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

Monoclonal Abs

Anti-H-2K^b-specific mAbs 5F1-2-14 (5F1), EH144, Y3, and 28.13.3 were used to monitor the expression level of H-2K^b on the R8 APCs as previously described (35). Anti-mouse CD8α mAbs 53.6.72, 19/178, H59, YTS105, and YTS169 as well as anti-mouse CD8β mAb YTS156.7 were used to monitor the expression of these CD8 proteins. Anti-mouse TCR Vβ5.2 mAb MR9.4 were used to verify the surface expression of TCR (36). Anti-mouse CD8α mAb 53.6.72 or anti-mouse CD8β mAb YTS156.7 was used for immunoprecipitation of each CD8 subunit. Anti-mouse CD3ε 145.2C11 was used to stimulate the N15 hybridoma cells as a positive control for IL-2 production (37).

Flow cytometric analysis

Indirect flow cytometric analysis was performed on the parental N15CD8⁻ cells (negative for CD8 expression), and N15 cells expressing CD8 variants for the surface expression of TCR, CD8α, and CD8β molecules by incubating each cell line with a specific Ab, followed by a secondary Ab (anti-murine or anti-rat) conjugated with FITC (Caltag, San Francisco, CA). A minimum of 5000 cells were analyzed by FACS for each sample.

Mutant H-2K^b APC

APC R8 is a pre-B lymphoma cell line that was transformed with Abelson leukemia virus and was used to present the VSV8 peptide in the context of wild-type H-2K^b. R8.161 is a cell line derived from the parental R8 line that lacks the expression of H-2K^b (35); it was transfected with cDNAs encoding H-2K^b variants to generate the corresponding APCs. To generate alanine substitution mutations in H-2K^b, the H-2K^b cDNA was used as a template for a PCR-based mutagenesis. A single alanine mutation was introduced in the cDNA of H-2K^b corresponding to residues Lys¹⁹⁸ (K198), Gln²²⁶ (Q226), or Asp²²⁷ (D227). Mutated cDNAs were ligated into the pCRII vector (Invitrogen, San Diego, CA) for sequence confirmation. The verified constructs were subcloned into the pSH-xs expression vector, which carries the hygromycin resistance gene (37). The individual cDNA construct was then transfected into the R8.161 line to generate H-2K^b mutant lines, R8.K198A, R8.Q226A, or R8.D227A, as previously described (35). These transfectants were sorted by FACS and selected for clones whose H-2K^b expression levels are similar to that of parental R8 cells.

CD8α constructs

To generate the CD8α^R8A mutant, mCD8α was subjected to mutagenesis using a PCR method as described above. The cDNAs encoding the mutated CD8α (CD8α^R8A) were then subcloned into pSH-xs. The chimeric CD8α (CD8α-β-α-α) construct was generated by ligation of three cDNA fragments: the XbaI-BsrI fragment encoding the Ig-like domain of CD8α, the BsrI-NspI fragment encoding the stalk region of the CD8β, and the NspI-SacI fragment encoding the transmembrane segment (TM) and the cytoplasmic domain of CD8α. The XbaI-BsrI fragment and BsrI-NspI fragment were obtained by restricted digestion of the cDNA construct encoding the wild-type CD8α. The BsrI-NspI fragment was obtained by PCR from the cDNA construct encoding the wild-type CD8β using a 5′ primer, which contains the BsrI site encoding the very C terminus of the CD8α Ig-like domain fused with a small segment of DNA encoding the N-terminal portion of the CD8β stalk region, and a 3′ primer, which contains a small DNA segment encoding the C-terminal portion of the CD8β stalk region, followed by the NspI site-containing segment encoding for the N-terminal portion of the TM domain of CD8α. The cDNA was sequence-verified and subcloned into the pSH-xs vector. The construct encoding the chimeric CD8α^R8A (CD8α^R8A-β-α-α) was similarly generated, except that the XbaI-BsrI fragment encoding the Ig-like region was released by restriction digestion from the cDNA construct of CD8α^R8A.

N15 transfectants expressing CD8α variants

To generate the cell line expressing homodimeric CD8α^R8Aα^R8A, ∼10⁷ parental N15CD8⁻ cells were electroporated with 10 μg of SalI-linearized pSH-CD8α^R8A at 250 V and 800 μF using a cell electroporator (Life Technologies, Gaithersburg, MD). After 48 h the cells were plated in complete RPMI 1640 medium containing 2 mg/ml of hygromycin at a density of 2 × 10⁴ cells/well in 24-well plates and cultured for 2 wk. Hygromycin-resistant clones were analyzed for CD8α expression by FACS. A minimum of 20 positive clones were pooled and sorted for similar levels of CD8 expression. To generate the cell lines expressing the CD8α^R8Aβ heterodimer, the same method was used, except the mutant CD8α^R8A and wild-type CD8β were cotransfected into the parental N15CD8⁻ cells. The resulting transfectants were tested for the cell surface expression of TCR, CD8α, and CD8β by FACS. The coreceptor functions of these cell lines were examined by IL-2 production triggered by R8 APCs loaded with various concentrations of VSV8 peptide. The expression of functional TCR in each N15 transfectant was positively verified by IL-2 production induced by cross-linking of TCR with an anti-CD3ε Ab, 2C11.

IL-2 production assay and inhibition of coreceptor function by anti-CD8α mAb Abs

The IL-2 production of the CD8-expressing N15 transfectants was quantified using the MTT assay (37). The parental R8 APC or mutant APCs expressing H-2K^b variants R8.K198A, R8.Q226A, or R8.D227A were irradiated (3300 rad) and loaded with VSV8 peptide at concentrations ranging from 10⁻⁹ to 10⁻⁴ M at 37°C for 2 h. Approximately 1 × 10⁵ transfectants were then incubated with an equal number of APCs in a total volume of 200 μl of complete RPMI supplemented with 10 ng/ml of PMA in 96-well plates for 24 h. Supernatants were harvested and stored at −70°C. CTLL-20 cells, an IL-2-dependent cell line, were incubated with serially diluted supernatant in 96-well plates (10⁴ cells/well) for 48 h. The MTT substrate (3-[4,5-dimethylthiazol-z-yl]2–5-diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis) was added 4 h before harvesting by dissolving cells in SDS/isopropanol/HCl. The solubilized MTT product was measured at 570 nm using the SpectraMax 250 spectrophotometer (Molecular Devices, Sunnyvale, CA). The IL-2 concentrations in the supernatants were determined based on the standard curve generated by purified recombinant human IL-2 (Chiron, Emeryville, CA).

Inhibition of coreceptor function by anti-CD8α mAbs was analyzed similarly to the IL-2 production assay, except that 10 μg/ml of affinity-purified anti-CD8α mAb, 53.6.72, YTS169, or YTS169 Fab in RPMI medium was added to 1 × 10⁶ N15 cells expressing CD8α variants for 30 min before incubation with peptide-loaded APCs.

SDS-PAGE analysis of CD8 proteins

The cell surface CD8 proteins were analyzed by immunoprecipitation of lysates prepared from the CD8 transfectants (20). Briefly, ∼10⁷ cells were surface biotinylated and solubilized by lysis buffer containing TBS (10 mM Tris and 150 mM NaCl, pH 7.6), 1% Triton X-100, 1% aprotinin, 1 mM PMSF, 2 mM EDTA, and 5 μg/ml leupeptin. Postnuclear supernatants were precleared and immunoprecipitated using anti-CD8α mAb, 53.6.72; anti-CD8β mAb, YTS156; or control mAb, 2H11, prebound to γ Bind Plus beads (Amersham Pharmacia Biotech, Arlington Heights, IL). After washing the beads four times with lysis buffer, beads were treated with Laemmli sample buffer, boiled, and resolved by a 12.5% SDS-PAGE under reducing conditions. Proteins were blotted onto nitrocellulose membranes and probed with HRP-conjugated streptavidin, followed by visualization using the ECL substrates (Amersham Pharmacia Biotech).

Comparison of the stalk regions of CD8α and CD8β

Both CD8α and CD8β contain a stalk region that is directly adjacent to the Ig-like domain and may therefore play a role in mediating CD8-MHCI interaction. To investigate the role(s) of the stalk region, we compared the polypeptide sequences of both CD8α and CD8β stalk regions of murine, rat, and human proteins. In Fig. 1⇓A, polypeptide sequences are aligned by a pair of conserved cysteine residues. The Cys¹⁵¹ and Cys¹⁶⁶ of the murine CD8α are aligned with Cys¹³⁷ and Cys¹⁵⁰ of the murine CD8β. This alignment reveals that for the region before the first cysteine residue (Asn¹²³ to Asp¹⁵⁰ in murine CD8α and Asp¹¹⁶ to Gln¹³⁶ in murine CD8β) CD8α is less conserved than CD8β, with 28% (8 of 28) and 66% (14 of 21) identity, respectively. For the region between the aligned cysteine residues, CD8α and CD8β have a similar degree of conservation between species, but have no obvious homology to each other within the same species. Overall, compared with CD8α, CD8β has a shorter (35 vs 44 residues for murine proteins) and more conserved (66 vs 28%, among murine, rat, and human proteins) stalk region. Differences revealed by this alignment suggest that the stalk region of CD8β may have a role distinct from the stalk region of CD8α.

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FIGURE 1.

The β stalk region enhances the Ag sensitivity of the CD8αα coreceptor. A, Comparison of the protein sequences of the stalk regions of mouse (m), rat (r), and human (h) CD8α and -β. The alignment is based on the conserved cysteine residues amino terminal to the TM domains of CD8α and -β, with identical residues among the species boxed. Note the remarkable conservation of the amino acids between 116–150 (m) of the β-chain, which contains five conserved potential O-linked glycosylation sites (open circle lollipop). Known sites of O-linked glycosylation in rat CD8α and proposed glycosylation in human CD8α are circled (45 ,46 ). A potential N-linked glycosylation site in murine CD8α is marked as a triangle lollipop, and a potential O-linked glycosylation site is marked as open circle lollipop. Note that the potential glycosylation sites of CD8α among the species are not well conserved. No sequence homology between the CD8α and -β stalks is evident in this comparison. B, Schematic representation of wild-type CD8α and chimeric CD8α. Wild-type CD8αα is composed of two α subunits held together by two disulfide bonds at the stalk regions. Each α subunit includes four domains: one Ig-like domain (aa 1–122) containing two N-linked glycosylation sites (open triangle lollipop), one 44-residue (aa 123–166) long stalk region containing one potential N-linked glycosylation site and multiple O-linked sites, one TM (aa 167–192), and one cytoplasmic tail (Cyto; aa 193–220) containing an lck-binding motif. The chimeric CD8α, (α-β-α-α)₂, is phenotypically a CD8αα homodimer, but has a β stalk region instead of an α stalk region. The most notable difference is that one potential N-linked and a cluster of O-linked glycosylation sites are missing immediately adjacent to the Ig-like domain. C, FACS analysis of the N15 CD8 transfectants. The surface expression of TCR, CD8α, and CD8β epitopes on the transfectants was detected by indirect fluorescence staining with specific mAbs, and species-specific secondary Abs. The filled curve represents specific staining with mAb, and the open curve corresponds to background staining (FITC-labeled secondary Ab alone). Histograms were compiled from a minimum of 5000 cells. In these cells only CD8αβ wild type expresses the CD8β Ig domain on the cell surface. D, IL-2 production assay of the transfectants expressing CD8 variants. CD8α transfectants were activated with H-2K^b R8 APC pulsed with 10⁻⁹–10⁻⁴ M VSV8 peptide. IL-2 production curves of the transfectants are shown as an open circle for CD8αα-wt, a triangle for CD8αβ-wt, and a filled circle for the chimeric CD8α.

Stalk region of CD8β subunit enhances the coreceptor function of CD8αα

To examine the role of the CD8β stalk, we created a chimeric CD8α (CD8α-β-α-α) subunit by replacing the stalk region of wild-type CD8α (CD8α-α-α-α) with the stalk region of CD8β (Fig. 1⇑B). The chimeric CD8α is now nine residues shorter than the wild-type CD8α and lacks the N-linked and a cluster of O-linked carbohydrates present in the wild-type protein directly following the Ig-like domain. The chimeric CD8α was then transfected into a T cell hybridoma N15TCRCD8⁻ cell line whose TCR is specific for the VSV8 peptide Ag (vesicular stomatitis virus nucleocapsid protein residues 52–59, RGYVYQGL) in the context of H-2 K^b, but lacks the expression of the CD8 protein (37). As shown in Fig. 1⇑C, the chimeric CD8α is able to express, on the cell surface, a comparable level of N15TCR as detected by MR 9.4, and CD8α molecules, detected by 53.6.72, and H59 Ab to the CD8αα, or CD8αβ wild-type cell lines. Only CD8αβ wild type has detectable CD8β on the surface, as detected by CD8β Ig domain-specific Ab, YTS156 (36). With the introduction of the chimeric CD8α coreceptor, this cell can be triggered by VSV8 peptide Ag to produce IL-2 (Fig. 1⇑D). Interestingly, at concentrations of peptide Ag <10⁻⁶ M, the chimeric CD8α-expressing cells are 40–100 times more sensitive than the wild-type CD8α-expressing cells, while at higher Ag concentrations such a difference was no longer observed. The IL-2 production curve of chimeric CD8α is almost identical with that of CD8αβ wild type (Fig. 1⇑D). Thus, the CD8β stalk region alone is capable of enhancing the coreceptor function of CD8α.

Wild-type CD8β restores the Ag sensitivity of a functionally inactive CD8α mutant, Arg8Ala

The x-ray structure of the H-2K^b/mCD8αα or HLA-A2/hCD8αα complex revealed that Arg⁸ (R8) of murine CD8α or Arg⁴ (R4) of human CD8α forms multiple hydrogen bonds and salt bridges with the β₂-microglobulin (β₂M) (38, 39). To verify that these interactions between Arg⁸ and β₂M are functionally relevant, we generated a murine CD8α carrying an alanine residue in this position (CD8α^R8A) and tested for its coreceptor function in the N15 T cell hybridoma. Fig. 2⇓A, upper panel, shows that the N15 cells expressing CD8α^R8A require ∼1000-fold higher concentrations of peptide Ag to induce a level of IL-2 production similar to that of wild-type CD8α-expressing cells, indicating that the Arg⁸-β₂M interaction is important for the coreceptor function of CD8αα homodimers. Interestingly, coexpression of CD8β rescues the coreceptor function of CD8α^R8A (Fig. 2⇓A, lower panel). This result suggests that the interaction between Arg⁸ located within the Ig-like domain of CD8α and β₂M is not required for the coreceptor function of CD8αβ heterodimers.

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FIGURE 2.

The β stalk region restores the Ag sensitivity of a functionally defective mutant, CD8α^R8A. A, R8 residue of CD8α1 subunit is critical for coreceptor function of CD8αα, but not CD8αβ. Upper panel, IL-2 production curves of cell lines expressing wild-type CD8αα and CD8α^R8Aα^R8A. Lower panel, IL-2 production curves of the wild-type CD8αβ and CD8α^R8Aβ. Units of IL-2 production include the mean ± SD of triplicate samples. B, FACS analysis of the CD8α and chimeric CD8α N15 T cell transfectants. The surface expression of TCR and CD8α epitopes on the transfectants was detected by indirect fluorescence staining as described previously. The cells express a comparable level of TCR and CD8α molecules. The R8A mutation abolishes H59 Ab binding. C, IL-2 production curves of transfectants expressing chimeric CD8 variants. IL-2 production curves are represented with a filled circle for CD8α^R8A, an open circle for chimeric CD8α^R8A, a filled triangle for chimeric CD8α, and an open triangle for CD8αβ-wt. IL-2 production was detected at 10⁻⁹ M VSV8 peptide in the cell line CD8α^R8A-β-α-α, which is a 1000-fold decrease, in terms of the amount of peptide concentration required, relative to the CD8α^R8A-α-α-α mutant.

Stalk region of CD8β restores the Ag sensitivity of a functionally defective CD8α^R8A Ig-like domain

The results in Fig. 2⇑A raise the question of whether the Ig-like domain and/or the stalk region of CD8β are responsible for restoring the function of CD8α^R8A. The availability of the chimeric CD8α allows us to test whether the stalk region of CD8β plays a role in the function of this CD8α^R8A mutant. We introduced the Arg8Ala mutation into the chimeric CD8α to generate the chimeric CD8α^R8A. Cells expressing the wild-type CD8α, the chimeric CD8α, the chimeric CD8α^R8A, or the wild-type CD8αβ were compared for their sensitivities to peptide Ag. To eliminate the possibility that the difference in IL-2 production is due to a difference in the expression of TCR and/or CD8 proteins, cells were analyzed by FACS for surface expression of these proteins. Fig. 2⇑B shows that the N15TCR expression is quite comparable in these cell lines, as monitored by the Vβ5-specific Ab, MR9.4. Expression levels of CD8α on these cell lines were evaluated by staining with five anti-CD8α Abs: 53.6.72, 19/178, H59, YTS105, and YTS169. Fig. 2⇑B shows that the staining patterns of all Abs, except H59, are very similar, indicating comparable expression levels of CD8α proteins. H59 detects the wild-type CD8α, but not the CD8α^R8A mutant or the chimeric CD8α^R8A. It appears that Arg⁸ is part of the epitope for H59 Ab. Thus differential staining detected by H59 and other Abs also allow us to verify the identity of the CD8α^R8A variant. Importantly, since chimeric CD8α^R8A retains antigenic epitopes of all four anti-CD8α Abs, it appears that the introduction of the CD8β stalk region did not alter the overall structure of the Ig-like domain of the chimeric CD8α protein.

The functional impact of the replaced β stalk in the context of CD8α^R8A was evaluated by IL-2 production triggered by peptide Ag. Fig. 2⇑C shows that, to our surprise, the N15 cells expressing chimeric CD8α^R8A are ∼1000-fold more sensitive to peptide Ag than those expressing CD8α^R8A with the α stalk. The peptide Ag sensitivity of chimeric CD8α^R8A-expressing N15 cells is actually comparable to that observed in cells expressing either chimeric CD8αα homodimers or wild-type CD8αβ heterodimers (Fig. 2⇑C). Thus, not only can the functional defect caused by the Arg8Ala mutation in the CD8α Ig-like domain be fully rescued by the replacement of the β stalk region, but it can also enhance sensitivity to a level similar to that of CD8αβ wild type. These results suggest that the introduction of the CD8β stalk region enhanced the coreceptor function of CD8α, presumably by altering the interaction with MHCI such that the Arg⁸-β₂M interaction is no longer required for the coreceptor function. Importantly, these results extend our previous observation and show that, without formally eliminating the role of the Ig-like domain of the CD8β, the stalk region of CD8β is sufficient for rescuing the functional defect in CD8α^R8A.

MHCI employs similar structural determinants to interact with CD8α and chimeric CD8α^R8A

Since the CD8αArg⁸-β₂M interaction is no longer needed for coreceptor function of the chimeric CD8α homodimer, we questioned whether MHCI employs different structural determinants to interact with the chimeric CD8α. To address this, we mutated three key residues (Lys¹⁹⁸ on the AB loop, Gln²²⁶ and Asp²²⁷ on the CD loop) in the α3 domain of H-2K^b which, based on the crystal structure of CD8αα/K^b complex (Fig. 3⇓A), form hydrogen bonds with wild-type CD8α (39). We reasoned that if the Ig-like domains of wild-type CD8α and chimeric CD8α^R8A are oriented differently, their interactions with the α3 domain of H-2K^b are likely to be different. It is conceivable that a change(s) in the CD8α-H-2K^b interaction will affect the efficiency of peptide Ag presentation and, hence, IL-2 production. APCs expressing each of these H-2K^b mutants were generated and used to present VSV8 peptide in the IL-2 production assay. Fig. 3⇓B, upper panel, shows the response of N15 cells expressing wild-type CD8α to peptide Ag presented by wild-type or mutant H-2K^b. APC expressing Gln226Ala H-2K^b are ∼10 times less efficient than APC expressing wild-type H-2K^b, and APC expressing Asp227Ala H-2K^b exhibit activity similar to APC expressing wild-type H-2K^b. Interestingly, APC expressing Lys198Ala H-2K^b exhibit a subtle change in Ag presentation; at lower concentrations of peptide Ag they are more efficient, but at higher concentrations they are less efficient, than the APC-expressing, wild-type H-2K^b. Most importantly, very similar profiles were observed when APC cells expressing each of these three MHCI variants were used to present peptide Ag to N15 cells expressing chimeric CD8α^R8A (Fig. 3⇓B, middle panel), including the unique profile observed in the APC expressing Lys198Ala H-2K^b (a characteristic crossing over between K^b-wild type (wt) and K^b Lys198Ala). Besides the similar profile of dose-response curves, the Ag sensitivity of chimeric CD8α^R8A is enhanced by ∼10- to 40-fold when stimulated by APC expressing wild-type K^b or by Lys198Ala or Asp227Ala K^b variants compared with that of CD8αα-expressing cells (Fig. 3⇓B, middle panel). When wild-type CD8αβ transfectants were stimulated with these APCs expressing K^b variants, similar profiles of IL-2 production curves were observed, except that greater amounts of IL-2 were produced and higher sensitivities of Ag recognition were observed at peptide concentrations <10⁻⁶ M (Fig. 3⇓B, lower panel). These observations are in agreement with our findings that the β subunit, or β stalk region, enhances the coreceptor function of CD8. Mutations at residues where MHCI contacts the CD8 Ig-like domain have a similar impact on the coreceptor function of all three cells, wild type CD8α, chimeric CD8α^R8A, and CD8αβ-wt. These results strongly suggest that the H-2K^b employs similar structural determinants for its interactions with these CD8α variants.

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FIGURE 3.

Chimeric CD8α^R8A contacts the same key K^b residues to mediate coreceptor function as CD8αα-wt. A, The crystal structure-based model of mCD8αα/H-2K^b. The mCD8αα/H-2K^b is a RASTER three-dimensional-rendered MOLSCRIPT drawing (47 ,48 ) In the figure the K^b heavy chain is in blue, β₂M is in green, CD8α1 is in yellow, and CD8α2 is in red. The side chains of the R8 residue on both subunits of CD8α are labeled, and the positions of K198, Q226, and D227 in the K^b molecule are indicated. B, IL-2 production curves of transfectant expressing wild-type CD8αα (upper panel), chimeric CD8α^R8A (middle panel), and wild-type CD8αβ (lower panel) that were stimulated with mutant APC loaded with the indicated amounts of VSV8.

Introduction of CD8β stalk compromises the blocking activity of an anti-CD8α Ab

Since the CD8α Arg⁸-β₂M interaction is not required for the coreceptor function of chimeric CD8α, but structural determinants of MHCI employed to interact with the Ig-like domain of CD8α and chimeric CD8α remained the same, we questioned whether introduction of the β stalk leads to a topological alteration in the complex of β₂M and the Ig-like domain of chimeric CD8α. To investigate such a possibility, we analyzed the inhibitory effects of a series of anti-CD8α Abs that were used previously for the evaluation of surface expression of CD8α (Fig. 2⇑B). Each of these Abs recognizes an epitope within the Ig-like domain, and several of them are capable of blocking the coreceptor function of CD8αα and CD8αβ (data not shown). We reasoned that if the overall topology of the CD8α-MHCI complexes were altered due to introduction of the β stalk, the inhibitory activity of one or more of these blocking Abs might be compromised. As shown in Fig. 4⇓A, upper panel, in cells expressing wild-type CD8α homodimer, anti-CD8α mAbs 53.6.72 and YTS169 completely block IL-2 production within a comparable range. However, in the cell line expressing chimeric CD8α^R8A homodimer (Fig. 4⇓A, lower panel), the anti-CD8α mAb 53.6.72 blocks IL-2 production completely, but Ab YTS169 blocks IL-2 production 10- to 100-fold less effectively. Furthermore, this result is similar to the Ab blocking experiment performed on the cells expressing CD8αβ-wt heterodimer (Fig. 4⇓B). Since the differential sensitivity to the YTS169 Ab blocking is not due to any difference in the expression levels of these CD8α variants (Fig. 2⇑B, anti-CD8α staining) or TCR (Fig. 2⇑B, anti-TCR staining), nor is it due to a loss of the antigenic epitope of Ab YTS169 in chimeric CD8α^R8A (Fig. 2⇑B, YTS169 staining), we hypothesized that the less bulky Fab of YTS169 should have more profound differential effects in blocking these two cell lines. Based on the results of these experiments, as shown in Fig. 4⇓A, the Fab of YTS169 is 100 times more effective in blocking the coreceptor function of wild-type CD8α than chimeric CD8α^R8A. Since the antigenic epitope is present in the Ig-like domain of CD8α (36), the lesser effectiveness of YTS169 Ab in blocking IL-2 production on chimeric CD8α-expressing cells suggests that the introduction of the β stalk leads to a change in the interaction between the Ig-like domain of CD8α and MHCI, such that the binding of YTS169 is better tolerated with respect to the coreceptor function. This is consistent with the observation that Arg8Ala is not critical for CD8αβ coreceptor function. These results indicated that introduction of the β stalk does not affect the overall structure of the CD8α Ig-like domain, but does alter the topology of the CD8α-MHCI complex.

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FIGURE 4.

Anti-CD8α Abs block the chimeric CD8α^R8A homodimer differently. A, IL-2 production curves of cell lines expressing homodimer wild-type CD8αα (upper panel) or chimeric CD8α^R8A (lower panel) in the presence or the absence of anti-CD8α mAbs or Fab. B, IL-2 production curves of cell lines expressing heterodimer CD8αβ-wt in the presence or the absence of the blocking Abs.

Chimeric CD8α^R8A retains the glycosylation patterns of the CD8β subunit

The stalk regions of CD8α and CD8β exhibit different glycosylation patterns. The O-linked glycosylation of CD8β is regulated upon differentiation or activation, suggesting that these carbohydrates may play a role in the function of CD8β. We therefore investigated whether the characteristic glycosylation pattern of CD8β is retained in the chimeric CD8α. We showed that surface-expressed CD8β migrates as a set of bands with apparent molecular mass ranging from 30–36 kDa on SDS-PAGE (Fig. 5⇓, lanes 2 and 5), and CD8α migrates as a more discrete band with an apparent molecular mass of ∼42 kDa (Fig. 5⇓, lanes 1 and 2). These patterns are consistent with previous reports, which showed that the glycosylation on CD8α and CD8β is significantly different (20, 32). The cell surface-expressed chimeric CD8α^R8A migrates as a series of bands similar to those of CD8β proteins observed in the CD8αβ complexes (Fig. 5⇓, lane 4 vs lane 2). Thus, the replaced β stalk lowers the apparent overall molecular mass of chimeric CD8α^R8A. Importantly, the broad range of its apparent molecular mass suggests that the β stalk retains its glycosylation patterns in the chimeric CD8α^R8A. Similar heterogeneous bands are observed on the surface of cells expressing chimeric CD8α (data not shown). These results indicate that the chimeric CD8α^R8A is glycosylated similarly to the wild-type CD8β.

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FIGURE 5.

SDS-PAGE analysis of surface CD8α and chimeric CD8α^R8A on T cell transfectants. After surface biotinylation, the CD8α or CD8β subunits were immunoprecipitated from the designated cell lines using mAb 53.6.72 for CD8α or YTS 156 for CD8β. The immunoprecipitates were run on 12.5% SDS-PAGE gels under reducing conditions and blotted onto nitrocellulose membranes, and proteins were detected with ECL. The α subunits of the wild-type homodimer CD8αα and CD8α^R8Aα^R8A were run at 40 kDa as indicated (lanes 1 and 3). Anti-CD8α Ab immunoprecipitated both CD8α and CD8β from transfectants expressing CD8αβ (lane 2). Under reducing conditions, α and β subunits migrate at 40 and 30–36 kDa, respectively. The heterogeneous migration of the CD8β subunit is due to variable glycosylation at O-linked sites on the β stalk region. The same result is observed on the anti-CD8β IP (lane 5). In lane 4, the immunoprecipitation of surface chimeric CD8α^R8A-β-α-α molecules using the anti-CD8α mAb reveals at least two major bands and one minor band with a molecular mass range of 32–38 kDa. This result indicates that the chimeric CD8α^R8A coreceptor shows the heterogeneous mobility of the wild-type β stalk region in SDS-PAGE.