Expression, Purification, and Functional Analysis of Murine Ectodomain Fragments of CD8αα and CD8αβ Dimers

Abstract

Soluble mouse CD8␣␣ and CD8␣␤ 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␣␣ f for H-2K b as measured by BIAcore revealed a ϳ65 M K d , similar to that of CD8␣␤ f . Consistent with this result, CD8␣␣ f as well as CD8␣␤ 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␣␤ versus CD8␣␣ co-receptor function on T cells is linked to differences within their membranebound stalk regions and/or intracellular segments. As recently shown for sCD8␣␣, 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.CD8 has been shown to function in mediating signal transduction and adhesion on a subset of cells within the T cell compartment and is critically involved in the development of T cells expressing MHC class I-restricted T cell receptor (TCR) 1 (1). CD8 is encoded by two distinct genes, termed CD8␣ (or Lyt 2) and CD8␤ (or Lyt 3) and expressed on the cell surface as a mixture of disulfide-linked CD8␣␣ homodimers and CD8␣␤ heterodimers (2-7). CD8␣ is a 34 -37-kDa transmembrane glycoprotein whose extracellular segment contains a compact 122-amino acid (aa) N-terminal Ig-like domain and an extended 48-residue stalk region. A 28-aa cytoplasmic domain, including a cysteine motif responsible for interaction with p56 lck , follows a canonical hydrophobic transmembrane anchor. CD8␤ is a 32-kDa glycoprotein sharing a similar architecture as CD8␣ but with Ͻ20% sequence identity (4, 5). The stalk regions of the CD8␣ and ␤ chains are quite different in length, with the CD8␤ stalk being 10 -13 residues shorter than that of CD8␣. Interestingly, the sialic acid content of O-linked glycans adducted to CD8␤ selectively decreases on thymocytes and activated T cells compared with that found on resting T cells. For its cell surface expression, CD8␤ requires association with the CD8␣ subunit, forming a CD8␣␤ heterodimer (6, 8). Moreover, CD8 genes are selectively expressed. Although CD8␣␤ heterodimers are predominantly found on the surface of TCR␣␤ T cells and thymocytes, CD8␣␣ homodimers are additionally expressed on a subset of ␥␦ T cells, intestinal intraepithelial lymphocytes and natural killer cells (9, 10). Hence, it is safe to conclude that the two sets of CD8 co-r...

Essential Role of CD8 Palmitoylation in CD8 Coreceptor Function

“…Similar findings have been obtained using soluble multimeric MHC-peptide complexes and flow cytometry (13). Surprisingly, however, neither CD8␣␣ nor soluble CD8␣␤ increase TCR ligand binding, even though they bind to MHC class I molecules with similar affinities (12,14,15). Another aspect of the role of CD8␤ in CD8 coreceptor function emerged from studies by Hoeveler and Malissen (4) and Irie et al (5) showing that CD8␣␤ associates more efficiently with lck and induces higher lck activation on cross-linking with anti-CD8 Ab, as compared with CD8␣␣ or CD8␣␤ lacking the cytoplasmic tail of CD8␤.…”

“…The reason for this difference is unknown, because in solution CD8␣␣ and CD8␣␤ bind to MHC class I molecules with similar affinities (15). Moreover, the binding site of CD8 for lck resides in the cytoplasmic tail of CD8␣ (3,4).…”

A role for “self” in T-cell activation

“…It is possible that the activation mechanism in CD4 + and CD8 + T-cells is to some extent different. Several groups have measured the affinity of CD8 binding to MHC class I [26][27][28][29] , whereas binding of CD4 to MHC class II has been much more difficult to demonstrate, suggesting that the affinity of the CD4/MHC class I interaction is at least 25-fold lower 30 . Furthermore, even though CD8 and CD4 have similar biological roles, they have little structural similarity 31,32 .…”

Stalk Region of β-Chain Enhances the Coreceptor Function of CD8

“…Anti-mouse CD8␣ mAbs 53.6.72, 19/178, H59, YTS105, and YTS169 as well as anti-mouse CD8␤ mAb YTS156.7 were used to monitor the expression of these CD8 proteins. Anti-mouse TCR V␤5.2 mAb MR9.4 were used to verify the surface expression of TCR (36). Anti-mouse CD8␣ mAb 53.6.72 or anti-mouse CD8␤ mAb YTS156.7 was used for immunoprecipitation of each CD8 subunit.…”