Expression, Purification, and Functional Analysis of Murine Ectodomain Fragments of CD8aa and CD8ab Dimers*

Soluble mouse CD8aa and CD8ab dimers corresponding to the paired ectodomains (CD8f) 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 CD8aaf for H-2K b as measured by BIAcore revealed a ;65 mM Kd, similar to that of CD8abf. Consistent with this result, CD8aaf as well as CD8abf 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 CD8ab versus CD8aa co-receptor function on T cells is linked to differences within their membranebound stalk regions and/or intracellular segments. As recently shown for sCD8aa, 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.