Expression, purification, and functional analysis of murine ectodomain fragments of CD8alphaalpha and CD8alphabeta dimers.
Soluble mouse CD8alphaalpha and CD8alphabeta 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 CD8alphaalpha(f) for H-2K(b) as measured by BIAcore revealed a approximately 65 microM K(d), similar to that of CD8alphabeta(f). Consistent with this result, CD8alphaalpha(f) as well as CD8alphabeta(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 CD8alphabeta versus CD8alphaalpha co-receptor function on T cells is linked to differences within their membrane-bound stalk regions and/or intracellular segments. As recently shown for sCD8alphaalpha, 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.
Kern
P
P
Laboratory of Immunobiology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
Hussey
R E
RE
Spoerl
R
R
Reinherz
E L
EL
Chang
H C
HC
D000818:Animals; D016827:CD8 Antigens / Q000737:chemistry*; D016466:CHO Cells; D006224:Cricetinae; D019281:Dimerization; D007700:Kinetics; D016350:Leucine Zippers; D051379:Mice; D010446:Peptide Fragments / Q000302:isolation & purification*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012995:Solubility
D016827:CD8 Antigens; D010446:Peptide Fragments; D011994:Recombinant Proteins