CD3δ Establishes a Functional Link between the T Cell Receptor and CD8*

EXPERIMENTAL PROCEDURES

Antibodies

The following monoclonal antibodies were purchased from BD Pharmingen (San Diego, CA): anti-CD8α 53.6.72 (FITC or Cy5), anti-CD8β H35–17 (PE), anti-TCRβ H57 (allophycocyanide), anti-CD3ε 17A2 (PE), anti-CD4 GK 1.5 (FITC or Cy5), and anti-Thy-1.2 III/5 (Cy5). The anti-CD8β KT112 mAb (32) was purified and labeled with Cy5. For Western blotting antibodies specific for CD3ε (M-20, Santa Cruz Biotechnology, Santa Cruz, CA), CD3δ (17), CD8α tail (33), ζ chain (H146) (American Tissue Culture Collection, Manassas, CA), Thy-1 (rabbit IgG; gift from Dr. C. Bron, University of Lausanne, Switzerland), and CD45 (Transduction Laboratories, San Diego, CA) were used. FITC-labeled rabbit anti-rat antibody was from BD Biosciences. For blocking of CD8 binding to K^d Fab′ fragments of anti-K^d mAb SF1–1.1.1 (SF′) or anti-CD8β mAb H35 were used (32).

Cell Cultures and T1 TCR Transgenic Mice

The CD8⁻, CD8αα⁺, and CD8αβ⁺ T cell hybridomas were obtained and cultured as described previously (29). Hybridomas expressing the αCPM variant TCR (αIV/βIII) were obtained by transfection of CD8⁻ or CD8αβ⁺58 T cell hybridomas as described previously (34) with wild type T1 TCRα and β chains or T1 TCRIVα chain, in which the sequence 225–270 of the TCRα chain was replaced with the corresponding sequence of the TCRδ chain and T1 TCRIIIβ chain in which the sequence 270–300 of the TCRβ chain was replaced with the corresponding sequence of the TCRγ chain (16). Transfected hybridomas were FACS-sorted for high TCR expression and cultured in DMEM containing 2% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 50 μm β-mercaptoethanol, 10 μg/ml G418 (Calbiochem, San Diego, CA), 4 μg/ml puromycin (Calbiochem), and 0.5 mg/ml hygromycin (Calbiochem). To generate T1 TCR transgenic mice (H-2^d/d) cDNA encoding the T1 TCRα and β chains were separately cloned into the transgenic expression vector pHSE3′ using standard methods. XhoI fragments encoding the T1 TCRα and β chains were co-injected into BDF2 zygotes. T1 TCR transgenic BDF2 mice were backcrossed to Balb/c, RAG knockout mice. Backcrossing of the T1 TCR transgene to Balb/c, RAG knockout mice was repeated for over six generations. Surface expressions of TCR and CD8 of the cells under study were determined by FACS by using anti-CD8α mAb 53.6.72 (FITC), anti-CD8β mAb H35 (PE), and anti-TCR mAb H57 (allophycocyanide), respectively. The mean fluorescence intensities (in parentheses) were as follows: for the previously described CD8⁻ T cell hybridomas (3, 5, 134), for CD8aa⁺ hybridomas (492, 8, 82), and for CD8ab⁺hybridomas (453, 427, 72). These cells were used only in FRET analysis. For the hybridomas obtained from transfection of 58 cells, the following mean fluorescence intensities were measured as follows: for CD8αβ⁺ T1 TCR⁺ hybridomas (342, 68, 22), for CD8⁻ T1 TCR⁺ hybridomas (4, 5, 28), for CD8αβ⁺ T1 TCRαIV/βIII⁺ cells (305, 61,19), for CD8⁻ T1 TCRαIV/βIII⁺ hybridomas (4, 5, 25), and for T1 thymocytes (494, 1283, 128).

FACS and FRET Analyses

T cell hybridomas and T1 thymocytes were washed and resuspended at 2 × 10⁶ cells/ml in serum-free Opti-MEM medium (Invitrogen, Merebeke, Belgium) containing 1% BSA and 0.02% NaN₃. For FACS analysis aliquots of 25-μl cell suspension were incubated in a 96-well plates with fluorescence-labeled antibodies (5 μg/ml) for 20 min at 4 °C. After two washes in the same medium, fluorescence associated with live cells was measured on a FACSCalibur (BD Biosciences, Erembodegen, Belgium). For FRET analysis, 50-μl aliquots of cells (2 × 10⁶ cells/ml) were incubated in the same medium in 96-well plates with 5 μg/ml Cy5-labeled anti-CD8α 53.6.72, anti-CD8β KT112, anti-CD4 GK 1.5, or anti-Thy-1 antibody; PE-labeled anti-CD3ε 17A2 mAb; or 50 nm of PE-coupled K^d-PbCS(ABA) multimers. After 45 min of incubation at 4 °C, the cells were washed and fixed with paraformaldehyde (3% w/v in PBS) for 10 min at room temperature, and cell-associated fluorescence was assessed on a FACSCalibur at 580 nm upon excitation at 488 nm (E1), at 670 nm after excitation at 630 nm (E2), and at 670 nm after excitation at 488 nm (E3). The transfer of fluorescence was calculated as FRET units as follows: FRET unit = [E3_both −E3_none] − [(E3_Cy5 −E3_none) × (E2_both/E2_Cy5)] − [(E3_PE − E3_none) × (E1_both/E1_PE)]. The different fluorescence values (E) were measured on unlabeled cells (E _none), or cells labeled with PE (E _PE), Cy5 (E _Cy5), or Cy5 and PE (E _both).

Soluble K^d-peptide Complexes

K^d-¹²⁵“IASA”-YIPSAEK(ABA)I complexes (about 2000 Ci/mmol) were prepared as described previously (31). Non-radioactive K^d-PbCS(ABA) complexes were obtained by refolding of K^d heavy chain and human β2 microglobulin, produced in Escherichia coli using the dilution method (35, 36). The refolded monomers were biotinylated, purified, and reacted with PE-labeled extravidin (Sigma) as described previously (35, 36).

Intracellular Calcium Mobilization

P815 mastocytoma cells (1 × 10⁶ cells/ml) were pulsed with graded amounts of IASA-YIPSAEK(ABA)I for 2 h at 37 °C and UV-irradiated at ≥350 nm for 90 s to cross-link the peptide to K^d. T1 TCR hybridomas or T1 thymocytes (1 × 10⁶ cells/ml) were incubated with 5 μm Indo-1 (Sigma, Buchs, Switzerland) at 37 °C for 45 min, washed in DMEM, and incubated with P815 cells for 1 min at 37 °C at an E/T ratio of 1/3. Calcium dependent Indo-1 fluorescence was measured on a FACStar^TM as described (37).

TCR Photoaffinity Labeling

T1 TCR hybridomas or T1 TCR thymocytes (7 × 10⁶ cells/ml) were incubated in a 12-well plate with K^d-¹²⁵“IASA”-YIPSAEK(ABA)I (0.5–1.5 × 10⁷ cpm/3 × 10⁶ cells). After 1 h of incubation at 26 °C and UV irradiation at 312 ± 40 nm for 30 s with 90 watts, the cells were washed twice and lysed for ≥60 min on ice in 1 ml of PBS containing 50 mm n-octylglucoside and a mixture of protease inhibitors (Roche Molecular Biochemicals, Rotkreuz, Switzerland). TCR was immunoprecipitated using Sepharose-conjugated mAb H57. The immunoprecipitates were resolved on SDS-PAGE (10% reducing), and radioactivity was quantified by phosphorimaging analysis using a Fuji BAS1000 (29-32).

Isolation of Lipid Rafts

T1 TCR hybridomas or T1 TCR thymocytes (5 × 10⁷ cells) photoaffinity-labeled with K^d-¹²⁵“IASA”-YIPSAEK(ABA)I were lysed in 1 ml of MN buffer (25 mm MES, pH 6.5, 150 mmNaCl) containing 0.5% Brij96 (Sigma) and protein inhibitors (Roche Molecular Biochemicals) for 30 min on ice. The lysates were homogenized with a Dounce homogenizer (10 strokes) and fractionated on sucrose density gradients as described (29, 38). The sucrose gradients were fractionated from the top in ten fractions of 500 μl. Aliquots of the fractions were resolved on SDS-PAGE (10%, reducing) and either analyzed by phosphorimaging or Western blotted using antibodies specific for Thy-1 or CD45.

Confocal Microscopy

T1 TCR hybridomas or T1 thymocytes were washed with DMEM and incubated with 10 mmmethyl-β-d-cyclodextrin at room temperature, washed again with medium, and incubated in PBS containing 1% BSA for 30 min at room temperature or for 40 min at 4 °C with PE-labeled K^d-PbCS(ABA) multimers (50 nm). Following two washes with PBS, cells were analyzed using an LSM510 Zeiss confocal microscope. Median sections of cells were recorded. Alternatively, TCR hybridomas were incubated for 20 min at room temperature with anti-CD8β mAb KT112 and anti-TCR mAb H57 mAb in PBS containing 1% BSA, washed, and incubated with FITC-labeled anti-rat IgG antibody, and the distribution of CD8αβ was examined.

Co-immunoprecipitation and Western Blotting

T1 TCR hybridomas or T1 thymocytes (1.5 × 10⁷ cells) were lysed on ice for 2 h in Tris (20 mm, pH 8.0) containing 0.3% Triton X-100 and protease inhibitors (Roche Molecular Biochemicals). The lysates were spun at 10,000 × g for 10 min, and the supernatants were immunoprecipitated using monoclonal antibodies specific for CD8β (KT112), CD8α (53.6.72), or TCR (H57). The immunoprecipitates were washed twice with Tris buffer, pH 8.0, containing n-octylglucoside (50 mm) (Sigma) or as indicated with Tris buffer, pH 8.0, containing Triton X-100 (0.15%), supplemented with EDTA (5 mm), ethylmaleimide (5 mm) (Sigma), or NaCl (0.5m). Immunoprecipitates were resolved on SDS-PAGE (15%, reducing), transferred onto a nitrocellulose membrane, and Western blotted using anti-CD3δ, anti-CD3ε, or anti-CD8α antibodies. For detection the enhance chemiluminescence (ECL) (Amersham Biosciences) was used as described (29).

Modeling of CD3εδ

An homology model of the CD3εδ complex was built based on the CD3εγ 3D structure and CD3δ sequence alignment (5), using the MODELLER program (39). The conformations of the connecting loops of the immunoglobulin fold were refined using an ab initio method based on simulated annealing (40).

RESULTS

CD8αβ and Wild Type TCR αCPM Are Required for Efficient Intracellular Calcium Mobilization

To examine the role of CD8 and the TCR αCPM for T cell activation we first assessed intracellular calcium mobilization in T cell hybridomas expressing CD8 and wild type T1 TCR or T1 TCR in which the αCPM was replaced with the corresponding sequence of TCRδ (T1 TCR αIV/βIII). As shown in Fig. 1 A, hybridomas expressing the wt TCR and CD8αβ exhibited strong calcium mobilization upon incubation with P815 cells pulsed with 10⁻⁶ to 10⁻⁵ m IASA-YIPSAEK(ABA)I. This calcium flux was stable over the assayed period of 15 min. By contrast, no significant calcium mobilization was observed in the presence of the CD8β blocking antibody H35. Similarly, on hybridomas expressing the T1 TCR αIV/βIII, only scant calcium mobilization was observed, which was reduced to background levels in the presence of mAb H35 (Fig.1 B).

Using the same method we next examined thymocytes from T1 TCR transgenic mice. These mainly DP cells exhibited strong and stable intracellular calcium mobilization upon incubation with IASA-YIPSAEK(ABA)I-pulsed P815 cells (Fig. 1 C). Maximal response was observed at 10⁻⁷ mIASA-YIPSAEK(ABA)I. The stronger calcium responses observed on T1 thymocytes, as compared with CD8⁺ T1 T cell hybridomas, is explained, at least in part, by the higher surface expression of TCR and CD8 (see “Experimental Procedures”). In the presence of mAb H35, no marked calcium mobilization was observed, indicating that CD8 was required for this response (Fig. 1 C). Taken together these results indicate that, for efficient calcium mobilization, CD8αβ and CD3δ⁺ TCR are required, which is in accordance with previously studies showing that CD8 and the αCPM are crucial for efficient TCR signaling and positive selection of CD8 SP T cells (16-22).

CD8 Increases MHC-peptide Binding on Cells Expressing Wild Type but Not αCPM Variant TCR

TCR photoaffinity labeling with soluble monomeric K^d-¹²⁵“IASA”-YIPSAEK(ABA)I complexes allows direct assessment of TCR-ligand binding and its dependence on CD8 (30-32). Using this technique we compared TCR-ligand binding on T cell hybridomas expressing the wild type T1 TCR or the T1 TCR αIV/βIII. As shown in Fig.2 A, TCR photoaffinity labeling was reduced by over 6-fold in the presence of Fab′ fragments of anti-K^dα3 mAb SF1–1.1.1, which block CD8 binding to K^d (32). A slightly larger inhibition was observed on T1 thymocytes and on cloned T1 CTL (Fig. 2 B and Ref. 32). The same reductions were observed upon blocking of CD8 with anti-CD8β mAb H35 (data not shown).

Remarkably, on hybridomas expressing T1 TCR αIV/βIII, K^d-PbCS(ABA) binding was over 4-fold lower than on cells expressing wild type T1 TCR and blocking of CD8 binding to K^d caused only a small reduction. The nonspecific labeling, as seen in the presence of anti-K^dα1 mAb 20–8-4S, which blocks binding of K^d to TCR (32), was in the range of 3% on the hybridomas and below 1% on thymocytes. Upon blocking of CD8 binding to K^d, TCR photoaffinity labeling was slightly lower on hybridomas expressing T1 TCR αIV/βIII, as compared with hybridomas expressing the wild type T1 TCR (Fig. 2 A). Because the TCR expression is slightly lower on the former as compared with the latter cells (see “Experimental Procedures”), it seems that both TCR bind K^d-PbCS(ABA) with very comparable efficiency. This is consistent with the fact that both TCR have the same variable and constant domains and argues that the poor TCR photoaffinity labeling observed on T1 TCR αIV/βIII is accounted for by inefficient participation of CD8 in TCR ligand binding. Because CD8-mediated increase in TCR-ligand binding relies on association of CD8 with TCR·CD3 (29), this argues that the T1 TCR αIV/βIII associates poorly with CD8.

Wild Type but Not αCPM Variant TCR Associates with CD8 on Intact Cells

To validate this conclusion, we assessed the proximity of TCR·CD3 and CD8 by FRET. To this end we stained hybridomas expressing CD8 and wild type T1 TCR in the cold with Cy5-labeled anti-CD8β mAb KT112 and PE-labeled K^d-PbCS(ABA) multimers and measured the FRET value from PE to Cy5 by FACS. As shown in Fig.3 A, on CD8⁺hybridomas expressing the wild type T1 TCR, FRET was 2.2 units but only 0.4 unit on hybridomas expressing the T1 TCR αIV/βIII. The nonspecific signal, as recorded on the corresponding CD8⁻hybridomas, was about 0.03 unit. When using PE-labeled anti-CD3ε mAb 17A2, slightly less efficient FRET was observed (1.5 units, Fig.3 B). Remarkably, this FRET value was enhanced very little when soluble K^d-PbCS(ABA) monomers were present in the incubation at saturating concentration, indicating that the proximity of CD8 and TCR·CD3 on these hybridomas was not induced by MHC-peptide, i.e. it was constitutive.

To investigate which chain of CD8 was important for coupling of CD8 with TCR·CD3, we performed FRET experiments on hybridomas expressing CD8αβ or CD8αα. Upon staining of CD8αβ⁺hybridomas with PE-labeled K^d-PbCS(ABA) multimers and Cy5-labeled anti-CD8β KT112, a 4.3-fold stronger FRET was observed than on CD8αα⁺ hybridomas stained with Cy5-labeled anti-CD8α mAb 53.6.72 (Fig. 3 C). The nonspecific signal in this experiment, as assessed on CD8⁻ hybridomas, was 0.04 unit.

Strong FRET was observed on T1 thymocytes following staining with PE-labeled K^d-PbCS(ABA) multimers and Cy5-labeled anti-CD8β mAb KT112 (37 units, Fig. 3 D). About one-third lower FRET (25 units) was recorded when using Cy5-labeled anti-CD8α mAb 53.6.72. Because thymocytes express only CD8αβ, but CD8-transfected T cell hybridomas always express high levels of CD8αα (see “Experimental Procedures” and Refs. 29 and 33), the over 4-fold reduced FRET observed on CD8αα⁺ T cell hybridomas indicates that CD8αβ couples with TCR·CD3 more extensively than does CD8αα (Fig. 3 C). This is consistent with the finding that CD8αβ is co-immunoprecipitated with the TCR more efficiently as compared with CD8αα (29). In the presence of anti-CD8β mAb H35, this FRET was reduced to 6.2 units, indicating that mAb H35 impedes the association of TCR and CD8. Moreover, faint FRET (5 units) was observed when using Cy5-labeled anti-CD4 mAb GK1.5 as acceptor, which was about 2-fold above background, as recorded when using Cy5-labeled anti-Thy-1 antibody (Fig. 3 D). This is consistent with the observation that CD4 also associates with TCR·CD3 (25, 26). This FRET was not reduced in the presence of mAb H35, indicating that this antibody does not impair the PE K^d-PbCS(ABA) multimer staining. The differences in FRET values observed in the different experiments in Fig. 3 are accounted for in part by variations in TCR and CD8 expression of the different cells (see “Experimental Procedures”). Together these results indicate that CD8 and TCR·CD3 are in close proximity in T1 thymocytes and in T1 T cell hybridomas, given they express CD3δ⁺ TCR and CD8αβ.

Wild Type but Not αCPM Mutant T1 TCR Docks to Raft-associated CD8

Because wild type T1 TCR, but not variant T1 TCR αIV/βIII, associates with CD8αβ (Figs. 2 and 3) and CD8αβ is a raft constituent (33, 36), we examined the raft association of these two TCR. To this end we TCR photoaffinity-labeled CD8αβ⁺ T cell hybridomas expressing wild type or T1 TCR αIV/βIII, lysed the cells in Brij96, and fractionated the detergent-soluble and -insoluble components on sucrose gradients. A significantly larger fraction of K^d-¹²⁵“IASA”-YIPSAEK(ABA)I-labeled T1 TCR was found in the detergent-insoluble light fractions 2–4 on hybridomas expressing the wild type T1 TCR as compared with hybridomas expressing the T1 TCR αIV/βIII (Fig.4, A and B). To verify that our fractionation procedure was correct, we assessed the distribution of CD45 and Thy-1, which are known markers for the detergent-soluble and detergent-insoluble raft fractions, respectively (41, 42). Indeed CD45 was found exclusively in the detergent-soluble dense gradient fractions (fractions 6–10), whereas glycosylphosphatidylinositol-linked Thy-1 was detected only in the detergent-insoluble light fractions (fractions 2–4) (Fig. 4,E and F).

In T1 thymocytes an appreciable fraction of photoaffinity-labeled TCR was found in the light fractions (Fig.4 C). This fraction was greatly diminished when TCR photoaffinity labeling was performed in the presence of the CD8β-blocking mAb H35 (Fig. 4 D). These findings indicate that a fraction of TCR is raft-associated due to association of TCR·CD3 with raft-resident CD8. This is consistent with the findings that αCPM variant TCR (18) or TCR from CD3δ knockout mice (12) exhibit impaired raft-association and argues that CD3δ couples TCR·CD3 with CD8αβ and hence with lipid rafts.

Cross-linking of TCR·CD8 Adducts Results in the Formation of Large TCR·CD8 Aggregates

We next investigated what consequences TCR·CD3 cross-linking has on TCR aggregation. As assessed by confocal microscopy, incubation of T cell hybridomas expressing CD8αβ and wild type T1 TCR with PE-labeled K^d-PbCS(ABA) multimers at room temperature resulted in extensive patch formation and internalization (Fig. 5 A). The aggregate formation, but not the internalization, also took place when the incubation was performed in the cold (Fig. 5 B), suggesting that it does not require cell activation. Strong TCR·CD8 aggregate formation was also seen on T1 thymocytes upon incubation with K^d-PbCS(ABA) multimers and on CD8αβ⁺, T1 TCR⁺ hybridomas after incubation with anti-TCR, and anti-CD8β antibodies (Fig. 5, A and C). By contrast, on hybridomas expressing T1 TCR αIV/βIII, aggregate formation and internalization were greatly reduced. The same diffuse, mainly surface staining, was also observed when CD8αβ⁺, T1 TCR⁺ hybridomas, or T1 thymocytes were pretreated with methyl-β-cyclodextrin, which destabilizes lipid rafts (Fig.5 A). Taken collectively, these findings demonstrate that co-cross-linking of TCR·CD3 and CD8 results in formation of large TCR·CD8 aggregates and that for this to occur CD8αβ, lipid rafts, and CD3δ⁺ TCR are required.

CD3δ Associates with CD8

We next examined whether CD3δ co-immunoprecipitates with CD8. To this end, we lysed T1 thymocytes in Triton X-100 and analyzed their lysate as well as TCR and CD8 immunoprecipitates by SDS-PAGE and Western blotting. In the CD8 immunoprecipitate, CD3δ, but neither CD3ε nor ζ chains, was highly enriched; especially when compared with the TCR immunoprecipitates, the preferential co-precipitation of CD3δ was striking (Fig. 6 A and data not shown).

To obtain further information on the association of CD3δ with CD3ε and CD8, we washed the CD8 immunoprecipitates twice with different buffers and assessed the amount of co-precipitated CD3δ and CD3ε by Western blotting. The association of CD3δ with CD8 was substantially reduced (to 36%) upon washing with ethylmaleinimide, which alkylates free cysteines (Fig. 6 B). A smaller reduction (to 55%) was observed upon washing with EDTA-containing buffer, which chelates divalent cations. About 40% reduction was noted upon washing withn-octylglucoside, which disrupts association of transmembrane proteins (41). Strikingly, washing with 0.5 mNaCl had no marked effect, suggesting that the association of CD3δ with CD8 is not ionic in nature. By contrast, washing with this buffer removed most of the CD3ε from the immunoprecipitates. The other buffers affected the co-precipitation of CD3ε in the same way as the co-precipitation of CD3δ.

In CD8αβ⁺, T1 TCR⁺ T cell hybridomas, similar as with T1 thymocytes (Fig. 6 A), anti-CD8β antibody efficiently co-immunoprecipitated CD3δ but little CD3ε (Fig. 6 C). In contrast to the T1 thymocytes, there was less CD8 co-immunoprecipitated with the TCR, especially when compared with the amount of CD8 in the lysate. However, although thymocytes express only CD8αβ, the hybridomas express CD8αβ heterodimers and CD8αα homodimers (29, 33), which accounts for the large amount of CD8α detected in the lysate. Taken together these results indicate that association of CD3δ with CD8 is remarkably strong and resists washing with n-octylglucoside and high salt, which combined effectively disrupt the association of CD3δ with CD3ε. On the other hand, the association of CD8 with CD3δ (and CD3ε) is sensitive to alkylation or chelating of divalent cations, suggesting that it involves free cysteines and chelate complexes.

DISCUSSION

A key finding of the present study is that CD3δ mediates a functional link between TCR·CD3 and CD8 and that this is crucial for efficient TCR triggering and activation of CD8⁺ T cells. CD3δ knockout mice or mice expressing an αCPM variant TCR, which lacks the δ chain in their CD3 complex, exhibit strongly impaired positive selection and TCR-mediated activation of CD8 SP T cells (Fig.1 and Refs. 12, 15, 18). The same findings were obtained on CD8β knockout mice (20, 21) and in a milder form, on mice expressing tailless CD8β (22, 23, 29). The present study shows that these signaling defects are explained by the same molecular mechanism, namely that CD3δ couples the TCR with the coreceptor CD8. Several observations support this conclusion. Using TCR photoaffinity labeling with soluble monomeric K^d-PbCS(ABA) complexes, we find that on cells expressing the αCPM variant T1 TCR, CD8 fails to markedly increase TCR-ligand binding (Fig. 2). It is known that CD8 increases the avidity of TCR-ligand interactions by binding to TCR-associated MHC complexes and that this coordinate binding requires association of TCR and CD8 (29, 32, 43). For example, CD8αα or CD8αβ lacking the tail of CD8β (CD8αβ′), poorly associate with TCR·CD3 and therefore inefficiently increase TCR-ligand binding (29). Because T1 TCR αIV/βIII lack the δ chain of their CD3 complexes (17), our TCR photoaffinity labeling experiments argue that CD3δ mediates association of TCR·CD3 with CD8. Consistent with this are our FRET data showing that in T cell hybridomas wild type T1 TCR is in close proximity to CD8 whereas T1 TCR αIV/βIII is not (Fig. 3).

Moreover, because CD8αβ is palmitoylated and partitions in lipid rafts, a fraction of T1 TCR·CD3 is raft-associated, due to its association with CD8 (Fig. 4 and Ref. 29). Several observations indicate that raft association of TCR·CD3 is mediated by CD3δ and CD8αβ. First, TCR lacking CD3δ, due either to disruption of the CD3δ gene (12) or to αCPM replacement, exhibit no or little raft association (Fig. 4 and Ref. 18). Second, no significant TCR raft association was observed on T cell hybridomas lacking CD8β or on thymocytes upon blocking of CD8 (Fig. 4 and Ref. 29). Third, CD3δ was selectively co-immunoprecipitated with CD8 (and CD4) (Fig. 6 and Ref.26). Because CD3δ is known to associate with CD3ε (4, 5), this implies that the association of CD3δ with the coreceptor is stronger than with CD3ε, i.e. is remarkably avid.

How does CD3δ associate with the coreceptor ? Because CD8αα poorly associates with TCR·CD3 (Fig. 3 and Ref. 29), CD8β is important for this interaction. It has been shown that the tail of CD8β is involved in coupling CD8 with TCR·CD3 (29), but it is unclear what other portions of CD8 are involved in this interaction and in what way. Moreover, CD3δ can also be selectively immunoprecipitated with CD4 (26), which is surprising, given the striking structural differences between CD4 and CD8. Because CD4 and CD8 have in common that they associate with Lck and LAT (44), the question arises whether these may be involved in coupling the TCR with the coreceptor. This possibility, however, seems unlikely because mice expressing tailless CD3δ exhibit nearly normal positive selective of SP T cells (12), arguing that the tail of CD3δ is not required for its interaction with the coreceptor. Because Lck is intracellular and LAT has only nine extracellular residues, it seem inconceivable that they could interact with tailless CD3δ. Moreover, D3 and D4 of CD4 have been shown to be critical for coupling the TCR with CD4 (25).

Our co-immunoprecipitation experiments indicate that the association of CD8αβ with CD3δ is sensitive to EDTA and even more to alkylation of cysteines by ethylmaleinimide (Fig. 6 B). It is interesting to note that CD3γ, δ, and ε and CD8α and β all have two free cysteines in their extracellular membrane proximal regions (Fig. 7 A). It has been shown that those of the CD3 chains are engaged in chelate complexes with Zn²⁺, which contribute to their dimer formation (4,5). Although for CD8 two of these cysteines form an interchain disulfide bond, the other two may participate in such chelate complexes and thus strengthen its association with CD3δ (Fig. 7 A). The transmembrane portions of CD8 and CD3δ may also contribute to their association, as suggested by the destabilizing effect ofn-octylglucoside (Fig. 6 B), which disrupts the association of spanning proteins (41). Further studies are clearly needed to elucidate how CD3δ associates with the coreceptor, in particular what role its Ig domain plays. Based on the 3D structure of CD3εγ (5), straightforward homology modeling of the Ig domain of CD3δ is possible. Such modeling suggests that the outer surfaces of CD3δ and CD3γ (opposite the interface with CD3ε), which are most likely to interact with the coreceptor, are strikingly different. The outer surface of CD3δ is flatter and much less polar than the one for CD3γ, which is consistent with the finding that the association of CD3δ with CD8 is only slightly ionic in nature (Figs.6 B and 7 B).

What are the implications of CD3δ-mediated TCR·CD8 coupling for TCR signaling? TCR·CD3, lacking CD3δ, exhibits defective signaling such as impaired activation of kinases like Lck, p59_fyn, ZAP-70, and Erk and reduced phosphorylation of LAT (12, 18, 19). This has been attributed to their poor association with lipid rafts, which by concentrating kinases and their substrates and by excluding phosphatases are privileged sites for the induction of TCR signaling (12, 18, 36, 41, 42). Our results demonstrate that raft association of TCR is mediated by binding of CD3δ to raft-resident CD8 or, more precisely, with CD8/Lck, because CD8 associates with Lck in rafts (29,33). Although they are small in resting cells rafts, they dramatically increase in size upon TCR triggering, which greatly increases the separation of kinases and phosphatases and hence the efficiency of TCR signaling (41, 42). Our confocal studies show that co-cross-linking of TCR and CD8 by soluble MHC-peptide multimers or anti-TCR·CD3 and CD8 antibodies results in the formation of large aggregates of TCR and CD8 (Fig. 5). This was also observed under conditions where cell activation is prevented, e.g. in the cold or in the presence of Src kinase inhibitors (Fig. 5 and Ref.45).2 This aggregate formation was also inhibited by methyl-β-cyclodextrin or similar agents, which disrupt lipid rafts, and was not observed on cells expressing the CD3δ⁻ αCPM variant TCR (Fig. 5). Taken together these findings argue that cross-linking of raft-associated TCR·CD3 adducts with CD8/Lck results in strong TCR aggregation and the formation of large rafts and that this is essential for efficient TCR signal induction. Consistent with this is the observation that disruption of rafts greatly diminishes multimer staining of CD8⁺ CTL, because TCR and CD8 aggregate formation increases the binding of soluble MHC-peptide complexes (45). Furthermore, in cells lacking CD8αβ or upon blocking of CD8, raft association of TCR is diminished and TCR cross-linking-mediated TCR aggregation is strongly impaired, just as it is in cells expressing CD3δ⁻ TCR·CD3 or upon disruption of rafts (Fig. 5 and Refs. 12, 18, 29, 36, and 45).

In conclusion, the present study shows that CD3δ serves to establish a functional link between the TCR and the coreceptor CD8 and that this is essential for efficient TCR signaling, which in turn is needed for activation and positive selection of CD8 SP T cells. A similar conclusion was reached in a related study using a different approach (50). Even though strikingly different in structure, CD4 seems also to associate with CD3δ, thus forming a similar link with the TCR (25,26). Indeed, mice expressing CD3δ⁻ TCR αIV/βIII also exhibit impaired positive selection of CD4 SP T cells (16, 17). In accordance with this is the finding that Lck plays a crucial role in T cell development and that for positive selection of CD4 and CD8 SP T cells, Lck must be associated with the coreceptors CD4 and CD8, respectively (1, 2, 46). Finally, it has been shown that the negative selection of CD8 (and CD4) SP T cells is normal in mice expressing CD3δ⁻ TCR (16-18). It is interesting to note that TCR-mediated apoptosis of CD8⁺ cells is CD8-independent,i.e. in contrast to cell activation and positive selection it is not impaired by the lack of CD8 co-engagement (47, 48).