CD8β Endows CD8 with Efficient Coreceptor Function by Coupling T Cell Receptor/CD3 to Raft-associated CD8/p56^lck Complexes

Antibodies and Cell Culture.

The following antibodies were obtained from American Type Culture Collection: anti-CD8α mAb 53.6.72, anti-CD8β mAb H35–17, anti-CD8β mAb 53.5.81, anti-TCRCβ mAb H57, anti-TCRCα mAb H28, and anti-CD3ζ mAb HAM146. Anti-phospho-tyrosine (pY) mAb 4G10 and anti-lck mAb 3A5 were from Upstate Biologicals. Anti-LAT, anti-lck, and anti-CD3ε polyclonal antibodies were from Santa Cruz Biotechnology, Inc. Transfected Chinese hamster ovary (CHO) cells were maintained in Glasgow minimum essential medium (Life Technologies) supplemented with 25 μM methionine sulfoximine and 5% dialyzed FCS, as described (14). T cell hybridomas were cultured in DMEM (Life Technologies) supplemented with 5% FCS (Life Technologies), penicillin/streptomycin/neomycin (PSN; Life Technologies), and 50 μM β-mercaptoethanol.

Soluble CD8αβ, T1 TCR, and Biotinlyated K^d-PbCS(ABA).

Soluble T1 TCR and CD8 were prepared essentially as described (14). In brief, the cDNA encoding the extracellular portions of the T1 TCR α and β chains were fused to the sequences encoding the basic and acidic units of a leucine zipper, respectively. The constructs were cloned into the pEE14GS vector using the EcoRI site and stably transfected into CHO-K1 cells with yields of ∼8 mg/l. sT1 TCR was purified on H28-Sepharose, using acid elution (pH 3.1). The extracellular portions of murine CD8α (residues 1–156) and CD8β (residues 1–146) were fused to basic and acidic leucine zipper, cloned into pEE14GS, and stably expressed in CHO-K1 cells, with yields of ∼10 mg/l. sCD8 was purified on H35–17 Sepharose, using acid elution (pH 3.1). Correct folding of the purified sCD8, was confirmed by ELISA using five different anti-CD8 monoclonal antibodies (anti-CD8α mAb 53.6.72, 19/178 and anti-CD8β mAb 53.5.8, KT112, H35–17). Monomeric covalent K^d-¹²⁵”iodo-4-azidosalicylic acid (IASA)”-YIPSAEK (ABA) I complexes (∼2,000 Ci/mMol) were prepared as described (6, 9, 20). Biotinylated K^d-SYIPSAEK(ABA)I was obtained by refolding of K^d heavy chain and human β 2-microglobulin from Escherichia coli as described (20). The monomers were biotinylated, purified, and oligomerized by reaction with streptavidin (Molecular Probes) as described (6, 20).

CD8 Transfections of T1.4 Hybridomas.

T1.4 T cell hybridomas and their CD8α transfectants have been described previously (6, 10). The CD8β cDNA, cloned in the pMI vector, which contains human tailless CD2 (24), was mutated at position 178 (Tyr → Stop) or 179 (Cys → Ala) resulting in CD8β′ and CD8β′′, respectively, using the Quick-Change Mutagenesis Kit (Stratagene). For the chimeric CD8α construct α_β the CD8α extracellular and transmembrane cDNA (residues 1–190) was fused with the cytoplasmic CD8β cDNA (residues 176–194). The cDNA of the chimeric CD8β construct β_α was obtained by fusing the CD8β extracellular and transmembrane coding regions (residues 1–175) with the cytoplasmic CD8α cDNA (residues 191–220). The different cDNA were cloned in the pMI vector (24) and transfected into CD8β⁻, CD8α⁺ T1.4 hybridomas by retroviral gene transfer using the Phoenix packaging cell line (24). Phoenix cells were transfected using calcium phosphate precipitation. After 4 d the cells were sorted and then cloned for high CD2 expression using PE-labeled anti-CD2 mAb (BD PharMingen) and a FACStar™ (Becton Dickinson).

Calcium Measurements and TCR Photoaffinity Labeling.

P815 cells (10⁶/ml) were pulsed or not with 1 μM of IASA-YIPSAEK(ABA)I or IASA-YISSAEK(ABA)I (P255S) and then UV irradiated at >350 nm as described (6, 25). T cell hybridomas (10⁶/ml) were incubated with 5 μM Indo-1/AM (Sigma-Aldrich) at 37°C for 45 min, washed in DMEM, and incubated with P815 cells at an E/T ratio of 1/3 for 2 min. Calcium-dependent Indo-1 fluorescence was measured on a FACStar™ as described (25). TCR photoaffinity labeling of T1.4 hybridomas was performed as described (9–11, 20). In brief, hybridomas (10⁷/ml) were incubated with K^d-¹²⁵IASA-YIPSAEK(ABA)I for 2 h at 0–4°C. After UV-cross-linking at 312 ± 20 nm the cells were washed and lysed in RIPA buffer (50 mM HEPES [pH 7.4], 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM PMSF, 10 μg/ml leupeptin, 10 μM pepstatin A, 5 mM benzamidine, 2 μg/ml aprotinin) for 30 min on ice. The detergent soluble fractions were immunoprecipitated with H57-Sepharose and the samples analyzed by SDS-PAGE (10%, reducing) and PhosphorImaging using a Fuji BAS1000 PhosphorImager.

Isolation of DIM.

T cell hybridomas (5 × 10⁷) were lysed for 30 min on ice in MNE buffer (25 mM MES [pH 6.5], 150 mM NaCl, 5 mM EDTA) containing 1% Triton X-100 (Sigma-Aldrich). Alternatively, 0.5% Brij58 (Fluka) without EDTA was used (21). The lysates were homogenized with a Dounce homogenizer (10 strokes) and fractionated on sucrose density gradients as described (6). The gradients were fractionated from the top in 10 fractions. In some experiments the fractions 2–4 and 6–9 were pooled and referred to as DIM and detergent soluble membrane (M) fractions, respectively. Surface biotinylation was performed as described (6). For immunoprecipitation the fractions were incubated with Sepharose-conjugated anti-CD8α mAb 53.6.72, anti-CD8β mAb H35–17, or anti-TCR mAb H57 for 3 h at 4°C. The immunoprecipitates were resolved on SDS-PAGE (10%, reducing), Western blotted with streptavidin-horseradish peroxidase (HRP; GIBCO BRL) or anti-CD8α antiserum and revealed by enhanced chemoluminescence (ECL) as described (6, 20).

Coimmunoprecipitations.

To assess association of CD8 with lck, hybridomas (10⁷) were lysed in lysis buffer (1 ml; 50 mM HEPES [pH 7.4], 150 mM NaCl, 1% Brij96 [Fluka], and protease inhibitors) for 60 min on ice. After centrifugation for 20 min at 12,000 g and 4°C, the supernatants were incubated for 3 h at 4°C with anti-CD8 mAb 53.6.72 or H35–17 conjugated to Sepharose 4B (6). After two washes with lysis buffer, the samples were analyzed by SDS-PAGE (10%, reducing) and Western blotting using anti-lck mAb 3A5. To detect association of CD8 with TCR/CD3, cells (2 × 10⁷) were lysed likewise in 1 ml lysis buffer containing 0.3% NP-40 (Sigma-Aldrich) instead of Brij96. After one wash with lysis buffer, the immunoprecipitates were analyzed by SDS-PAGE (10%, reducing) and Western blotting using anti-CD8α antiserum.

Analysis of lck and CD3ζ Tyrosine Phosphorylation.

T1.4 hybridomas (5 × 10⁷) were incubated in DMEM (5 ml), supplemented with 1% BSA and 10 mM HEPES (pH 7.4) with 50 nM of K^d −SYIPSAEK(ABA)I tetramer for 20 min at 26°C followed by 2 min at 37°C. The cells were lysed in 1 ml of MNE buffer containing 1% Triton X-100, protease inhibitors (see above), 1 mM Na₃VO₄, and 10 mM NaF for 30 min on ice. The DIM fractions were lysed in 1 ml RIPA buffer supplemented with protease and phosphatase inhibitors. The soluble fractions were incubated for 3 h at 4°C with anti-CD3ζ HAM146-Sepharose or Protein A Sepharose, preincubated with anti-lck antiserum. After two washes with lysis buffer, the immunoprecipitates were analyzed by SDS-PAGE and Western blotting with anti-pY mAb 4G10.

BIAcore Measurements.

Surface plasmon resonance (SPR) studies were performed on a BIAcore 2000 (BIAcore AB) as described previously (15, 26). In brief, streptavidin (Sigma-Aldrich) was covalently coupled to Research Grade CM5 sensor chips using the Amine Coupling Kit (BIAcore). Coupling levels ranged from 6,000 to 8,000 response units (RU). Biotinylated K^d-SYIPSAEK(ABA) or, as control, soluble human CD4 (sCD4), were immobilized via streptavidin. To eliminate aggregates, sCD8αβ and sT1 TCR were purified by gel-filtration on a Superdex S-200 column (Amersham Pharmacia Biotech) and used within 24 h. Experiments were performed at 25°C using HBS buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3.4 mM EDTA, and 0.005% Surfactant P20; BIAcore) and a flow-rate of 10 μl/ml.

Soluble CD8αβ and T1 TCR Bind Independently to K^d-PbCS(ABA).

To assess whether the extracellular portion of CD8αβ affects TCR-ligand binding, we prepared soluble CD8αβ and T1 TCR. Purified soluble sT1 TCR was tested for ligand binding by TCR photoaffinity labeling with soluble K^d-¹²⁵“IASA”-YIPSAEK(ABA)I (9–11). After UV irradiation it exhibited on reducing SDS-PAGE gels one labeled specie of 80–90 kD corresponding to the complex of sK^d-PbCS (ABA) and one chain of the T1 TCR. This material was immunoprecipitated with anti-TCRCβ mAb H57 or anti-TCRCα mAb H28, confirming the heterodimeric nature of the sT1 TCR. To ascertain the biological activity of the soluble CD8αβ, it was tested as inhibitor in a cytolytic assay using PbCS(ABA) pulsed P815 cells as targets. Increasing concentrations of sCD8αβ inhibited the lysis of P815 cells by cloned PbCS(ABA)-specific S14 CTL, with a half maximal inhibition at 0.66 mg/ml (10 μM), and maximal inhibition of 80% at 2 mg/ml (data not shown), indicating that sCD8αβ competes with cell-associated CD8αβ.

SPR studies were performed to assess the interactions of sT1 TCR and sCD8αβ with immobilized K^d-PbCS(ABA). As shown in Fig. 1 A, sT1 αβ bound to K^d-PbCS(ABA) in a dose-dependent manner with an equilibrium constant K_D of 4 ± 0.4 μM. No binding was detectable when HLA-A2-flu matrix peptide complexes or CD4 were used instead of K^d-PbCS(ABA) or sT1 TCR (data not shown). sCD8αβ bound also in a dose-dependent manner to immobilized K^d-PbCS(ABA) (Fig. 1 B), but with low affinity (K_D 99 ± 10 μM), which is in agreement with published values (13, 14). To determine whether CD8αβ affects TCR-ligand binding, sT1 TCR was tested for binding to immobilized K^d-PbCS(ABA) in the presence or absence of 80 μM of sCD8αβ. As shown in Fig. 1 C, sCD8 did not increase the binding of sT1 TCR to K^d-PbCS(ABA). Consistent with this we found that sCD8αβ has no effect on TCR photoaffinity labeling on CD8⁻ T1.4 cell hybridomas (data not shown). These findings demonstrate that sCD8αβ does not affect the interaction of T1 TCR with K^d-PbCS(ABA). This is in accordance with a study showing that CD8αα has no effect on TCR-ligand binding (15), but not with another study claiming that soluble CD8αβ does (13).

The CD8β Transmembrane and Cytoplasmic Portions Are Required for Efficient Intracellular Calcium Mobilization in CD8-transfected T1.4 Hybridomas.

To evaluate the role of CD8β transmembrane and cytoplasmic portions for CD8 coreceptor function, we transfected CD8α⁺ T1.4 hybridomas (6, 10) with CD8β or the CD8β variants shown in Fig. 2. While the T1 CTL clone expresses only CD8αβ, the hybridomas express substantially more CD8α than CD8β, i.e., express homodimeric CD8αα and heterodimeric CD8αβ. The different hybridomas were tested for intracellular calcium mobilization upon incubation with P815 cells sensitized with IASA-YIPSAEK(ABA)I. CD8αβ cells exhibited higher calcium mobilization than the other cells (Fig. 3 A). More striking differences among the different cells emerged, when the peptide variant IASA-YISSAEK(ABA)I (P255S) was used, which is a weak agonist for T1 CTL (25). In this case, the CD8αβ⁺ cells displayed nearly the same calcium mobilization as observed for the wild-type peptide, whereas no change was observed in CD8⁻ or CD8αα⁺ cells. Hybridomas expressing CD8αβ′ or CD8αα_β responded less efficiently than those expressing CD8αβ′′ or CD8αβ_α.

We also measured intracellular calcium mobilization of the indicated hybridomas after incubation with K^d-SYIPSAEK(ABA)I tetramer. As shown in Fig. 3 B, only cells expressing CD8αβ, but not those expressing no CD8, CD8αα, or CD8αβ′ exhibited significant increase in intracellular calcium upon incubation with tetramer. As assessed by staining with Cy5 labeled tetramer, binding was maximal on all cells under the indicated conditions (data not shown).

The CD8β Tail Is Essential for CD8 Partitioning in Rafts and Efficient Association with lck.

As CD8 association with lck seems to take place preferentially in rafts (6), we examined the partitioning of the different CD8 molecules in rafts. To this end the different cells were fractionated in Triton X-100 soluble (M) and insoluble (DIM) fractions and their CD8 content assessed. As shown in Fig. 4 A, CD8αβ and C8αα_β, efficiently partition in DIM, whereas CD8αβ′ and CD8αβ′′ did not. Thus, mutation of Cys179 of CD8β impaired CD8 association with rafts as dramatically as deletion of the CD8β tail. Interestingly CD8αβ_α was significantly more raft-associated as compared with CD8αβ′ and CD8αβ′′, indicating that the transmembrane portion of CD8β is also involved in the recruitment of CD8 to rafts. Essentially the same results were obtained, whether or not EDTA was present in the lysis buffer, i.e., whether or not CD8 could associate with lck (data not shown). These results show that raft-association of CD8 is essentially mediated by the CD8β tail and this mainly due to palmitoylation of its Cys 179.

We next examined the association of CD8 with lck by coimmunoprecipitation. For simplicity we used here total Brij96 lysates, rather than M and DIM fractions, as this detergent has been used previously to assess CD8 association with lck (6–8). As the CD8 transfectants under study express CD8αα and CD8αβ (Fig. 2), we performed immunoprecipitation with anti-CD8α mAb, which precipitates CD8αα and CD8αβ (Fig. 4 B), as well as with anti-CD8β mAb, which precipitates only CD8αβ (Fig. 4 C). We find that CD8αβ and CD8αα_β associate more efficiently with lck, compared with CD8αα, CD8αβ′, CD8αβ′′, and CD8αβ_α. The superior lck association with CD8αβ was especially visible in anti-CD8β immunoprecipitates. This is so, because these cells express substantially higher amounts of CD8αα than CD8αβ. Thus, even though the association of CD8αα with lck is weak, it is well visible due to its high expression. The amounts of CD8 in anti-CD8α or CD8β immunoprecipitates were comparable for the different hybridomas (Fig. 4, D and E). These results demonstrate that efficient association of CD8 with lck takes place in rafts.

As it has been reported that CD8 associates with LAT, in a manner similar as it associates with lck (5, 23), we Western blotted the same CD8 immunoprecipitates with anti-LAT antibody. Even though LAT was well detectable in total lysates, no LAT was found in the CD8 immunoprecipitates (data not shown).

CD8β Tail Is Required for CD8-mediated Increase in TCR-ligand Binding.

To assess the ability of the various CD8 constructs to strengthen TCR-ligand binding, we TCR photoaffinity labeled the different cells with soluble, monomeric K^d-¹²⁵”IASA”-YIPSAEK(ABA)I. As shown in Fig. 5, T1 TCR photoaffinity labeling was highest on CD8αβ⁺ cells. In the presence of anti-CD8β mAb H35–17, which blocks binding of CD8 to TCR-associated K^d-PbCS(ABA) (10), TCR photoaffinity labeling was reduced by 12-fold, to the same low levels observed on CD8⁻ cells. CD8αα was quite unable to increase TCR-ligand binding, which is consistent with previous findings in a related system (4). Importantly, deletion of the cytoplasmic domain of CD8β (CD8αβ′) or substitution with the one of CD8α (CD8αβ_α) caused a significant (67%) reduction in TCR photoaffinity labeling, demonstrating that the CD8β tail is required for efficient CD8-mediated increase of TCR-ligand binding. By contrast, mutation of CD8β Cys 179 (CD8αβ′′) caused only a modest decrease in TCR photoaffinity labeling. As this variant poorly associates with lck (6; Fig. 4), it appears that CD8/lck supports TCR-ligand binding somewhat better than CD8αβ alone. It is noteworthy that CD8αβ′ and CD8αβ_α exhibit partially increased TCR photoaffinity labeling as compared with CD8αα, indicating that the extracellular and/or transmembrane regions of CD8β also play a role in enhancing TCR interactions with MHC-peptide.

The CD8β Tail Mediates Constitutive Association of CD8 with TCR/CD3.

Our results so far indicate that CD8αβ increases the avidity of TCR-ligand binding on cells, but not in solution and that the tail of CD8β is needed for this effect (Figs. 1 and 5). We next examined whether this is so, because on cells CD8 associates with the TCR. To this end, we lysed hybridomas expressing no CD8, CD8αα, CD8αβ, CD8αβ′, or CD8αβ′′ in 0.3% NP-40 and immunoprecipitated TCR. As shown in Fig. 6 A, the amount of CD8α coimmunoprecipitated with TCR was substantially higher on CD8αβ⁺ or CD8αβ′′⁺ cells, than on CD8αα⁺ or CD8αβ′⁺ cells. These differences were not accounted for by variations of TCR or CD8 expression, as all cells expressed comparable levels of CD3ε, CD8α, and CD8β (Fig. 6, B and C).

To find out whether K^d-PbCS(ABA) complexes strengthen association of CD8 with TCR, CD8αβ⁺ cells were photo–cross-linked with soluble K^d-SYIPSAEK(ABA)I complexes and analyzed as described in the previous paragraph. No significant increase in CD8 association with TCR/CD3 was observed (Fig. 6 D), indicating that the interaction of CD8αβ with TCR/CD3 is constitutive and not induced by MHC-peptide. Moreover, the coimmunoprecipitation was not impaired by the SH2 peptide pYEEI (data not shown), which has been shown to disrupt activation-induced coupling of CD8 to TCR/CD3 via lck and ZAP-70 (27). Constitutive association of CD8 (and CD4) with TCR/CD3 has been reported previously and low concentrations of NP-40 or Triton X-100 have been recommended to preserve these interactions (28–30). Essentially the same findings where obtained when Brij78 was used, which unlike NP-40 and Triton X-100 solubilizes lipid rafts, indicating that raft integrity was not required for this interaction (data not shown).

CD8αβ Mediates Raft-association of TCR/CD3.

As the previous experiments indicated that CD8β mediates association of CD8 with lck in rafts (Fig. 4), as well as with TCR/CD3 (Fig. 6), we examined whether therefore CD8β induces raft-association of TCR/CD3. To this end we TCR photoaffinity labeled T1.4 T cell hybridomas that expressed CD8αβ, CD8αβ′, CD8αβ′ or no CD8 with soluble K^d-¹²⁵”IASA”-YIPSAEK(ABA)I and fractionated their Brij58 lysates on sucrose density gradients. Brij58 was used rather than Triton X-100, because it better preserves weak molecular interaction of membrane proteins (21). This difference becomes especially apparent after long periods of incubation, such as fractionation on sucrose density gradients. As shown in Fig. 7, the DIM-containing light fractions exhibited substantial amounts of photoaffinity labeled TCR on CD8αβ⁺ cells, but not on cells expressing CD8αβ′, CD8αβ′′, or no CD8. Cells expressing CD8αβ_α also lacked significant amounts of photoaffinity labeled TCR in the light fractions (data not shown). The gradient fractions displayed the expected distributions of thy-1, which is a marker of rafts and CD45, which is excluded from rafts (data not shown, and references 17–21). These findings indicate that the association of TCR/CD3 with CD8αβ promotes association of TCR/CD3 with rafts, and that tail of CD8β is required for this.

CD8αβ⁺ Hybridomas Exhibit Strong lck and CD3ζ Phosphorylation in Response to K^d-PbCS(ABA) Tetramer.

To correlate the intracellular calcium mobilization with activation of signaling molecules, we assessed lck phosphorylation in the different CD8 transfectants. The cells were stimulated with K^d-SYIPSAEK(ABA)I tetramer and lck tyrosine phosphorylation assessed in DIM fractions. CD8αβ cells displayed strong lck tyrosine phosphorylation upon incubation with K^d-SYIPSAEK(ABA)I tetramer, while cells expressing no CD8 or CD8αα, CD8αβ′, or CD8αβ′′ cells exhibited only background lck phosphorylation (Fig. 8, A and B). In accordance with this, CD3ζ tyrosine phosphorylation was highest in CD8αβ⁺ cells activated by K^d-SYIPSAEK(ABA)I tetramer, in particular of the pp23 hyperphosphorylated form (Fig. 8 C). In the M fractions the same pattern of tyrosine phosphorylated lck was discernible in the presence, but not absence of phosphatase inhibitors (data not shown).