Surface expression of alternative forms of the TCR/CD3 complex.

Abstract

T-cell antigen receptor (TCR) heterodimers of both the a13 and y8 types are expressed at the surface of T cells only in association with a complex of invariant chains called CD3. The requirement for individual CD3 components to achieve TCR surface expression was examined by cotransfection of a non-T-cell line with TCR a and 13, as well as CD38, e, 'y, and C, cDNAs. Both transient and stable transfectants expressing TCR and CD3 epitopes at the cell surface were generated. (3)(4)(5). Each of the components is also generally thought to be essential for efficient assembly and transport of the complex to the cell surface. This assumption is based on studies of a limited range of T-cell mutant lines lacking expression of one or another TCR/CD3 component (6-11).The alternative direct approach of de novo reconstitution of the TCR/CD3 complex at the surface of non-T cells by cotransfection with the genes encoding the putative components is hampered by the complexity of the receptor and the resulting low likelihood of coexpressing all required gene products within the same cell. We have overcome this problem by using the highly sensitive technique of immunomagnetic selection to identify and purify both transient and stable transfectants present at very low frequency (12). We show that TCR/CD3 surface expression can, indeed, be reconstituted in non-T cells-i.e., no T-cell-specific components other than TCR and CD3 subunits are required. MATERIALS AND METHODS Plasmids. Full-length cDNAs encoding TCR a and ( chains expressed in the cytotoxic T lymphocyte (CTL) line 2C (13), as well as CD3 chains 8, E, 'y, and C (14-17), were cloned in the sense orientation into the expression vector CDM8 (18) between the HindIII or Sac I sites at the 5' end and the Xba I site at the 3' end. In most cases insertion into the vector CDM8 involved the prior attachment of linkers encoding appropriate restriction sites at one or both ends ofthe cDNAs to generate the required complementary overhangs.Transfection andc Immunomagnetic Selection. Equal amounts of each cDNA of interest, to a total of 20 ug per transfection, were introduced by a modified CaPO4 coprecipitation procedure (19) into HeLa cells grown to 70%o confluency on a standard tissue culture plate. For detection of transient surface expression the cells were removed from the plates 60 hr after transfection with 0.3% EDTA and rosetted with magnetic microspheres (Nichols Institute, San Juan Capistrano, CA) coated with the clonotypic monoclonal antibody (mAb) 1B2 (20). For generation of stable transfectants positive cells purified at the transient stage were repeatedly retransfected with all six TCR and CD3 cDNAs and reselected with mAb 1B2 until clones were derived that maintained expression in culture permanently. The use of immunomagnetic selection to purify transfectants expressing exogenous surface products is described in more detail in ref. 12.Fluorescent Staining. HeLa cells and transfectants were removed from tissue culture plates as described above. All cells were washed...

The Stalk Domain of NKp30 Contributes to Ligand Binding and Signaling of a Preassembled NKp30-CD3ζ Complex

“…Therefore, Arg-143 more likely provides a switch to induce CD3 signaling than promotes assembly of the NKp30-CD3 complex. This idea is compatible with previous reports on the TCR-CD3 complex demonstrating that CD3 is loosely associated with the TCR in ground state (39) and that CD3 is essential for plasma membrane expression of the TCR (22,40). Previous studies showed that in the presence of lipid, the cytoplasmic tail of CD3 is folded, thereby preventing ITAM phosphorylation, whereas in aqueous solution it loses its conformation and can be phosphorylated.…”

“…The functional importance of this interaction is illustrated by the finding that NK cells from knock-out mice lacking CD3 and Fc⑀RI␥ showed reduced cytolytic activity toward a large number of tumor cell lines (20). Moreover, previous studies showed a reduced number of T cell receptor (TCR) molecules on the surface of T cells due to intracellular retention of TCRs in the absence of CD3 (21)(22)(23). The NMR structure of a disulfidestabilized transmembrane helix dimer of CD3 has been solved (PDB code 2HAC; Ref.…”

Conversion of CTLA-4 from Inhibitor to Activator of T Cells with a Bispecific Tandem Single-Chain Fv Ligand

“…5b). These results explain the requirement for TCR expression for 24:26-induced activation because expression of TCR-␤ is critical for TCR-expression on the T cell surface (29). Therefore, we concluded that binding of 24:26 to CTLA-4 triggers a TCR-dependent signaling pathway that is qualitatively similar to that seen following TCR ligation with agonist ligands.…”

“…To address this issue, we used the Jurkat T cell subclone JRT3 that lacks expression of the ␤-chain of the TCR complex (28). This defect translates into a lack of expression of TCR heterodimer and of TCR--and CD3 chains on the cell surface (29). For an optimal window of response, the following experiments were performed using Y165F CTLA-4 transfectants.…”

Biochemical Evidence for the Presence of a Single CD3δ and CD3γ Chain in the Surface T Cell Receptor/CD3 Complex

“…Thus, a small number of TCR/CD3 complex expressed on T cell surface in either CD3␥ Ϫ/Ϫ or CD3␦ Ϫ/Ϫ mice could contain more than one molecule of CD3␦ or CD3␥, respectively. Similarly, cell line studies have shown that CD3␥⑀ and CD3␦⑀ heterodimers could associate with either of the TCR chains, further supporting the multivalent structure of the TCR/CD3 complex (26,27). Irrespective of whether CD3␥ and CD3␦ chains associate preferentially or randomly with TCR chains, if there are two molecules each of TCR␣ and -␤ chains, there exists a possibility that there could be two molecules each of CD3␥ and CD3␦ in a given TCR/CD3 complex.…”