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J Biol Chem, Vol. 274, Issue 49, 35119-35128, December 3, 1999

Conformational and Biochemical Differences in the TCR·CD3 Complex of CD8⁺ Versus CD4⁺ Mature Lymphocytes Revealed in the Absence of CD3^*

David A. Zapata^§, Alberto Pacheco-Castro^¶, Pilar S. Torres, Almudena R. Ramiro^**, Ester San José^**, Balbino Alarcón^**, Laeticia Alibaud, Bent Rubin, María L. Toribio^**, and José R. Regueiro^§§

From  Inmunología, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain, the ^** Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Cientificas, Facultad de Biología, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain, and the  Centre d'Immunopathologie et de Génétique Humaine, Centre National de la Recherche Scientifique, Centre Hospitalaire Universitaire de Purpan, 31300 Toulouse, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mature CD4⁺ and CD8⁺ T lymphocytes are believed to build and express essentially identical surface T-cell receptor-CD3 (TCR·CD3) complexes. However, TCR·CD3 expression has been shown to be more impaired in CD8⁺ cells than in CD4⁺ cells when CD3 is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR·CD3 complex in human CD3-deficient CD8⁺ and CD4⁺ T cells. The results indicated that the membrane TCR·CD3 complex of CD8⁺ T lymphocytes was conformationally different from that of CD4⁺ lymphocytes in the absence of CD3. In addition, CD8⁺, but not CD4⁺, CD3-deficient T lymphocytes were shown to contain abnormally glycosylated TCR proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCR). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4⁺ and CD8⁺ T lymphocytes in the intracellular control of TCR·CD3 assembly, maturation, or transport that are revealed when CD3 is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mature T lymphocytes recognize pathogen-derived peptides on antigen-presenting cells by means of the multimeric membrane protein ensemble termed the T-cell receptor (TCR)¹·CD3 complex. This TCR·CD3 complex includes two clonally distributed variable chains that directly interact with antigens (TCR and TCR) and four invariant polypeptides that regulate assembly and signal transduction (CD3, CD3, CD3, and ) (1). The assembly of complete TCR·CD3· complexes takes place in a highly ordered manner within the endoplasmic reticulum: first CD3 chains, then TCR chains, and finally  chains. Further conformational maturation, including carbohydrate processing, occurs in the Golgi apparatus before exportation of mature complexes to the T cell surface. The biochemical machinery involved in the assembly, processing, and exportation of TCR·CD3 complexes is assumed to be shared by all T-lineage cells. Thus, CD4⁺ and CD8⁺ are believed to build biochemically and conformationally identical antigen receptors, although differences in the numbers that reach or remain at the cell surface have been noted (2). Therefore, the lack of any CD3 chain would be expected to affect to a similar extent the assembly and exportation of TCR·CD3 complexes by mature CD4⁺ and CD8⁺ T cells. However, this was not the case in several murine and human CD3 deficiencies (reviewed in Ref. 3). In particular, it has been consistently shown that in the absence of CD3 or CD3, TCR·CD3 expression (or conformation) is more impaired in mature peripheral CD8⁺ cells than in their CD4⁺ counterparts, both in human and in murine deficiencies (3-7). Three other observations suggested the existence of CD8⁺ cell-specific defects in human CD3 deficiency: first, the proband died after a viral infection (a cytolytic T-cell-dependent function) despite normal antibody responses (helper T-cell-dependent) (8); second, the peripheral blood CD8⁺ T cell subset was more strongly reduced (5-fold) than the CD4⁺ T cell subset (only 2-fold) (4); and third, the scanty peripheral CD3-deficient CD8⁺ cells, but not CD4⁺ cells, failed to grow in vitro under optimal stimuli (PHA and allogeneic feeder cells) (4). This paradox may be the reflection either of subset-specific defective maturation of CD3^low to CD3^high thymocytes in these mutants (3) or of hitherto unrecognized biochemical differences in the assembly or maturation of TCR·CD3 complexes between peripheral CD8⁺ and CD4⁺ T lymphocytes, revealed only when certain CD3 chains are absent. Alternatively, as suggested previously (4), the peripheral CD8⁺ T cells of such CD3 deficiencies may belong to a minor population (in CD3-sufficient individuals) that was relatively expanded when either CD3 chain was absent (9).

The recent availability of Herpesvirus saimiri C-488 (HVS)-immortalized mature peripheral CD3-deficient CD8⁺ lymphocytes (5) has allowed us to address this paradox in the case of human CD3 deficiency, by studying phenotypically and biochemically the TCR·CD3 complex of human CD3-deficient CD8⁺ T cells in comparison with CD4⁺ T cells lacking CD3 and with appropriate -sufficient controls. The results support the notion that, indeed, there are differences in the way mature CD8⁺ and CD4⁺ T lymphocytes assemble or process TCR·CD3 complexes, which are revealed when CD3 is absent.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Immortalization Procedures-- PBLs isolated either from healthy donors or from a CD3-deficient () individual named D. S. F. were immortalized as described (5, 10, 11). The Herpesvirus saimiri-exposed T cells, hereafter referred to simply as HVS cells, had been cultured for 5 years (DSF4, a CD4⁺ HVS cell line), 4 years (CTO, CD8⁺⁺, and DSF8, CD8⁺), 3 years (D8EDTA, CD8⁺, and AGU, CD4⁺⁺), or 2 years (RHE, CD4⁺⁺, and ANZ, CD8⁺⁺) when the experiments reported here were performed. Cells have always been grown in parallel.

Flow Cytometry Analyses-- The expression of different surface markers was studied by flow cytometry according to a standard procedure (12). The antibodies used in these studies are listed in Table I. All commercial antibodies were fluorescein isothiocyanate- or phycoerythrin-conjugated and, for the rest, a second fluorescein isothiocyanate- or phycoerythrin-conjugated antibody (anti-mouse IgG or IgM and anti-rabbit IgG, from Caltag) was used.

Intracellular stainings were done following the protocol described in Ref. 15. In all cases, cell viability was determined by the expression of CD45, and intracellular markers were analyzed within CD45⁺ cells. Anti-CD1a (OKT6, 1:1000) and anti-CD74 (VICY1, 1:100) ascites were used as negative and positive controls, respectively, for intracellular stainings with ascitic fluids. For intracellular stainings with rabbit antisera, preimmune serum was used as a negative control. Epstein-Barr virus-transformed lymphoblastoid B cells were used as specificity controls.

For comparative stainings the mean fluorescence intensity (MFI) was used, which is defined as the average fluorescence value of the corresponding mAb referred to the logarithmic scale of fluorescence intensity along the x axis of the histograms.

Calcium Flux Measurement-- Intracellular Ca²⁺ release was induced in PBLs loaded with the fluorescent dye Fluo3AM (Sigma) according to a standard procedure (12).

Radiolabeling and Immunoprecipitation-- For metabolic labeling, at least 10⁷ cells were washed once with DPBS (0.14 M NaCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 2.7 mM KCl). Then cells were left to become resting by incubation in DPBS supplemented with 1% fetal calf serum for 1 h at 37 °C/5% CO₂. After one more washing with DPBS, cells were resuspended in 300 µl of methionine/cysteine-free RPMI medium (Sigma), supplemented with 5% fetal calf serum, containing 170 µCi of a [³⁵S]Met and [³⁵S]Cys mixture (Amersham Pharmacia Biotech). Cells were then incubated for 30 min at 37 °C/5%CO₂ and afterward an equal amount of "cold" RPMI medium (without [³⁵S]Met and [³⁵S]Cys) was added to the labeling tubes. Cells were incubated in these conditions for 1 h more at 37 °C/5% CO₂. Then cells were washed twice in DPBS and subsequently lysed on ice (45 min incubation) in 1% digitonine-containing lysis buffer (1% digitonine (RBI, Natick, MA), 50 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl₂, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (Sigma) and 8 mM iodoacetamide (Sigma), pH 7.6). Cell debris was removed by centrifuging the tubes at 2000 × g for 10 min. Then supernatants were collected and centrifuged for 30 min at 14,000 × g to eliminate the smallest cell debris. Again, supernatants, hereafter referred to as lysates, were collected and transferred to new tubes.

The lysates were precleared twice by incubation with Sepharose beads (Amersham Pharmacia Biotech) containing 1% digitonine, followed by a 2-min centrifugation at 12,000 × g in an Eppendorf centrifuge at 4 °C. The precleared supernatants were subsequently incubated for 1.5 h at 4 °C with 0.5 ml of OKT3 or APA1/2 supernatants (anti-CD3/ or anti-CD3, respectively) coupled to protein G-Sepharose beads (Amersham Pharmacia Biotech). The beads were afterward washed five times in lysis buffer. For deglycosylation with endo--N-acetylglucosaminidase H (Endo H) (Roche Molecular Biochemicals), immunoprecipitates were resuspended, after the last wash, in 45 µl of denaturing buffer (5% SDS, 10% 2-mercaptoethanol) and boiled for 10 min. After a 2-min/12,000 × g centrifugation, supernatants were pipetted to new Eppendorf tubes, dividing each immunoprecipitate in two tubes, one to be deglycosylated (Endo H⁺) and the other to be used as non-deglycosylation control (Endo H). 4.5 µl of 10× Endo H buffer (0.5 M sodium citrate, pH 5.5) and 1 µl of Endo H (1000 IU/ml) were added to the Endo H⁺ samples, which were in turn incubated overnight at 37 °C. The Endo H tubes were meanwhile kept at 4 °C. Samples were then resuspended in Laemmli sample buffer and boiled for 5 min before a short spin at 12,000 × g. SDS-polyacrylamide gel electrophoresis was performed on 10% or 12% polyacrylamide gels (see legends to Figs. 5 and 6), and the samples were analyzed by autoradiography or in a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA). The films were electronically scanned (Bio-Rad Geldoc 2000 analyzer) to determine the relative intensity and molecular weight of each protein.

For surface labeling, at least 10⁷ cells were washed twice with phosphate-buffered saline and resuspended in 150 µl of phosphate-buffered saline. Then cells were ¹²⁵I-labeled using the lactoperoxidase method by adding 2 mCi of Na¹²⁵I (Amersham Pharmacia Biotech), 30 µl of a 140 IU/ml lactoperoxidase solution (Sigma), and 10-µl aliquots of a 0.06% H₂O₂ solution four times at 5-min intervals. To stop the labeling reaction, a solution of 20 mM KI and 1 mM tyrosine phosphate-buffered saline was added to the tubes (Sigma). The samples were then lysed in a lysis buffer containing 1% Brij 96 (Sigma), 150 mM NaCl, 20 mM Tris-HCl, pH 7.8, 10 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each leupeptin and aprotinin. The lysates were precleared twice with Sepharose beads as described above. Immunoprecipitation was performed with 2-4 µg of APA 1/1 (anti-CD3) coupled to protein A-Sepharose beads (Amersham Pharmacia Biotech). This antibody has been shown to coprecipitate CD3 and CD3 chains (12), which can be then resolved by deglycosylation. Samples were then divided in two, and after five washings with lysis buffer, one part was digested overnight with N-glycosidase F (N-Gly⁺, Roche Molecular Biochemicals), whereas the other was left undigested (N-Gly). Briefly, N-Gly⁺ samples were denatured by boiling for 2 min in 7 µl of a solution containing 0.5% SDS and 0.8% 2-mercaptoethanol. After cooling the samples on ice, 15 µl of N-Gly buffer (250 mM Na₂PO₄, pH 8.8, 1% Nonidet P-40, 10 mM EDTA) were added to all tubes, and then 1 µl/sample of 200 unit/ml N-Gly was added to the N-Gly⁺ samples. N-Gly⁺ tubes were incubated overnight at 37 °C, whereas N-Gly tubes were maintained at 4 °C. Finally, all samples, N-Gly⁺ and N-Gly, were resuspended in Laemmli sample buffer, boiled for 5 min before a short spin at 12,000 × g, and resolved by SDS-polyacrylamide gel electrophoresis on 12% polyacrylamide gels.

Northern Blot Analysis-- Total RNA was isolated with Ultraspec (Biotecx, Houston, TX) following the manufacturer's instructions, and then at least 10 µg of RNA were run on 1% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized as described elsewhere (Ref. 16 and references therein) with ³²P-labeled cDNA probes corresponding to the TCR C, C (17), C (18), C (19), and pT. The same blot was subsequently stripped and hybridized with a -actin probe.

V Usage Analysis by Reverse Transcription-PCR-- Total RNA (2 µg) was reverse-transcribed into cDNA using an oligo-dT primer according to the manufacturer's protocol (Roche Molecular Biochemicals). cDNA from HVS-immortalized cells or from PBLs was amplified by PCR using 22 different 5' V-specific primers (V1-V20; see Fig. 7) together with a common 3' C-specific primer (20). Amplified products were electrophoresed, transferred to nylon membranes, and analyzed by Southern blot hybridization with a C probe (Jur2, 17).

Statistical Analysis-- Student's t test was used for all comparisons. Only p values below 0.05 were considered significant. Data are presented as mean ± S.D.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peripheral Blood CD8⁺ Lymphocytes from a Human CD3 Deficiency Were Functional T Cells with a Normal V Repertoire-- The barely detectable surface expression of certain TCR·CD3 epitopes in CD8⁺ lymphocytes from human CD3 deficiency (Fig. 1A and Ref. 4), together with the fact that they did not grow in optimal T-cell culture conditions (4), could be interpreted as an indication that they were not T lymphocytes at all but rather NK-lineage cells. Our previous phenotypic analysis of such CD8⁺ lymphocytes did not support that hypothesis for two reasons: 1) most CD8⁺ cells in CD3 deficiency were CD8^bright (that is, CD8⁺), whereas CD8⁺ NK cells are normally CD8^dull (CD8⁺); and 2) NK markers (CD16, CD56, and CD57) were not overrepresented within the CD8⁺ cell subset in CD3 deficiency (Ref. 4). Nevertheless, an additional phenotypical and functional assay was performed to further ascertain the T-lineage descent of CD3-deficient CD8⁺ cells and to rule out their putative NK-lineage descent. First, V usage within CD8^bright cells was assayed by cytofluorometry and found to be normal (Fig. 1B). Second, an early TCR·CD3-dependent functional response, namely calcium flux, was tested and found also to be normal (Fig. 2). This ruled out the existence of an early signal transduction defect in CD8⁺ cells as a cause of the observed selective growth defect. Taken together, these results suggested that peripheral blood CD8⁺ cells from human CD3 deficiency were polyclonal, functionally competent T lymphocytes.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Phenotypical analysis of fresh peripheral blood CD3-deficient () T lymphocytes. A, representative reactivity patterns of an anti-CD3 mAb (Leu4) with T cells (filled histograms) as compared with normal + T cells (open histograms), either CD4+ (left) or CD8bright (right). The profiles are shown as logarithm of relative fluorescence versus cell number. The vertical line in each panel indicates the upper limit of background fluorescence using an isotype-matched irrelevant mAb. B, comparative TCR-V usage by T cells (filled bars) as compared with normal + controls (open bars), either CD4+ (left) or CD8bright (right). Data are given as the mean percentage of positive cells ± S.D. of three different experiments for each indicated V-specific antibody.

View larger version (47K): [in this window] [in a new window]   Fig. 2.   Functional analysis of fresh peripheral blood CD3-deficient () T lymphocytes. CD3-mediated calcium mobilization by T cells (bottom panels) as compared with normal + T cells (top panels), either CD4+ (left) or CD8bright (right). Calcium levels, expressed as relative fluorescence intensity (y axis) were measured consecutively: first basally, second after addition of an anti-CD3 mAb (M) (IOT3b, 100 µl at 12.5 µg/ml, from Immunotech), and third after cross-linking the mAb (X) (human-adsorbed goat anti-mouse IgG (H+L), 40 µl at 1.25 mg/ml, from Caltag). Each manipulation is marked by a vertical white line lasting approximately 0.5 min. A representative experiment of two independent assays is shown.

The Membrane TCR·CD3 Complex of Immortalized CD3-deficient CD8⁺ T Lymphocytes Was Conformationally Different from That of CD3-deficient CD4⁺ Lymphocytes-- The limited number of peripheral blood CD8⁺ lymphocytes in CD3 deficiency and our inability to obtain CD8⁺ T cell lines (4) precluded further studies on the CD8⁺ T cell subset. This prompted us to try HVS immortalization. An HVS-immortalized -deficient T cell line termed DSF8 (CD8⁺, data not shown) was obtained and characterized phenotypically with a large panel of TCR·CD3-specific mAbs (Fig. 3). For comparative purposes, the results for each mAb are depicted as the MFI ratio of ⁺ cells to cells, either CD8⁺ (Fig. 3A, black columns) or, for comparison, CD4⁺ (Fig. 3A, shaded columns) (5). A ⁺: ratio close to 1 (indicated by the horizontal dotted line) reflects an equivalent expression of the epitope detected by that particular mAb in and ⁺ cells (see, for example, CD2 and CD4 or CD8). ⁺: ratios above 1 reflect an impaired expression of the detected epitope by cells relative to ⁺ controls (impaired 2-, 3-, and 4-fold for ratios of 2, 3, and 4, respectively, and so forth). By this criterion, three conclusions emerged. First, all anti-CD3 anti-TCR mAbs used stained both CD4⁺ and CD8⁺ cells poorly relative to ⁺ controls (⁺: MFI ratios 2; Fig. 3A). Second, there were, however, clear relative staining differences among different mAbs, which were shared by CD4⁺ and CD8⁺ cells. Thus, the ⁺: MFI was around 2 when SPV.T3b was used, but it reached 4 when RW2-8C8, X35, UCHT-1, or OKT3 were analyzed (see Fig. 3A and representative stainings of the two patterns in Fig. 3B). These results suggest that in addition to displaying lower overall levels of TCR·CD3 complexes, CD3 cells were more deficient in the relative expression of certain TCR·CD3 epitopes. The relative expression of other T cell surface molecules was not affected in cells (e.g. CD2, Fig. 3, A and B). A mAb hierarchy of CD3-dependence may be established based on these binding results, as shown in Fig. 3A. In general, TCR-specific mAbs bound T cells more poorly than CD3-specific mAbs, relative to ⁺ controls. It has been shown that TCR·CD3 recognition by certain mAbs may be influenced by glycosylation (e.g. WT31, Ref. 21), and we have reported previously that the lack of CD3 affects TCR·CD3 assembly and glycosylation in CD4⁺ T cells (12, 22). Therefore, the impaired binding observed in Fig. 3 may in fact be a reflection of the higher or lower glycosylation of the particular epitope recognized by each mAb. Third, as observed in PBLs (Fig. 1A), TCR·CD3 binding was significantly more impaired in CD8⁺ cells than in CD4⁺ cells, relative to ⁺ controls, with three mAbs: Leu4, 2Ad2, and BMA031 (see representative reactivity patterns in Fig. 3B).

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Cell surface TCR·CD3 expression in CD8+ as compared with CD4+ HVS-immortalized T cells. A, comparative expression of several TCR·CD3 epitopes in immortalized T cells, either CD8+ (black bars) or CD4+ (shaded bars). Data are given as MFI ratios ± S.D. of + relative to cells with the indicated mAbs. MFI ratios above 1 (indicated by the horizontal dotted line) reflect an impaired expression of TCR·CD3 on cells with that particular mAb. The equivalent expression of CD2 and CD4 or CD8 is shown for comparison. n indicates the number of independent experiments performed. B, representative reactivity patterns of selected anti-TCR·CD3 mAbs with immortalized T cells (filled histograms) as compared with + controls (open histograms), either CD4+ (top row) or CD8+ (bottom row). The profiles are shown as logarithm of relative fluorescence versus cell number. The vertical line in each panel indicates the upper limit of background fluorescence using isotope-matched irrelevant mAbs. The reactivity pattern with an anti-CD2 mAb is shown for comparison.

These findings showed that, in the absence of CD3, immortalized CD8⁺ cells expressed essentially the same number of membrane TCR·CD3 complexes as CD4⁺ cells (as shown with SPV.T3b), which were, however, different from those of CD4⁺ cells (as shown with Leu4, 2Ad2, and BMA031). These results are concordant with PBLs and thus validate the immortalized cells as a model system to study such differences. The general decrease in TCR·CD3 staining observed in cells (Fig. 3) could be due to the lack of intracellular subunits (other than CD3) to build TCR·CD3 complexes (due, for instance, to degradation), or to poor transport of the TCR·CD3 complex to the surface. To distinguish between these two possibilities, intracellular stainings of permeabilized and ⁺ T cells, both CD4⁺ and CD8⁺, were performed and compared as above. The results indicated that cells had slightly more intracellular CD3, CD3, and TCR relative to ⁺ controls (Fig. 4). Therefore, these data are consistent with the hypothesis that the decreased cell surface TCR·CD3 expression seen in T cells (Fig. 3) is likely due to poor transport of the TCR to the cell surface.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Intracellular expression of TCR·CD3 chains in CD8+ as compared with CD4+ HVS-immortalized T cells after permeabilization. A, comparative intracellular expression of several TCR or CD3 chains in immortalized T cells, either CD8+ (black bars) or CD4+ (shaded bars). Data are given as in Fig. 3. MFI ratios above or below 1 (indicated by the horizontal dotted line) reflect decreased or increased intracellular levels of the chain detected with that particular antibody, respectively. The expression of CD74 is shown for comparison. n indicates the number of independent experiments performed. B, representative reactivity patterns of selected anti-TCR and anti-CD3 antibodies with immortalized T cells (filled histograms) as compared with + controls (open histograms), either CD4+ (top row) or CD8+ (bottom row). The profiles are shown as logarithm of relative fluorescence versus cell number. The vertical line in each panel indicates the upper limit of background fluorescence using isotype-matched irrelevant mAbs or preimmune serum.

Taken together, the phenotypic data suggested that HVS-immortalized CD8⁺ cells lacking CD3 built surface TCR·CD3 complexes, which were conformationally different from those of CD4⁺ cells, despite the apparent intracellular availability of several relevant TCR and CD3 components other than CD3. This was found within the background of a poor transport of TCR·CD3 complexes to the surface of both cell types. Such CD8⁺ cell-specific features could be due to: 1) a change in TCR·CD3 subunit composition; 2) a change in TCR·CD3 subunit glycosylation; or 3) a change in accessibility of certain TCR·CD3 epitopes (due to steric hindrance). Further studies were thus undertaken to explore the first two possibilities.

Immortalized CD3-deficient CD8⁺, but Not CD4⁺, Lymphocytes Lacked Normal TCR Chains-- Unimmortalized CD3-deficient CD4⁺ cells had previously been analyzed by immunoprecipitation (12, 22) and immunohistochemistry (23) and had been found to contain all TCR and CD3 chains except CD3. Thus, the impaired expression of their mutant TCR·CD3 complex could be explained as a simple consequence of the lack of CD3 on several CD3-dependent TCR·CD3 epitopes. The unexpected finding that some of the same epitopes were conformationally different in CD8⁺ cells prompted us to perform biosynthetic studies on these cells.

First, a [³⁵S]methionine/cysteine pulse-chase immunoprecipitation was done using OKT3 (anti-CD3, Fig. 5A) and nonreducing conditions. The results confirmed the lack of CD3 in CD8⁺ cells and an impaired association of ₂ to the mutant complex, as described previously in unimmortalized CD4⁺ cells (12, 22). Interestingly, whereas in ⁺ cells, the immature complex (i) progressively gave rise to the mature form (m), in cells, the complex had an intermediate size throughout the experiment. Furthermore, the CD3-associated TCR single chains, either totally (tg) or partially (pg) glycosylated, were not detected in cells. Single TCR chains (tg), in contrast, were present and were apparently more stable in cells. However, an additional labeled protein running slightly faster than tg was observed only in cells. This protein could be a partially glycosylated form of the TCR protein, which would be consistent with the hypothesis that the transport and processing of the TCR chain is slowed in the absence of CD3. Alternatively, the additional labeled protein could represent a new, unidentified protein (see below). Therefore, the TCR heterodimer in CD8⁺ cells was smaller in size than the normal mature form found in ⁺ cells and lacked the maturation-associated (glycosylation) size changes found in normal TCR heterodimers. In addition, a lack of CD3-associated TCR chains and CD3 was prominent, and an additional protein, probably partially glycosylated TCR, was detected in CD8⁺ cells.

View larger version (55K): [in this window] [in a new window]   Fig. 5.   Biochemical analyses of CD3-deficient TCR·CD3 complexes by metabolic labeling. A, CD8+ cells, and CD8++ cells as a control, were pulsed with [35S]methionine/cysteine for 1.5 h, lysed in a 1% digitonine-containing lysis buffer, and chased for the time periods indicated, before immunoprecipitation with OKT3 (anti-CD3/). Electrophoresis was done under nonreducing conditions (so that TCR dimers were preserved) in a 10% polyacrylamide gel. Positions of the expected proteins are indicated by the arrows. m, mature; i, immature; tg, totally glycosylated; pg, partially glycosylated. B, cells shown in A were labeled, lysed, and precipitated as above with OKT3 or with an anti-CD3 antibody (APA1/2). Half of each immunoprecipitate was digested with Endo H (+) or left untreated () before electrophoresis (12% polyacrylamide) under reducing conditions. Positions of the expected glycosylated (tg and pg) and deglycosylated (dg) proteins are indicated by arrows. The asterisk marks the abnormal chain observed in cells after Endo H treatment. C, CD4+ cells, CD4++ cells as controls, and CD8+ cells for comparison were labeled with [35S]methionine/cysteine, lysed, precipitated with OKT3, and digested, where indicated, before electrophoresis (12% polyacrylamide) under reducing conditions. D, left, CD8+ cells were labeled with [35S]methionine/cysteine, lysed, precipitated with the indicated TCR-specific antibodies, and electrophoresed (12% polyacrylamide) under reducing conditions. CD8++, CD4+, and CD4++ cells were included for comparison. For immunoprecipitations with H36, an independently derived HVS-immortalized CD8+ cell line termed D8EDTA (sample 2) was analyzed in parallel with DSF8 (normally termed , but in this particular immunoprecipitation termed 1). Right, CD8+ and CD4+ cells ( and +) were [35S]methionine/cysteine-labeled, lysed, immunoprecipitated with the TCR-specific antibody F1, and digested with Endo H (where indicated) before electrophoresis under reducing conditions in a 12% polyacrylamide gel.

Second, a more detailed analysis of [³⁵S]methionine/cysteine-labeled CD3-associated TCR chains was performed under reducing conditions (Fig. 5B), with or without Endo H treatment to remove pre-Golgi N-linked oligosaccharides. The results using two different anti-CD3 mAbs (OKT3 and APA1/2) clearly confirmed that the TCR composition of CD8⁺ cells was different from that of ⁺ controls. Indeed, it involved not only the loss of normal TCR (dg) as observed in Fig. 5A, but also the addition of another, unidentified 32-kDa protein (Fig. 5B, *). By contrast, TCR (dg) was present and normal in size in CD8⁺ cells. There were also some notable differences between Endo H lanes and ⁺ Endo H lanes: 1) as shown in Fig. 5A, totally glycosylated TCR (tg) chains were absent or very reduced in cells; 2) as in Fig. 5A, totally glycosylated TCR (tg) was present in cells, and an additional labeled protein running slightly faster than tg (probably partially glycosylated TCR) was observed only in cells; and 3) a very strongly labeled 38-kDa protein was prominent in cells, coincident in apparent molecular mass with partially glycosylated TCR (pg). This protein could represent the glycosylated form of the unidentified 32-kDa protein (Fig. 5B, *). However, it may also contain some abnormally glycosylated forms of TCR, because the labeling intensity of deglycosylated TCR (dg) apparently exceeds the combined labeling intensity of normal glycosylated TCR (tg) and the additional labeled protein mentioned above. For comparative purposes, immortalized CD4⁺ cells, both ⁺ and , and CD8⁺ cells were [³⁵S]methionine/cysteine-labeled and analyzed under reducing conditions using OKT3, with or without Endo H (Fig. 5C). The results indicated that immortalized CD4⁺   cells contained normal-sized TCR (tg; pg is not detected in this particular gel) and TCR (tg) proteins and an additional minor labeled protein running slightly faster than tg (again, probably partially glycosylated TCR). CD8⁺ cells, in contrast, lacked normal TCR (tg) but showed normal TCR (tg; partially glycosylated TCR is not apparent in this particular gel), and the strongly labeled 38-kDa protein seen in Fig. 5B, which perhaps is the glycosylated form of the unknown 32-kDa protein (Fig. 5C, *), and some glycosylated form of TCR.

Finally, direct immunoprecipitations of TCR chains were carried out in [³⁵S]methionine/cysteine-labeled cells and analyzed under reducing conditions (Fig. 5D). The results using an anti-TCR mAb (F1, Ref. 13) confirmed the absence of normal TCR in CD8⁺ cells but not in CD8⁺⁺ controls or in CD4⁺ cells (⁺ or ). The TCR chain of the CD8⁺⁺ sample had slightly smaller (~1 kDa) relative molecular mass than in CD4⁺ cells, perhaps due to genetic variation (the specific transcripts were also slightly smaller in this particular cell line, see below and Fig. 8). Similar results (i.e. lack of normal TCR in CD8⁺ cells, sample ₁ ) were obtained with an unrelated antiserum against TCR (H36, Ref. 14). The lack of TCR was confirmed with H36 in an independently derived HVS-immortalized CD8⁺ cell line termed D8EDTA (sample ₂ in Fig. 5D). By using an anti-TCR mAb (F1, Ref. 13), totally glycosylated TCR (tg) was detected in CD8⁺ cells as well in their CD8⁺⁺ controls, and also in immortalized CD4⁺ cells and their ⁺ controls (Fig. 5D). Interestingly, in addition to tg, two highly labeled 40- and 42-kDa proteins were observed in CD8⁺ cells. The proteins were also present in CD8⁺⁺ controls, although at clearly lower relative levels, despite the fact that the number of cells used for the analysis was similar (5 × 10⁶ in all lanes). Such excess of F1-reactive proteins in CD8⁺ cells may explain the significant increase of intracellular F1 binding observed in CD8⁺ cells, as compared with CD4⁺ cells (relative to ⁺ cells, Fig. 4A). The two additional proteins are probably glycosylated forms of TCR, because they resolved into a single protein with the apparent molecular mass of deglycosylated TCR (dg) after Endo H digestion. Indeed, at least one of these proteins may be present in the very strongly labeled 38-kDa CD3-associated protein observed in CD8⁺ cells (Fig. 5, B and C). Alternatively, they may represent unidentified proteins that pair with TCR in some T cell types. Similar additional proteins have also been observed in Jurkat cells using F1 (24).

Collectively, the results of the biosynthetic studies indicated that immortalized CD8⁺ cells lacking CD3 contained an abnormally small TCR heterodimer composed of a normal TCR chain, some abnormally glycosylated TCR chains and an additional unidentified chain that was smaller than normal TCR and could not be recognized by two unrelated TCR-specific antibodies. To ascertain whether the intracellular findings were representative of the surface mutant TCR·CD3 complex, CD8⁺ (and, as a control, CD8⁺⁺) cells were radioiodinated, lysed, and immunoprecipitated with APA1/1 (anti-CD3). Half of the precipitates were digested with N-Gly to remove all N-linked oligosaccharides before electrophoresis under reducing conditions. The results are shown in Fig. 6A. Clear differences were observed between and ⁺ cells, both in TCR and in CD3 chains. In contrast to biosynthetic results, the amount of CD3-associated TCR proteins was clearly lower (around 3-fold by electronic densitometry) in cells than in ⁺ cells, so that only major TCR proteins were visible in samples. With this limitation, the surface-labeled TCR of cells (Fig. 6A $) apparently lacked both normal (i.e. fully glycosylated) TCR and TCR. The lack of normal TCR in CD8⁺ cells confirmed previous biosynthetic results (see above, Fig. 5). The lack of normal TCR, in contrast, was not found in those biosynthetic experiments, in which TCR was present as a minor but detectable protein (Fig. 5, B and C). Thus, it cannot be ruled out that the surface TCR of CD8⁺ cells contained a small amount of fully glycosylated TCR that cannot be detected by radioiodination due to the impaired association of surface TCR chains to CD3 in cells. Two labeled TCR chains, with apparent molecular masses of 40 and 43 kDa, respectively, are discernable in cells before deglycosylation. Such proteins could in part represent abnormally glycosylated TCR, as they are reminiscent in size and intensity of the two highly labeled proteins that coprecipitated with normal TCR when F1 was used (Fig. 5D) and, in the case of the smaller one, of the strongly labeled 38-kDa protein coprecipitated with CD3 in Fig. 5, B and C. The fact that, upon deglycosylation, a major portion of those proteins had the same apparent molecular mass as normal deglycosylated TCR + TCR (( + ) dg) would lend support to this notion. In addition, a minor portion of the 40- or 43-kDa protein resolved upon deglycosylation into an abnormally small protein of about 30 kDa (Fig. 6A, *), which may be identical to the deglycosylated 32-kDa protein precipitated with anti-CD3 in Fig. 5, B and C. This unidentified protein may represent the protein that is associated with TCR in place of normal TCR in CD8⁺ cells. These results confirmed that the surface TCR composition of CD8⁺ cells was essentially similar to the aberrant intracellular complex characterized previously. However, in the extracellular complex, an impaired association of TCR chains to CD3 components was observed, and neither normal totally glycosylated TCR nor TCR was detectable. Rather, abnormally glycosylated TCR and an unidentified 30-kDa chain were observed. CD8⁺ showed also some clear differences in CD3 chains composition as compared with CD8⁺⁺ controls, which were not evident in biosynthetic experiments. Indeed, the sample had, in addition to normal CD3, one or more highly labeled proteins with an apparent molecular mass in the range of 25-30 kDa, within the size range of normal CD3 and CD3 (Fig. 6A, ?). Upon deglycosylation, the protein or proteins had the same apparent molecular mass as normal CD3 (dg). Therefore, the 25-30-kDa proteins may be abnormally glycosylated CD3. However, due to the increased amount of labeled protein in the deglycosylated sample, there could also be more than one protein with a size similar to that of CD3 present. To further substantiate that the highly labeled 25-30-kDa protein(s) of CD8⁺ cells was abnormally glycosylated CD3, it was eluted from the gel, deglycosylated with Endo H, and analyzed by electrophoresis under reducing conditions. Normal CD3 and CD3 were analyzed in parallel, as controls. The results are shown in Fig. 6B. The normal CD3 sample showed mature glycosylation, whereas normal CD3 showed about half mature and half immature glycosylation. The 25-30-kDa protein(s) from cells did not run at the same size as CD3 or CD3 from ⁺ cells, and, in contrast to normal CD3, showed no change in size upon Endo H treatment, consistent with mature glycosylation of surface-labeled proteins. Taken together, these results suggested that the surface TCR·CD3 complex of cells contained normal CD3 and an additional protein, which most likely represented abnormally glycosylated CD3. Experiments are in progress to prove the identity of such protein, by amino acid analysis and partial protease digestion of the deglycosylated form of CD3 in versus ⁺ cells.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   TCR·CD3 immunoprecipitation of radioiodinated CD8+ cells and CD8+ + cells as controls. A, CD8+ cells, either + (left) or (right), were surface-labeled with 125I, lysed in a 1% Brij96-containing buffer, precipitated with an anti-CD3 mAb (APA1/1) and digested, where indicated (+), with N-Gly before electrophoresis under reducing conditions in a 12% polyacrylamide gel. B, certain CD3 proteins were eluted from the experiment shown in A and digested with Endo H, where indicated (+), before electrophoresis under reducing conditions in a 12% polyacrylamide gel. The positions of the expected glycosylated (tg) or deglycosylated (dg) proteins are indicated by arrows. $ and ? mark the abnormal glycosylated CD3-associated TCR and CD3 chains of CD8+ cells, respectively. *marks the putative abnormal deglycosylated TCR chain of CD8+ cells.

The surface labeling experiments collectively indicated that immortalized CD8⁺ cells expressed an aberrant TCR·CD3 complex with fewer CD3-associated TCR proteins, lacking normal TCR and normally glycosylated TCR among them, and with what appeared to be abnormally glycosylated forms of TCR and CD3, together with an unidentified 30-kDa protein (in deglycosylated form), which may represent the protein paired with TCR on the cell surface. Some of these biochemical results were reminiscent of TCR⁺ cells (25), but the possibility that CD8⁺ cells were TCR⁺, rather than aberrant TCR⁺, was ruled out by staining with -specific monoclonals TCR1 (26) and 11F2 (27) (data not shown) and by Northern blot analysis (see below).

The aberrant TCR of CD8⁺ cells was not present in CD4⁺ cells or in ⁺ controls (CD4⁺ or CD8⁺). Therefore, it could explain the selective conformational differences detected in peripheral blood (4, 7) or immortalized (Ref. 5 and the present study) CD8⁺ cells by cytofluorometry. However, it was necessary to rule out the possibility that CD8⁺ cells were an aberrant clone. To this end, first, V usage was tested by reverse transcription-PCR and found to be comparable to matched controls (32% of the 22 V genes tested; normal range, 31-45%; Fig. 7, ₁ HVS). Second, the lack of TCR was confirmed on an independently derived HVS-immortalized CD8⁺ T-cell line termed D8EDTA (Fig. 5D, Sample ₂ HVS). This T-cell line was also oligoclonal (33% of the V genes tested, Fig. 7, ₂ HVS), but with a different repertoire than ₁ HVS. These patterns were compatible with the variable predominance of V gene usage in HVS-immortalized peripheral T cells previously described (28).

View larger version (79K): [in this window] [in a new window]   Fig. 7.   TCR V usage by HVS-immortalized CD8+ cells. 22 V-specific reverse transcription-PCR were performed as described under "Experimental Procedures," and reverse transcription-PCR products were subsequently hybridized with a 32P-labeled constant TCR probe. Two independently derived CD8+ T cell lines (1 or DSF8 and 2 or D8EDTA (see the legend to Fig. 5D)) were compared with normal CD8++ T cells (+ HVS). Fresh peripheral blood T cells, both ( PBL) and + (+ PBL) were analyzed in parallel and are included for comparison. The numbers indicate the particular V amplified in each lane.

TCR Gene Expression Was Normal in Immortalized CD3-deficient CD8⁺ Lymphocytes-- The lack of normal TCR in CD8⁺ cells suggested the possibility that they expressed alternative mature (⁺) or immature (pT⁺) TCR ensembles. To explore such possibilities, total RNA was isolated from CD8⁺ cells, and from appropriate controls, and probed for several TCR specific transcripts by Northern analyses (Fig. 8). The results demonstrated that CD8⁺ cells contained normal levels and sizes of mature TCR and TCR mRNA, whereas they lacked expression of TCR, TCR, and pT transcripts. In addition, they did not display germline transcription at the TCR locus and, therefore, T early  transcripts were absent (data not shown). These results support the conclusion that CD8⁺ cells are not NK cells, nor T cells. Interestingly, they express the TCR gene at the transcriptional level, although it is apparently not expressed at the protein level.

View larger version (74K): [in this window] [in a new window]   Fig. 8.   Analysis of TCR, TCR, TCR, TCR, and pT gene transcription in immortalized CD4+ + and or in CD8+ + and cell lines. Northern blots of total RNA isolated from the indicated cell sources were hybridized with specific 32P-labeled probes, as depicted. Wild type Jurkat cells (TCR+), Peer (TCR+), or SUP-T1 (immature pT-expressing lymphocytes (16)) were analyzed in parallel. In all cases, -actin mRNA expression served as an internal positive control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we have investigated TCR·CD3 assembly and expression in immortalized natural mutant CD8⁺ and CD4⁺ T lymphocytes lacking CD3. We were prompted to undertake this study by the unexpected observation that peripheral blood TCR·CD3 expression was more impaired in CD8⁺ than in CD4⁺ cells when CD3 is absent (Refs. 4 and 5 and Fig. 1A). Despite their phenotypical defect, CD8⁺ cells were functional (Fig. 2). Therefore, we tried to understand the mechanisms behind the feeble expression of surface TCR·CD3 on those cells. The unexpected finding was that, in addition to lacking CD3, immortalized CD8⁺ cells, but not CD4⁺ cells, lacked or expressed few mature TCR chains (see below).

The consequence of this biochemical finding was that membrane TCR·CD3 complexes from immortalized CD3-deficient CD8⁺ T cells were conformationally different from those of CD3-deficient CD4⁺ T cells or normal CD8⁺ and CD4⁺ T cells (Fig. 3). These conformational differences may explain the selective TCR·CD3 expression impairment detected previously in peripheral blood (4, 7)² or immortalized (Ref. 5 and present data) murine or human CD8⁺ T cells by cytofluorometry. Greater sensitivity of CD8⁺ cells than CD4⁺ cells to the absence of CD3 (in terms of TCR·CD3 surface expression) might be related to differences in the intracellular control of TCR assembly, maturation, and/or transport between the two lineages. The lower levels of surface TCR found in normal mature CD8⁺ T lymphocytes (2) may be the reflection of such biochemical differences (thus qualitative rather than quantitative) and perhaps relevant in the context of receptor-coreceptor interactions (see below). Indeed, the comparative staining of peripheral blood CD4⁺ and CD8⁺ cells using a broad panel of TCR·CD3-specific mAbs revealed that, in contrast to expectations, certain mAbs stained CD8⁺ cells better than CD4⁺ cells.³ A similar situation, i.e. increased sensitivity of CD8⁺ versus CD4⁺ cells in TCR·CD3 expression, has also been observed in mice lacking CD3 (3, 6). The biochemical basis, however, remained unexplored, perhaps due to the scant peripheral blood lymphoid compartment.

A further study of the influence of CD3 deficiency on expression of other TCR·CD3 components was performed. Pulse-chase experiments clearly showed the following points: 1) absence of CD3 chains, 2) absence of CD3 chain coprecipitation, and 3) absence of TCR heterodimer maturation in CD8⁺ T cells (Fig. 5A). In addition, TCR chains seemed more stable, a finding that, together with absence of CD3 coprecipitation and of TCR heterodimer maturation, strongly indicated that TCR·CD3 complexes (or at least most of them) stayed in the endoplasmic reticulum (29). The same results were obtained when several different anti-TCR·CD3 mAbs were used in the other immunoprecipitations.

Nonglycosylated TCR chains have a molecular mass of about 30 kDa, and partially or fully glycosylated TCR chains have molecular masses of about 40-45 kDa (up to six N-linked sugars). Nonglycosylated TCR chains have a molecular mass of 35 kDa, and partially or fully glycosylated TCR chains have a molecular mass of about 37-40 kDa. Deglycosylation experiments with Endo H demonstrated that deglycosylated TCR chains from the CD3-deficient CD4⁺ or CD8⁺ cells migrate to the same position. However, deglycosylated TCR chains from CD3-deficient CD8⁺ T cells have a molecular mass of 2-3 kDa lower than normal (32-34 versus 30-32 kDa; see Fig. 5, B and C). As TCR C cDNA sequences in CD4⁺ and CD8⁺ CD3-deficient T cells are the same as in normal T cells,⁴ the most logical explanation seems to be that TCR chains from CD3-deficient CD8⁺ T cells are more easily deglycosylated compared with CD3-deficient CD4⁺ T cells (or normal T cells). This may be due to the fact that TCR chains in CD3-deficient CD8⁺ T cells are more immature than those of CD3-deficient CD4⁺ T cells or normal T cells. It is likely that immature TCR loses recognition by F1 and other C-specific reagents. Indeed, the F1 monoclonal recognizes an exposed epitope spanning residues 141-159 of the C domain (13), including an Asn residue, which is one of the four potential N-glycosylation sites of the human C domain (30). Loss of recognition by F1 has been reported previously in a Jurkat variant lacking the transmembrane and cytoplasmic domains of TCR (31). Parallel studies on CD3-deficient Jurkat T cells have shown that 1) an inverse relationship exists between the amount of TCR chains coprecipitated and the amounts of CD3 chains produced in a T cell line, and 2) the production of only partially glycosylated CD3 chains influences TCR chain glycosylation.⁵

Abnormal assembly and glycosylation of the TCR·CD3 complex have been reported previously in CD4⁺ cells (12, 22). However, the differences in TCR observed in CD8⁺ versus CD4⁺ -deficient cells have not been reported previously. Nascent TCR chains have been shown to be uniquely unstable in immature murine T cells (32), perhaps because of altered processing of oligosaccharide side-chains in the endoplasmic reticulum (33). These differences are believed to cause the reduced TCR·CD3 expression observed in immature versus mature T lymphocytes (34) or in transfected eukaryotic cells (35). Similarly, our results suggest that human mature CD8⁺ and CD4⁺ T lymphocytes may differ in the intracellular control of TCR·CD3 assembly, maturation, or transport. Such differences would be revealed when CD3 is absent, particularly in TCR, which is the most unstable chain.

The most difficult data to interpret are those from external labeling of TCR·CD3 complexes from CD3-deficient CD8⁺ T cells: 1) few TCR molecules are labeled/coprecipitated (in proportion to CD3 chains precipitated), and 2) deglycosylated TCR chains have an apparent molecular mass lower than deglycosylated TCR chains from normal T cells (Fig. 6). In addition, CD3-deficient CD8⁺ T cells seem to express CD3 (and probably TCR) chains, which are more glycosylated than the normal counterparts. Thus, are the TCR·CD3 complexes on the surface of CD3-deficient CD8⁺ T cells normal, with expression of low levels of mature TCR chains? Or alternatively, are the TCR·CD3 complexes on the surface of CD3-deficient CD8⁺ T cells constructed with a TCR chain different from normal TCR chains?

Despite the observed biochemical differences in the TCR·CD3 complex of CD8⁺ versus CD4⁺ cells, their functional behavior was undistinguishable when several CD3-induced activation events were analyzed (5). Thus, it may well be that the peripheral T lymphocytes that we can analyze when CD3 is absent are those carrying TCR·CD3 complexes that are conformationally compatible with the corresponding coreceptor. A direct interaction of the TCR with CD4 and CD8 has been proposed before (36-39).

TCR·CD3-induced calcium flux was normal in CD4⁺ and CD8⁺ CD3-deficient PBLs (Fig. 2) and HVS-immortalized (5) T cells. However, it was partially impaired in an unimmortalized CD4⁺ T cell line (DIL2) derived from the same donor (12). This discrepancy may have been due to clonal variation of DIL2 or, alternatively, to differences in the selective pressures imposed by the culture conditions. DIL2 was grown with PHA, which is a strong TCR·CD3 stimulant, whereas HVS cells grow by TCR·CD3-independent interactions (11) and may therefore be more representative of PBL behavior.

	ACKNOWLEDGEMENTS

We thank Drs. A. Arnáiz-Villena and A. Corell for sharing DSF4 cells, Drs. C. Trigueros and A. Alvarez for technical support, and Drs. O. Acuto, J. Borst, M. Brenner, D. Jaraquemada, E. L. Reinherz, and J. E. de Vries for mAb samples. Hoffmann-LaRoche is gratefully acknowledged for the continuous supply of recombinant IL-2.

	FOOTNOTES

^* This work was supported by Ministerio de Educación y Cultura Grant PR112/96, Comunidad Autónoma de Madrid Grant 13/97, Comisión Interministerial de Ciencia y Tecnología Grant SAF96/119, Dirección General de Enseanza Superior e Investigación Cientifica Grant PM98/91, and Ministerio de Educación y Cultura Grant HF1996/0163 (to J. R. R.).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.

^§ Supported by the Ministerio de Educación y Cultura.

^¶ Supported by the Comunidad Autónoma de Madrid.

Supported by the Universidad Complutense de Madrid.

^§§ To whom correspondence should be addressed. Tel.: 34-91-3941642; Fax: 34-91-3941641; E-mail: regueiro@med.ucm.es.

² M. van Tol, personal communication.

³ P. S. Torres, unpublished data.

⁴ D. A. Zapata, unpublished data.

⁵ A. Huchenq, L. Alibaud, C. Bouchouata, C. Gouaillard, R. Llobera, A. Alcover, J. Arnaud, and B. Rubin, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: HVS, Herpesvirus saimiri C-488; MFI, mean fluorescence intensity; TCR, T-cell receptor; PCR, polymerase chain reaction; Endo H, endo--N-acetylglucosaminidase H; PBL, peripheral blood lymphocyte; mAb, monoclonal antibody; N-Gly, N-glycosidase F.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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