In vitro production and characterization of partly assembled human CD3 complexes.

Pairwise assembly of human CD3 chains takes place in the endoplasmic reticulum of T cells. Subsequently, the CD3 heterodimers form complexes with Tiα and Tis chains forming hexameric TiαβCD3γeδe complexes. Finally, association with the ζ2 homodimer occurs in Golgi apparatus before the fully assembled T-cell receptor is transported to the cell surface. To study the structural properties of the human CD3 chains, we have developed new methods to produce and fold the extracellular domains of CD3γ, CD3δ and CD3e. Proteins were expressed in Escherichia coli as denatured chains and de novo folded in vitro. CD3γ and CD3e folded as soluble monomers, whereas CD3δ did not yield any soluble proteins. When folding the chains pairwise, soluble CD3γe and CD3δe heterodimers could be isolated, whereas CD3γδ heterodimers were not produced. Using antibodies as structural probes, we identified two different types of antigenic epitopes that were dependent on heterodimerization. Our data indicate that CD3e undergoes a conformational change after dimerization with CD3γ or CD3δ. Furthermore, we demonstrated that the CD3γe heterodimer could be purified using immunoaffinity chromatography.