CD3delta Establishes a Functional Link between the T Cell Receptor and CD8*

Marie-Agnès DouceyDagger §, Laurence Goffin§, Dieter Naeher||, Olivier Michielin, Petra Baumgärtner, Philippe Guillaume, Ed Palmer||, and Immanuel F. Luescher**

From the Dagger  Institute for Biochemistry, University of Lausanne, Epalinges 1066, Switzerland, the  Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges 1066, Switzerland, and the || Laboratory of Transplantation Immunology and Nephrology, University Hospital Basel, Basel 4031, Switzerland

Received for publication, August 8, 2002, and in revised form, September 4, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

T cells expressing T cell receptor (TCR) complexes that lack CD3delta , either due to deletion of the CD3delta gene, or by replacement of the connecting peptide of the TCRalpha chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8beta or tailless CD8beta , we examined whether CD3delta serves to couple TCR·CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2Kd. We report that, in thymocytes and hybridomas expressing the T1 TCR·CD3 complex, CD8alpha beta associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8alpha alpha or a CD3delta - variant of the T1 TCR. CD3delta was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3delta . Because CD8alpha beta is a raft constituent, due to this association a fraction of TCR·CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3delta couples TCR·CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8+ T cells.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The differentiation of CD4 CD8 double-positive (DP)1 thymocytes into CD8 single-positive (SP) T cells requires appropriate signals from the TCR and the coreceptor CD8 (1, 2). DP thymocytes and CD8 SP peripheral T cells express TCRalpha beta that are associated with three signal-transducing units, namely CD3delta epsilon and CD3gamma epsilon heterodimers and a disulfide-linked zeta zeta chain homodimer (3-5). The CD3gamma delta and epsilon  subunits contain in their cytoplasmic tail a single immunoreceptor tyrosine-based activation motif, whereas the tail of the zeta  chain harbors three immunoreceptor tyrosine-based activation motifs. For surface expression of TCRalpha beta , their association with CD3epsilon , gamma , and zeta  but not with CD3delta is required (6-9). Accordingly, knockout of CD3epsilon , gamma , and zeta  chain arrests T cell development at early stages (6-11). By contrast, in CD3delta knockout mice T cell development proceeds to the DP stage, but positive selection of CD8 (and CD4) SP T cells is severely compromised (9, 12). During TCRalpha beta assembly the TCRbeta chain first associates with CD3epsilon gamma and the TCRalpha chain with CD3epsilon delta , and the resulting trimers then associate and the TCRalpha beta disulfide bond is formed (13). Although CD3delta is physically associated with the pre-TCR complex, it is not required for pre-TCR signaling, which is essential for the transition of double-negative (DN) to DP thymocytes (10, 14, 15).

A conserved motif in the TCRalpha chain-connecting peptide domain, which connects the transmembrane and the Ig domains, referred to as alpha CPM, plays a crucial role in positive selection of CD8 and CD4 SP T cells (16-18). The alpha CPM consists of seven highly conserved amino acids (FETDXNLN) and is present in TCRalpha beta but not in TCRgamma delta (16). In mice expressing TCR in which the alpha CPM is replaced by the corresponding sequence of the TCRdelta chain, positive selection of SP T cells is greatly impaired, whereas negative selection is normal (16-18). These variant TCRs, referred to as alpha IV/beta III (16) TCR, exhibit impaired association with CD3delta , zeta -chain phosphorylation, defective activation of p59Fyn and extracellular signal-regulated kinase, impaired phosphorylation and recruitment of ZAP-70, p56lck (Lck), and LAT to lipid rafts (17-19). Very similar findings were obtained in CD3delta knockout mice (12, 15), arguing that the defects observed for the alpha CPM variant TCR are mainly accounted for by their impaired association with CD3delta .

On the other hand, the beta  chain of CD8 plays a key role for positive selection of CD8 SP T cells. Although CD8 can be readily expressed as the CD8alpha alpha homodimer, the number of CD8 SP T cells in CD8beta knockout mice is greatly reduced (20, 21). In a milder form, positive selection is also compromised in mice overexpressing tailless CD8beta , and activation is impaired in CD8+ T cells expressing tailless CD8beta (22, 23). Given the similarity in impaired activation and positive selection of CD8 SP T cells in mice lacking CD8beta or CD3delta , we examined here whether this is accounted for by the same mechanism, i.e. whether CD3delta couples TCR with CD8alpha beta . Several studies indicated that CD8 (and CD4) associates with TCR·CD3. For example, the proximity between CD8 (and CD4) and the TCR has been demonstrated on cells by using fluorescence resonance energy transfer (FRET) (24, 25). In other studies, CD8 was co-immunoprecipitated with anti-TCR·CD3 antibodies (26-29). According to one of these studies the tail of CD8beta is involved in the association of CD8 with TCR·CD3 (29), and another suggested that CD3delta can be selectively co-immunoprecipitated with CD8 (and CD4) (26).

To investigate whether and how CD3delta establishes a functional link between the TCR·CD3 and CD8, we used thymocytes and T cell hybridomas expressing the T1 TCR. This TCR recognizes the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid on Lys-259 (PbCS(ABA)) in the context of Kd (30). Photoactivation of the ABA group results in cross-linking of the T1 TCR with Kd-PbCS(ABA), which permits direct assessment of TCR-ligand interactions by TCR photoaffinity labeling (30-32). Using TCR photoaffinity labeling, FRET, co-immunoprecipitation, and confocal microscopy, we find that CD8alpha beta , but not CD8alpha alpha , associates with the TCR via CD3delta and that this is required for efficient T cell activation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antibodies-- The following monoclonal antibodies were purchased from BD Pharmingen (San Diego, CA): anti-CD8alpha 53.6.72 (FITC or Cy5), anti-CD8beta H35-17 (PE), anti-TCRbeta H57 (allophycocyanide), anti-CD3epsilon 17A2 (PE), anti-CD4 GK 1.5 (FITC or Cy5), and anti-Thy-1.2 III/5 (Cy5). The anti-CD8beta KT112 mAb (32) was purified and labeled with Cy5. For Western blotting antibodies specific for CD3epsilon (M-20, Santa Cruz Biotechnology, Santa Cruz, CA), CD3delta (17), CD8alpha tail (33), zeta  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 Kd Fab' fragments of anti-Kd mAb SF1-1.1.1 (SF') or anti-CD8beta mAb H35 were used (32).

Cell Cultures and T1 TCR Transgenic Mice-- The CD8-, CD8alpha alpha +, and CD8alpha beta + T cell hybridomas were obtained and cultured as described previously (29). Hybridomas expressing the alpha CPM variant TCR (alpha IV/beta III) were obtained by transfection of CD8- or CD8alpha beta + 58 T cell hybridomas as described previously (34) with wild type T1 TCRalpha and beta  chains or T1 TCRIValpha chain, in which the sequence 225-270 of the TCRalpha chain was replaced with the corresponding sequence of the TCRdelta chain and T1 TCRIIIbeta chain in which the sequence 270-300 of the TCRbeta chain was replaced with the corresponding sequence of the TCRgamma 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 beta -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-2d/d) cDNA encoding the T1 TCRalpha and beta  chains were separately cloned into the transgenic expression vector pHSE3' using standard methods. XhoI fragments encoding the T1 TCRalpha and beta  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-CD8alpha mAb 53.6.72 (FITC), anti-CD8beta 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 CD8alpha beta + T1 TCR+ hybridomas (342, 68, 22), for CD8- T1 TCR+ hybridomas (4, 5, 28), for CD8alpha beta + T1 TCRalpha IV/beta III+ cells (305, 61, 19), for CD8- T1 TCRalpha IV/beta III+ 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-CD8alpha 53.6.72, anti-CD8beta KT112, anti-CD4 GK 1.5, or anti-Thy-1 antibody; PE-labeled anti-CD3epsilon 17A2 mAb; or 50 nM of PE-coupled Kd-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 = [E3both - E3none] - [(E3Cy5 - E3none) × (E2both/E2Cy5)] - [(E3PE - E3none) × (E1both/E1PE)]. The different fluorescence values (E) were measured on unlabeled cells (Enone), or cells labeled with PE (EPE), Cy5 (ECy5), or Cy5 and PE (Eboth).

Soluble Kd-peptide Complexes-- Kd-125"IASA"-YIPSAEK(ABA)I complexes (about 2000 Ci/mmol) were prepared as described previously (31). Non-radioactive Kd-PbCS(ABA) complexes were obtained by refolding of Kd heavy chain and human beta 2 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 Kd. 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 FACStarTM 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 Kd-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 Kd-125"IASA"-YIPSAEK(ABA)I were lysed in 1 ml of MN buffer (25 mM MES, pH 6.5, 150 mM NaCl) 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 mM methyl-beta -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 Kd-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-CD8beta 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 CD8alpha beta 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 CD8beta (KT112), CD8alpha (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 (0.5 M). Immunoprecipitates were resolved on SDS-PAGE (15%, reducing), transferred onto a nitrocellulose membrane, and Western blotted using anti-CD3delta , anti-CD3epsilon , or anti-CD8alpha antibodies. For detection the enhance chemiluminescence (ECL) (Amersham Biosciences) was used as described (29).

Modeling of CD3epsilon delta -- An homology model of the CD3epsilon delta complex was built based on the CD3epsilon gamma 3D structure and CD3delta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CD8alpha beta and Wild Type TCR alpha CPM Are Required for Efficient Intracellular Calcium Mobilization-- To examine the role of CD8 and the TCR alpha CPM 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 alpha CPM was replaced with the corresponding sequence of TCRdelta (T1 TCR alpha IV/beta III). As shown in Fig. 1A, hybridomas expressing the wt TCR and CD8alpha beta 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 CD8beta blocking antibody H35. Similarly, on hybridomas expressing the T1 TCR alpha IV/beta III, only scant calcium mobilization was observed, which was reduced to background levels in the presence of mAb H35 (Fig. 1B).


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  		Fig. 1.   Blocking of CD8 or replacement of the alpha CPM inhibits antigen recognition. Indo-1-labeled CD8+ T cell hybridomas expressing wild type (wt) (A), the alpha CPM variant T1 TCR alpha IV/beta III (B) T1 TCR, or thymocytes from T1 TCR transgenic mice (C) were incubated in the presence (circles) or in the absence (squares) of anti-CD8beta mAb H35 with P815 cells sensitized with graded concentrations of IASA-YIPSAEK(ABA)I, and calcium-dependent Indo-1 fluorescence was measured by FACS. Mean values and S.D. were calculated from three experiments.

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. 1C). Maximal response was observed at 10-7 M IASA-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. 1C). Taken together these results indicate that, for efficient calcium mobilization, CD8alpha beta and CD3delta + TCR are required, which is in accordance with previously studies showing that CD8 and the alpha CPM 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 alpha CPM Variant TCR-- TCR photoaffinity labeling with soluble monomeric Kd-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 alpha IV/beta III. As shown in Fig. 2A, TCR photoaffinity labeling was reduced by over 6-fold in the presence of Fab' fragments of anti-Kdalpha 3 mAb SF1-1.1.1, which block CD8 binding to Kd (32). A slightly larger inhibition was observed on T1 thymocytes and on cloned T1 CTL (Fig. 2B and Ref. 32). The same reductions were observed upon blocking of CD8 with anti-CD8beta mAb H35 (data not shown).


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  		Fig. 2.   CD8 strengthens TCR-ligand binding on cells expressing wild type but not alpha CPM variant TCR. T cell hybridomas expressing CD8alpha beta and wild type (wt) or alpha CPM variant T1 TCR alpha IV/beta III (A) or T1 thymocytes (B) were incubated at 26 °C for 30 min in the absence (-) or presence of Fab' fragments of anti-Kdalpha 3 mAb SF1-1.1.1 (SF') (20 µg/ml) or anti-Kdalpha 1 mAb 20-8-4S (10 µg/ml) with Kd-125"IASA"-YIPSAEK(ABA)I. After UV irradiation, the cells were lysed and TCR was immunoprecipitated and analyzed by SDS-PAGE (10%, reducing) and phosphorimaging. The TCR labeling observed in the absence of SF' was defined as 100%. Mean values and S.D. were calculated from two experiments.

Remarkably, on hybridomas expressing T1 TCR alpha IV/beta III, Kd-PbCS(ABA) binding was over 4-fold lower than on cells expressing wild type T1 TCR and blocking of CD8 binding to Kd caused only a small reduction. The nonspecific labeling, as seen in the presence of anti-Kdalpha 1 mAb 20-8-4S, which blocks binding of Kd to TCR (32), was in the range of 3% on the hybridomas and below 1% on thymocytes. Upon blocking of CD8 binding to Kd, TCR photoaffinity labeling was slightly lower on hybridomas expressing T1 TCR alpha IV/beta III, as compared with hybridomas expressing the wild type T1 TCR (Fig. 2A). 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 Kd-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 alpha IV/beta III 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 alpha IV/beta III associates poorly with CD8.

Wild Type but Not alpha CPM 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-CD8beta mAb KT112 and PE-labeled Kd-PbCS(ABA) multimers and measured the FRET value from PE to Cy5 by FACS. As shown in Fig. 3A, 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 alpha IV/beta III. The nonspecific signal, as recorded on the corresponding CD8- hybridomas, was about 0.03 unit. When using PE-labeled anti-CD3epsilon mAb 17A2, slightly less efficient FRET was observed (1.5 units, Fig. 3B). Remarkably, this FRET value was enhanced very little when soluble Kd-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.


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  		Fig. 3.   Wild type, but not alpha CPM variant TCR, associates with CD8. A, T cell hybridomas expressing wild type (wt) or alpha CPM variant T1 TCR (alpha IV/beta III) and CD8alpha beta (+) or not (-) were stained with PE-labeled Kd-PbCS(ABA) multimer and Cy5-labeled anti-CD8beta mAb KT112. B, alternatively, staining was performed with PE-labeled anti-CD3epsilon mAb 17A2 and Cy5-labeled mAb KT112 in the absence (-) or presence (+) of soluble Kd-PbCS(ABA) monomer (1 µM). C, T cell hybridomas expressing CD8alpha beta , CD8alpha alpha , or no CD8 were stained with PE-labeled Kd-PbCS(ABA) multimer and Cy5-labeled anti-CD8beta KT112 or anti-CD8alpha mAb 53.6.72. D, thymocytes from T1 TCR transgenic mice were stained in the absence (-) or presence (+) of anti-CD8beta mAb H35 (15 µg/ml) with PE-labeled Kd-PbCS(ABA) multimers and Cy5-labeled mAb KT11, 53.6.72, anti-CD4 mAb GK1.5 or anti-Thy-1 mAb III/5. Cells were analyzed by FACS using excitation at 488 and 630 nm. FRET units were calculated from the fluorescence emissions at 580 and 670 nm (see "Experimental Procedures"). Mean values and S.D. were calculated from two to four experiments, each performed in triplicate.

To investigate which chain of CD8 was important for coupling of CD8 with TCR·CD3, we performed FRET experiments on hybridomas expressing CD8alpha beta or CD8alpha alpha . Upon staining of CD8alpha beta + hybridomas with PE-labeled Kd-PbCS(ABA) multimers and Cy5-labeled anti-CD8beta KT112, a 4.3-fold stronger FRET was observed than on CD8alpha alpha + hybridomas stained with Cy5-labeled anti-CD8alpha mAb 53.6.72 (Fig. 3C). 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 Kd-PbCS(ABA) multimers and Cy5-labeled anti-CD8beta mAb KT112 (37 units, Fig. 3D). About one-third lower FRET (25 units) was recorded when using Cy5-labeled anti-CD8alpha mAb 53.6.72. Because thymocytes express only CD8alpha beta , but CD8-transfected T cell hybridomas always express high levels of CD8alpha alpha (see "Experimental Procedures" and Refs. 29 and 33), the over 4-fold reduced FRET observed on CD8alpha alpha + T cell hybridomas indicates that CD8alpha beta couples with TCR·CD3 more extensively than does CD8alpha alpha (Fig. 3C). This is consistent with the finding that CD8alpha beta is co-immunoprecipitated with the TCR more efficiently as compared with CD8alpha alpha (29). In the presence of anti-CD8beta 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. 3D). 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 Kd-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 CD3delta + TCR and CD8alpha beta .

Wild Type but Not alpha CPM Mutant T1 TCR Docks to Raft-associated CD8-- Because wild type T1 TCR, but not variant T1 TCR alpha IV/beta III, associates with CD8alpha beta (Figs. 2 and 3) and CD8alpha beta is a raft constituent (33, 36), we examined the raft association of these two TCR. To this end we TCR photoaffinity-labeled CD8alpha beta + T cell hybridomas expressing wild type or T1 TCR alpha IV/beta III, lysed the cells in Brij96, and fractionated the detergent-soluble and -insoluble components on sucrose gradients. A significantly larger fraction of Kd-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 alpha IV/beta III (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).


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  		Fig. 4.   Wild type, but not alpha CPM variant TCR, docks to raft-associated CD8. T cell hybridomas expressing CD8alpha beta and wild type (A, E, and F) or alpha CPM variant T1 TCR alpha IV/beta III (B) or thymocytes from T1 TCR transgenic mice (C and D) were incubated in the absence (A-C, E, and F) or presence (D) of anti-CD8beta mAb H35 (15 µg/ml) at 26 °C with Kd-125IASA-YIPSAEK(ABA)I complexes. After UV irradiation the washed cells were lysed in Brij96 (0.5%) and the lysates were fractionated on sucrose gradients. Ten fractions were collected from the top, aliquots were immunoprecipitated with anti-TCR mAb H57, and the precipitates were resolved on SDS-PAGE (10%, reducing) and evaluated by phosphorimaging (A-D). Aliquots of the fractions from A were analyzed by SDS-PAGE (10%, reducing) and Western blotted with antibodies specific for CD45 (E) or Thy-1 (F). Detergent-insoluble components were mainly found in fractions 2-4, and detergent-soluble components were in fractions 6-10. Each experiment was repeated at least once.

In T1 thymocytes an appreciable fraction of photoaffinity-labeled TCR was found in the light fractions (Fig. 4C). This fraction was greatly diminished when TCR photoaffinity labeling was performed in the presence of the CD8beta -blocking mAb H35 (Fig. 4D). 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 alpha CPM variant TCR (18) or TCR from CD3delta knockout mice (12) exhibit impaired raft-association and argues that CD3delta couples TCR·CD3 with CD8alpha beta 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 CD8alpha beta and wild type T1 TCR with PE-labeled Kd-PbCS(ABA) multimers at room temperature resulted in extensive patch formation and internalization (Fig. 5A). The aggregate formation, but not the internalization, also took place when the incubation was performed in the cold (Fig. 5B), suggesting that it does not require cell activation. Strong TCR·CD8 aggregate formation was also seen on T1 thymocytes upon incubation with Kd-PbCS(ABA) multimers and on CD8alpha beta +, T1 TCR+ hybridomas after incubation with anti-TCR, and anti-CD8beta antibodies (Fig. 5, A and C). By contrast, on hybridomas expressing T1 TCR alpha IV/beta III, aggregate formation and internalization were greatly reduced. The same diffuse, mainly surface staining, was also observed when CD8alpha beta +, T1 TCR+ hybridomas, or T1 thymocytes were pretreated with methyl-beta -cyclodextrin, which destabilizes lipid rafts (Fig. 5A). 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 CD8alpha beta , lipid rafts, and CD3delta + TCR are required.


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  		Fig. 5.   CD3delta and CD8alpha beta are required for cross-linking-induced formation of large TCR·CD8 aggregates. A, T cell hybridomas expressing CD8alpha beta and wild type (wt), T1 TCR (alpha IV/beta III), or thymocytes from T1 TCR transgenic mice, pretreated or not with methyl-beta -D-cyclodextrin (MCD), were incubated at room temperature for 20 min with PE-labeled Kd-PbCS(ABA) multimers and examined using confocal microscopy. B, alternatively, the staining of the hybridomas was performed for 40 min at 4 °C. C, the hybridomas were incubated for 20 min at room temperature with anti-CD8beta mAb KT112, anti-TCR mAb H57, and FITC-labeled rabbit anti-rat IgG antibody and analyzed by confocal microscopy. Representative images are shown from over ten cells analyzed per condition and from at least two different experiments.

CD3delta Associates with CD8-- We next examined whether CD3delta 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, CD3delta , but neither CD3epsilon nor zeta  chains, was highly enriched; especially when compared with the TCR immunoprecipitates, the preferential co-precipitation of CD3delta was striking (Fig. 6A and data not shown).


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  		Fig. 6.   CD3delta associates with CD8 on CD8ab+, T1 TCR+ cells. A, thymocytes from T1 TCR transgenic mice were lysed in cold Tris, pH 8, containing 0.3% Triton X-100, and the total lysate or immunoprecipitates with anti-CD8beta mAb KT112 or anti-TCR mAb H57 was resolved on SDS-PAGE (15%, reducing) and Western blotted with antibodies specific for CD3epsilon , CD3delta , and CD8alpha . B, the immunoprecipitates with anti-CD8alpha mAb 53.6.72 were either (i) washed twice with in Tris, pH 8, containing 0.15% Triton X-100 (lane 1) or 50 mM octylglucoside (lane 2) or (ii) washed twice with Tris, pH 8, containing 0.15% Triton X-100 and either 5 mM EDTA (lane 3), 5 mM ethylmaleinimide (lane 4), or 0.5 M NaCl (lane 5) and then Western blotted using antibodies specific for CD3delta , CD3epsilon , and CD8alpha , respectively. The Western blots with anti-CD3delta and -CD3epsilon from three experiments were quantified by densitometry and expressed in percentages, with 100% being the value recorded for lane 1. C, hybridomas expressing T1 TCR wild type and CD8alpha beta were analyzed as described for A.

To obtain further information on the association of CD3delta with CD3epsilon and CD8, we washed the CD8 immunoprecipitates twice with different buffers and assessed the amount of co-precipitated CD3delta and CD3epsilon by Western blotting. The association of CD3delta with CD8 was substantially reduced (to 36%) upon washing with ethylmaleinimide, which alkylates free cysteines (Fig. 6B). A smaller reduction (to 55%) was observed upon washing with EDTA-containing buffer, which chelates divalent cations. About 40% reduction was noted upon washing with n-octylglucoside, which disrupts association of transmembrane proteins (41). Strikingly, washing with 0.5 M NaCl had no marked effect, suggesting that the association of CD3delta with CD8 is not ionic in nature. By contrast, washing with this buffer removed most of the CD3epsilon from the immunoprecipitates. The other buffers affected the co-precipitation of CD3epsilon in the same way as the co-precipitation of CD3delta .

In CD8alpha beta +, T1 TCR+ T cell hybridomas, similar as with T1 thymocytes (Fig. 6A), anti-CD8beta antibody efficiently co-immunoprecipitated CD3delta but little CD3epsilon (Fig. 6C). 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 CD8alpha beta , the hybridomas express CD8alpha beta heterodimers and CD8alpha alpha homodimers (29, 33), which accounts for the large amount of CD8alpha detected in the lysate. Taken together these results indicate that association of CD3delta with CD8 is remarkably strong and resists washing with n-octylglucoside and high salt, which combined effectively disrupt the association of CD3delta with CD3epsilon . On the other hand, the association of CD8 with CD3delta (and CD3epsilon ) is sensitive to alkylation or chelating of divalent cations, suggesting that it involves free cysteines and chelate complexes.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A key finding of the present study is that CD3delta mediates a functional link between TCR·CD3 and CD8 and that this is crucial for efficient TCR triggering and activation of CD8+ T cells. CD3delta knockout mice or mice expressing an alpha CPM variant TCR, which lacks the delta  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 CD8beta knockout mice (20, 21) and in a milder form, on mice expressing tailless CD8beta (22, 23, 29). The present study shows that these signaling defects are explained by the same molecular mechanism, namely that CD3delta couples the TCR with the coreceptor CD8. Several observations support this conclusion. Using TCR photoaffinity labeling with soluble monomeric Kd-PbCS(ABA) complexes, we find that on cells expressing the alpha CPM 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, CD8alpha alpha or CD8alpha beta lacking the tail of CD8beta (CD8alpha beta '), poorly associate with TCR·CD3 and therefore inefficiently increase TCR-ligand binding (29). Because T1 TCR alpha IV/beta III lack the delta  chain of their CD3 complexes (17), our TCR photoaffinity labeling experiments argue that CD3delta 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 alpha IV/beta III is not (Fig. 3).

Moreover, because CD8alpha beta 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 CD3delta and CD8alpha beta . First, TCR lacking CD3delta , due either to disruption of the CD3delta gene (12) or to alpha CPM 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 CD8beta or on thymocytes upon blocking of CD8 (Fig. 4 and Ref. 29). Third, CD3delta was selectively co-immunoprecipitated with CD8 (and CD4) (Fig. 6 and Ref. 26). Because CD3delta is known to associate with CD3epsilon (4, 5), this implies that the association of CD3delta with the coreceptor is stronger than with CD3epsilon , i.e. is remarkably avid.

How does CD3delta associate with the coreceptor ? Because CD8alpha alpha poorly associates with TCR·CD3 (Fig. 3 and Ref. 29), CD8beta is important for this interaction. It has been shown that the tail of CD8beta 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, CD3delta 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 CD3delta exhibit nearly normal positive selective of SP T cells (12), arguing that the tail of CD3delta 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 CD3delta . 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 CD8alpha beta with CD3delta is sensitive to EDTA and even more to alkylation of cysteines by ethylmaleinimide (Fig. 6B). It is interesting to note that CD3gamma , delta , and epsilon  and CD8alpha and beta  all have two free cysteines in their extracellular membrane proximal regions (Fig. 7A). 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 CD3delta (Fig. 7A). The transmembrane portions of CD8 and CD3delta may also contribute to their association, as suggested by the destabilizing effect of n-octylglucoside (Fig. 6B), which disrupts the association of spanning proteins (41). Further studies are clearly needed to elucidate how CD3delta associates with the coreceptor, in particular what role its Ig domain plays. Based on the 3D structure of CD3epsilon gamma (5), straightforward homology modeling of the Ig domain of CD3delta is possible. Such modeling suggests that the outer surfaces of CD3delta and CD3gamma (opposite the interface with CD3epsilon ), which are most likely to interact with the coreceptor, are strikingly different. The outer surface of CD3delta is flatter and much less polar than the one for CD3gamma , which is consistent with the finding that the association of CD3delta with CD8 is only slightly ionic in nature (Figs. 6B and 7B).


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  		Fig. 7.   Concepts of CD3delta association with CD8. A, transmembrane and adjacent extracellular and cytoplasmic sequences of CD8alpha , CD8beta , CD3delta , CD3epsilon , CD3gamma , CD3zeta , and CD4. The sequences and definition of the transmembrane regions were taken from Swiss-Prot (available at www.expasy.ch/sprot). The spanning regions are shown in gray boxes, and the numbers indicate their N- and C-terminal residues. Cysteines are shown in boldface, basic residues in black boxes, and acidic ones are in ovals. B, the electrostatic potential of the outer surface (opposite to CD3epsilon ) of the CD3gamma structure (left) and the CD3delta model (right) shown from top to bottom. Acidic domains are shown in red, and basic ones are in blue. The images were produced using the software GRASP (49).

What are the implications of CD3delta -mediated TCR·CD8 coupling for TCR signaling? TCR·CD3, lacking CD3delta , exhibits defective signaling such as impaired activation of kinases like Lck, p59fyn, 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 CD3delta 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-beta -cyclodextrin or similar agents, which disrupt lipid rafts, and was not observed on cells expressing the CD3delta - alpha CPM 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 CD8alpha beta 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 CD3delta - TCR·CD3 or upon disruption of rafts (Fig. 5 and Refs. 12, 18, 29, 36, and 45).

In conclusion, the present study shows that CD3delta 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 CD3delta , thus forming a similar link with the TCR (25, 26). Indeed, mice expressing CD3delta - TCR alpha IV/beta III 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 CD3delta - 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).

    ACKNOWLEDGEMENTS

We are grateful for helpful discussion with Drs. Jean-Charles Cerottini and Pascal Batard and for expert technical assistance by Sandra Levrand.

    FOOTNOTES

* This work was supported by grants from the Swiss National Foundation (Grant 31-61946.00) and the Sandoz Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to the work.

** To whom correspondence should be addressed. Tel.: 41-21-692-5988; Fax: 41-21-653-4474; E-mail: iluesche@eliot.unil.ch.

Published, JBC Papers in Press, September 4, 2002, DOI 10.1074/jbc.M208119200

2 M.-A. Doucey, L. Goffin, D. Naeher, O. Michielin, P. Baumgärtner, P. Guillaume, E. Palmer, and I. F. Luescher, unpublished results.

    ABBREVIATIONS

The abbreviations used are: DP, CD4+CD8+ double-positive thymocytes; SP, single-positive; ABA, 4-azidobenzoic acid; alpha CPM, TCRalpha chain connecting peptide motif; DN, CD4-CD8- double-negative thymocytes; FRET, fluorescence resonance energy transfer; IASA, iodo-4-azidosalicylic acid; LAT, linker for activation of T cells; PbCS, Plasmodium berghei circumsporozoite; TCR, T cell receptor; TCR alpha IV/beta III, TCR in which residues 225-270 of the alpha  chain and residues 270-300 of the beta  chain are replaced with the corresponding sequences of the TCRdelta and gamma  chain, respectively; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorting; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; MHC, major histocompatibility complex.

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