Expression, purification, and functional analysis of murine ectodomain fragments of CD8αα and CD8αβ dimers

Soluble mouse CD8 alpha alpha and CD8 alpha beta dimers corresponding to the paired ectodomains (CD8(f)) or their respective component Ig-like domains (CD8) were expressed in Chinese hamster ovary cells or the glycosylation variant Lec3.2.8.1 cells as secreted proteins using a leucine zipper strategy. The affinity of CD8 alpha alpha(f) for H-2K(b) as measured by BIAcore revealed a similar to 65 mu M K-d, similar to that of CD8 alpha beta(f). Consistent with this result, CD8 alpha alpha(f) as well as CD8 alpha beta(f) blocked the effector function of N15 T cell receptor transgenic cytolytic T cells in a comparable, dose-dependent fashion. Furthermore, both Lec3.2.8.1-produced and Chinese hamster ovary-produced CD8 homodimers and heterodimers were active in the inhibition assay. These results suggest that the Ig-like domains of CD8 molecules are themselves sufficient to block the requisite transmembrane CD8-pMHC interaction between cytolytic T lymphocytes and target cells. Moreover, given the similarities in co-receptor affinities for pMHC, the findings suggest that the greater efficiency of CD8 alpha beta versus CD8 alpha alpha co-receptor function on T cells is linked to differences within their membrane-bound stalk regions and/or intracellular segments. As recently shown for sCD8 alpha alpha, the yield, purity and homogeneity of the deglycosylated protein resulting from this expression system is sufficient for crystallization and x-ray diffraction at atomic resolution.