Regions of the CD8 molecule involved in the regulation of CD2-mediated activation.

The CD8 molecule regulates T cell activation mediated via the CD3 T cell receptor and the adhesion molecule CD2. CD8 mAbs have been found to inhibit early (Ca2+ rise) as well as late events (cytotoxicity, proliferation, and lymphokine secretion) mediated via the CD2 pathway. A panel of eight anti-human CD8 mAbs was tested for inhibition of CD2-mediated Ca2+ rise in a cytotoxic T cell clone. The inhibition ranged from 5 to 53% independently of mAb isotype and affinity measured by half saturation binding. We then characterized these mAbs for their reactivity toward three mutants of the human CD8 alpha carrying amino acid sequence changes in the surface-exposed loops homologous to the immunoglobulin CDR1, 2, and 3. The mutations included replacement of the human CD8 alpha CDR1- and CDR2-like loops by the homologous mouse sequences and the insertion of a glycine in the middle of the CDR3-like loop. Thus, five mAbs were found to be affected by the mutation in the CDR2-like loop but not by alterations in the other CDR-like loops. Conversely, the other two mAbs (8E1.7 and B9.8) were affected only by mutations in the CDR1- and CDR3-like loops, respectively. Cross-inhibition experiments were essentially in agreement with these results. Interestingly, all the mAbs directed against the CDR2-like loop were potent inhibitors of CD2-mediated Ca2+ rise, with one exception probably due to poor affinity. Thus, in addition to being a site of interaction with major histocompatibility complex Class I as recent data have indicated, this region of the CD8 alpha subunit may play a role in regulating T cell activation.

Evidence for an extended structure of the T-cell co-receptor CD8 alpha as deduced from the hydrodynamic properties of soluble forms of the extracellular region.

We expressed soluble forms of the human T-cell coreceptor CD8 alpha extracellular region, CD8 alpha 161, and the amino-terminal immunoglobulin-like domain, CD8 alpha 114, in Chinese hamster ovary cells and Escherichia coli, respectively. Both molecules were readily purified to homogeneity in milligram amounts and were recognized by a large panel of monoclonal antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that approximately 70% of CD8 alpha 161 was secreted as a disulfide-linked dimer, but CD8 alpha 114 was not disulfide-linked. To investigate the structural features of CD8 alpha 161 and CD8 alpha 114 under native conditions, we performed gel filtration and sucrose gradient sedimentation analysis. In spite of being partially or totally noncovalently bound, both recombinant molecules were stably associated homodimers, as no monomers could be detected at a fairly low protein concentration (approximately 1 microM). This suggests that the CD8 alpha amino-terminal domain alone strongly contributes to chain association. Determination of the Stokes radius (Rs) and sedimentation coefficient (s20,w) gave results consistent with CD8 alpha 114 having a globular shape and CD8 alpha 161 being an asymmetric molecule. Taking into account the contribution of hydration to the frictional coefficient, we obtained for CD8 alpha 161 an axial ratio of approximately 5, when modeled as a prolate ellipsoid. These results indicate that the elongated structure of CD8 alpha 161 is essentially contributed by the hinge region and help to explain how the CD8 alpha is able to bridge the distance between the T-cell surface and its binding site in the alpha 3 domain of major histocompatibility complex class I molecules on the target cell.

A soluble form of the human CD8 alpha chain expressed in the baculovirus system: biochemical characterization and binding to MHC class I.

We have generated a soluble form of the CD8 molecule consisting of the entire extracellular domains of the human alpha chain, by expressing a mutated CD8 alpha cDNA in SF9 cells infected with a recombinant baculovirus. The truncated molecule was secreted into the medium mostly as a disulfide-linked homodimer in which a single cysteine residue in the hinge-like region (Cys143) was sufficient to assure covalent bonding. Soluble CD8 purified to homogeneity appears to be monodisperse as assessed by gel filtration analysis and contains only O-linked carbohydrates. To determine whether recombinant CD8 can interact with MHC class I molecules, we developed an assay that measures binding of MHC class I-bearing cell lines to purified CD8 adsorbed to plastic plates. The level of binding of cells to immobilized CD8 depended on the amount of CD8 bound to the plate and correlated with the levels of cell surface MHC class I expression. The binding was specifically inhibited by monoclonal antibodies directed either against CD8 or MHC class I molecules. This assay therefore provides a way to measure CD8 binding to MHC class I independently of other cell-cell interactions and should allow direct structure-function studies.