The antigen receptor on T cells is a multisubunit complex composed of a clonotypic heterodimer and four invariant signaling subunits, CD3γ, CD3δ, CD3
, and TCR-ζ. The generally accepted stoichiometry of the αβ TCR is non-γδ TCR-αβ, CD3γ
, and TCR-ζζ (1
). Previous data obtained from expanded populations of peripheral γδ T cells suggested a similar structure for the γδ TCR, except that the γδ TCR could contain the TCR-ζ family member, Fc
R1 γ (FcRγ), as a homodimer or as a heterodimer with TCR-ζ in lieu of the TCR-ζ homodimer (2
). However, recent data have called for a revision in this model of γδ TCR structure. For example, the observation that FcRγ is a component of the γδ TCR conflicts with the findings that FcRγ transcripts are not detected in purified thymic and peripheral γδ T cells (3
) and that γδ T cell development is unaffected in FcRγ−/−
). We extended these studies by analyzing the subunit composition of γδ TCR complexes on ex vivo γδ T cells from γδ TCR Tg and nonγδ-TCR Tg mice (6
). Biochemical analyses showed that TCR-ζ is a component of the γδ TCR on ex vivo γδ T cells, while FcRγ is not. However, after in vitro activation and expansion, FcRγ is expressed and incorporated into the γδ TCR complex (6
). These data show that activation and expansion induce changes in the γδ TCR complex and demonstrate that the subunit composition of γδ TCRs expressed by activated and expanded populations of γδ T cells does not accurately represent the subunit composition of TCR complexes expressed by primary γδ T cells.
A more striking inconsistency with the previous model of γδ TCR structure was the finding that the γδ TCRs expressed on ex vivo thymic and lymph node γδ T cells do not contain CD3δ (6
). This result appears to contradict previous studies, in which the CD3 subunit composition of the αβ TCR and γδ TCR was found to be identical (2
). As these previous studies used expanded populations of peripheral γδ T cells, γδ TCR+
intestinal intraepithelial lymphocytes (iIELs)*
and γδ T cell hybridomas as sources of γδ T cells, it is possible that activation and expansion induced a change in the subunit composition of the CD3 complex. To test this, we compared the subunit composition of TCR complexes expressed by γδ T cells before and after in vitro activation and expansion. Here, we report the detection of a protein, equivalent in mass to CD3δ, in the γδ TCR after activation and expansion. Biochemical analyses revealed that this protein was not CD3δ but a differentially glycosylated form of CD3γ. In addition, modification of the CD3γ subunit was observed in in vitro–activated αβ T cells and in iIELs.
Together, these results demonstrate that the protein initially thought to be CD3δ in the γδ TCR complex is, instead, a differentially glycosylated form of CD3γ and provide further substantiation for the conclusion that the αβ TCR and γδ TCR complexes differ in their subunit composition.