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

Abstract

CD8 glycoproteins are expressed as either αα homodimers or αβ heterodimers on the surface of T cells. CD8αβ is a more efficient coreceptor than the CD8αα for peptide Ag recognition by TCR. Each CD8 subunit is composed of four structural domains, namely, Ig-like domain, stalk region, transmembrane region, and cytoplasmic domain. In an attempt to understand why CD8αβ is a better coreceptor than CD8αα, we engineered, expressed, and functionally tested a chimeric CD8α protein whose stalk region is replaced with that of CD8β. We found that the β stalk region enhances the coreceptor function of chimeric CD8αα to a level similar to that of CD8αβ. Surprisingly, the β stalk region also restored functional activity to an inactive CD8α variant, carrying an Ala mutation at Arg8 (R8A), to a level similar to that of wild-type CD8αβ. Using the R8A variant of CD8α, a panel of anti-CD8α Abs, and three MHC class I (MHCI) variants differing in key residues known to be involved in CD8α interaction, we show that the introduction of the CD8β stalk leads to a different topology of the CD8α-MHCI complex without altering the overall structure of the Ig-like domain of CD8α or causing the MHCI to employ different residues to interact with the CD8α Ig domain. Our results show that the stalk region of CD8β is capable of fine-tuning the coreceptor function of CD8 proteins as a coreceptor, possibly due to its distinct protein structure, smaller physical size and the unique glycan adducts associated with this region.

CD8αβ Has Two Distinct Binding Modes of Interaction with Peptide-Major Histocompatibility Complex Class I

“…Considering that the stalk region of CD8␣ is longer than that of CD8␤ (44 versus 35 amino acids), which allows the Ig-like domain of CD8␣ to extend farther from the T-cell surface, we proposed a binding mode for mouse CD8␣␤⅐pMHCI interaction in which CD8␤ and CD8␣ occupy site 1 and site 2, respectively (23). Consistent with this model is our observation that co-expression of WT CD8␤ with the CD8␣ R8A variant resulted in co-receptor activity indistinguishable from that of WT CD8␣␤ (17). Because mouse CD8␣…”

“…mAbs and Flow Cytometry-All of the mAbs used in the indirect flow cytometric analyses and mAb inhibition assays are described previously (17,23).…”

“…␤ Occupies Site 1-We recently showed that CD8␣ R8A ␤ is functionally active (17,28). This suggests that Arg 8 residue of the CD8␣ subunit is dispensable for the co-receptor function of CD8␣ R8A ␤.…”

“…ϩ transfectants triggered by antigen stimulation was quantified by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay described previously (17). In brief, 1 ϫ 10 5 R8 antigen-presenting cells loaded with 10 Ϫ9 -10 Ϫ4 M vesicular stomatitis virus octapeptide was incubated with an equal number of N15 transfectants expressing CD8 variants plus 10 ng/ml phorbol myristate acetate for 24 h. Inhibition of co-receptor activity by anti-CD8 mAb was analyzed by the IL-2 assay.…”

“…Both the extracellular domain and the cytoplasmic tail of the CD8␤ subunit have been implicated in providing increased efficiency of CD8␣␤ (12)(13)(14)(15)(16). We reported previously that the extracellular domain of the CD8␤ subunit is critical for this enhanced efficiency (14) and that introduction of the CD8␤ stalk region is sufficient to confer a CD8␣␤-like co-receptor efficiency to the CD8␣␣ homodimer (17). In addition, the sialylation of the O-linked glycans in the CD8␤ stalk region is differentiation stage-dependent and may modulate the intrinsic activity of CD8␣␤ during the transition from double-positive (CD4 ϩ CD8 ϩ ) to single-positive (CD8 ϩ ) T cells (18,19).…”