A human TCR-Ig chimeric protein used to generate a TCR α chain variable region-specific mAb

Mo/ecu/ar Immunology, Vol. 27, No. I I, pp. 1127-I 136, 1990 Printed in Great Britain.

0161-5890/90 $3.00 + 0.00 % I990 Pergamon Press plc

A HUMAN TCR-Ig CHIMERIC PROTEIN USED TO GENERATE A TCR a CHAIN VARIABLE

REGION-SPECIFIC mAb*

GEORGES BISMUTH,~~ HBLZNE GouY,t ROY A. MARIUZZA,$ CATHERINE VAQUERO~~ IOANNIS THEoDoRout and PATRICE DEBRET

tLaboratoire d’Immunologie Cellulaire et Tissulaire, CNRS UA 186, HGpital de la Pitii-SalpZtri&re, Paris, France; §DCpartement d’Immunologie, Institut Pasteur, 75015 Paris, France; /)INSERM Ul52,

HBpital Cochin, 75014 Paris, France

(First received 23 February 1990; accepted in revised form 18 April 1990)

Abstract-In a recent report, a construction containing the a chain-variable region (Va) coding sequence of a cDNA clone derived from a diphtheria toxoid-specific human T cell (P28), fused to a human immunoglobulin kappa light chain constant region (Ck), was used stably to transfect a murine myeloma cell. In the present study, these transfected cells were employed as an immunogen to raise a mAb, termed ICSVa, specific both for the VaCk chimeric protein secreted by the transfectant and the P28 T cell antigen receptor-Va region. mAb lC5Va specifically immunoprecipitates the VaCk protein as a family of 32-35 kDa bands present in the 35S-methionine-labeled culture supematant from the transfected cells. It specifically binds clone P28. Surface molecules recognized by mAb 1CSVa are physically linked to the CD3 molecules since cell treatment with either lC5Va or anti-CD3 mAbs caused the simultaneous down- regulation of the CD3/TCR molecular complex. This link is further supported by immunoprecipitation experiments. Thus, both the ICSVa and the anti-CD3 mAbs precipitate the 16-28 kDa CD3 molecules and the disulfide-linked form of P28 TCR from 1Z51-labeled P28 T cells. Studies performed in order to define whether a stimulus directly acting on the TCR-Va region may trigger the intracellular events observed during human T cell activation showed that (a) mAb lC5Va efficiently triggers the phospholipase C transduction pathway revealed by an accelerated phosphoinositides turn-over and an increased production of phosphorylated derivatives of inositol phosphates; (b) mAb lC5Va induces an up-regulation of ILZR mRNA, accompanied by a slight increase of IL2 and IFNa mRNA transcripts evidently amplified in the presence of PMA; (c) soluble mAb lC5Va is strongly mitogenic together with PMA. These results provide the first evidence for the structural authenticity of a secreted water-soluble chimeric form of the variable region of a human TCR a chain. They further demonstrate that such chimeric proteins may be valuable tool to further dissect the various functional structure of the human TCR.

INTRODUCTION

The T cell antigen receptor for foreign antigen is a cell surface heterodimer composed of two different chains, u and j?, associated with the CD3 multimeric molecular complex (Acuto et al., 1983; McIntyre and Allison, 1983; Meuer et al., 1983; Samelson et al., 1985). Each chain of the TCR contains a variable (V) and a constant (C) region, involved in the recognition of antigenic determinants in the context of a self major histocompatibility complex product and anchorage to the T cell surface, respectively (Kappler et al., 1983; Kronenberg et al., 1986). V and C regions are encoded like immunoglobulin chains by separate,

*This work was supported by grants from the Institut National de la Sante et de la Recherche Medicale.

iAuthor to whom correspondence should be addressed at Laboratoire d’Immunologie Cellulaire et Tissulaire, CERVI, Hbpital de la Piti&Salp6triZre, 83 Boulevard de l’H6pita1, 75013 Paris, France.

Abbreviations: APC: Antigen Presenting Cells; PMA: phorbol 12-myristate 13-acetate; DT: diphtheria toxoid; PA: phosphatidic acid; PI: phosphatidylinositol; IPs: inositol phosphates; PLC: phospholipase C.

non-continuous gene segments in the germline, which rearrange to produce a functional gene (Chien et al.,

1984; Hedrick et al., 1984; Kimura et al., 1986; Yanagi et al., 1985). The human Vcr region is encoded by two distinct gene segments, Vu and joining (Ja), which rearrange during T cell differentiation to generate a continuous Vu gene (Caccia et al., 1988; Yoshikai et al., 1985).

Detailed informations on the structure on the TCR, mainly concerning the physical basis of the dual mechanism of T cell recognition, have not been clearly established. The development of expression systems capable of producing large amounts of secreted TCR variable domains appears therefore to be a starting point in the understanding of TCR structure. One reason for this approach was based upon the assumption that such secreted material should have some structural homology with the TCR and may serve as a basis for an initial structural analysis of this molecular complex. In this respect, in a recent study, a chimeric protein consisting of a human T cell clone-Va region fused to a human immunoglobulin kappa light chain constant region was obtained (Mariuzza and Winter, 1989). This

1127

1128 GEORGES BISMUTH er al.

protein was synthetized by a murine myeloma cell line transfected by a construction containing the GI chain variable region coding sequence of a cDNA clone derived from a diphtheria toxoid(DT)3-specific human T cell termed P28 (Triebel et al., 1988). In the present report we have used this material to obtain a monoclonal antibody recognizing both the VaCk chimeric protein and the T cell clone P28. In most cases, anti-human TCR mAbs were obtained by immunization with whole T cells or soluble immunoprecipitated cc//l heterodimers (reviewed in Kung, 1987). Such procedures made it difficult to assess and distinguish the signalling role of various a and /!I variable or constant region epitopes. The use of this Va-containing chimeric protein gave us the opportunity to raise by this approach a mAb against the Va region of a human TCR c( chain and to study the role of the Va region in the activation process of clone P28. These results ultimately provided the first evidence for the reliability of the secreted Va molecule with a functional domain of the original TCR a chain.

MATERIAL AND METHODS

Reagents

L-[3sS]methionine (> 1000 Ci/mmol), “‘Iodine, myo- [2-3H]inositol (10-20 Ci/mmol) and 32P-orthophosphoric acid (carrier free) were bought from Amersham International. [6-3H]thymidine (1 Ci/mmol) was obtained from ORIS, Gif sur Yvette, France. Phorbol 12-myristate 13-acetate (PMA) was obtained from LC Services Corporation, Woburn, MA. Diphtheria toxoid (DT, 5000 Lf/ml) was kindly provided by Dr Perquin, Institut Pasteur, Paris, France.

T-cell clone

All T cell clones were specific for DT. They were derived from an HLA-DR6/7 healthy individual and propagated by periodic restimulation with the antigen presented by an autologous Epstein Barr virus (EBV) transformed B cell line as previously described (Triebel et al., 1984).

T cell proliferation assays were performed in 96 well flat-bottomed microtiter plates in a final volume of 200 ~1 (5 x lo4 T cells/well). Proliferation was measured after a 3 day culture by a 16 hr pulse with 1 pCi/well of [‘Hlthymidine.

Transfectants

The production of J558L myeloma cells, transfected with a chimeric gene consisting of the Va region of the TCR of the DT-specific T cell clone P28 fused to a human immunoglobulin k light chain constant region, was previously described (Mariuzza and Winter, 1989). Mouse L cell fibroblasts transfectants L12.2 (DRa :DR7Pl) were produced and cultured as previously reported (Klohe et al., 1988).

Hybridoma production and mAbs

Spleen cells of BALB/c mice immunized with VaCk-transfected J558L cells were fused with mouse myeloma Sp2/0 in the presence of 45% polyethylene glycol 1500 as previously described (Galfre and Mil- stein, 1981). The secreting hybridomas were screened for their ability to stain the T cell clone P28 by indirect immunofluorescence with fluorescein isothiocyanate-conjugated affinity-purified goat anti-mouse IgG Fc fragments (Nordic Immunology, Tilburg, The Netherlands). Immunofluorescence analyses were performed on a Facstar cell sorter (Becton Dickinson, Mountain View, CA). Anti-CD3 mAb X35 (IgG2a), a kind gift of Dr Bourel, CNTS, Rennes, France and anti-CD2 mAbs X1 1 and D66, kindly provided by Dr A. Bernard, were previously described (Huet et al., 1986; Kurrle et al., 1986). All mAbs were used as ascitic fluid.

Immunoprecipitations

Radioiodination of T cell surface proteins was performed as previously described (Hubbard and Cohn, 1975) using the lactoperoxydase procedure. Briefly, 10’ to 2 x 10’ cells were labeled with l-2 mCi lzsI and solubilized for 1 hr at 4°C in a lysis buffer containing 10mM Tris-HCI (pH 7.2), 150 mM NaCl, 10 u/ml aprotinin and either 0.5% Triton Xl00 or 1% digitonin. The lysate was centrifuged at 12,000 x g and precleared for 30 min with 50 ,nl of 10% (w/v) Staphylococcus Cowan Strain I. The ‘251-labeled lysate was then incubated for 2 hr at 4°C with 3-5 ~1 ascitic fluid of the specified mAb. Immunoprecipi- tations were carried out with 30,ul packed protein A-Sepharose. For low-protein A binding mAb (IgGl), protein A-Sepharose was preincubated with rabbit anti-mouse IgG antiserum. The samples were washed four times with lysis buffer containing 0.1% Triton Xl00 and once with PBS. The immune complexes were then boiled in SDS sample buffer containing or not 2-mercaptoethanol and analyzed on 12.5% discontinuous polyacrylamide gels.

For immunoprecipitation of the VaCk secreted chimeric protein, lo6 J558L transfectants were incubated in 1 ml methionine-free RPM1 1640 containing 5% dialyzed FCS and 100 PCi L-[35S]methionine for 16 hr at 37°C. The 35S-labeled supernatant was immunoprecipitated as described above with sheep anti-human k light chain antiserum (Miles, ICN or mAb ascitic fluid followed by precipitation with protein A-Sepharose. The precipitated proteins were analyzed under reducing conditions by SDS-PAGE (12.5%).

Phospholipase C activity

12P labeling of phosphatidylinositol (PI) cycle- related phospholipids and measurement of inositol phosphates (IPs) production were performed as previously described (Bismuth et al., 1988a,b). Briefly, for phospholipids analysis, T cells were incubated

mAb to human TCR Va region-containing chimeric protein 1129

in phosphate-free medium with carrier free 32P- orthophosphoric acid (50 pCi/ml) for 5 min at 37°C. Aliquots of the cell suspension (100~1) were then stimulated with the different mAbs for 1 hr at 37°C. The reaction was stopped with concentrated HCl and the 32P-labeled phospholipids extracted, separated and quantified. For the IPs production assay, myo- [2-‘Hlinositol (5 nCi/ml) overnight-prelabeled T cells were stimulated with the different mAbs or with antigen-pulsed DR7/3 1 transfectants (50 fig/ml of DT overnight) for 30 min at 37°C. IPs were then quantified by anion-exchange chromatography on Dowex AG l-X8 in the formate form (Bio-Rad Laboratories, Richmond, CA).

Northern blot analysis

Total cellular RNA was extracted at the indicated times by the guanidinium isothiocyanate procedure (Vaquero et al., 1982). For each point equal amounts of total RNA (10 pg) were fractionated by glyoxalate agarose gel electrophoresis and transferred (Thomas, 1980) to gene screen filters (New England Nuclear, Boston, MA). Filters were then sequentially hybrid- ized at high stringency with riboprobes obtained after transcription of the T,T, Blue Scribe Vector (Vector Cloning System, San Diego, CA) containing the 1.35 kb Pst I-Barn HI fragment from hu IL2 receptor (IL2R)3 cDNA (Leonard et al., 1984) and 1 kb Hint II from hu y interferon (IFNy) cDNA (Paul Sondemeyer, Transgene and Vaquero et al., 1984). Riboprobes were used at 5 x 105cpm/ml of hybridization medium, and after high stringency washes, radioactivity bound to the filters was detected by autoradiography. The levels of ribosomal RNA were visualized for quantification and the sizes of mRNA estimated from relative position of the 28s and 18s RNA bands in ethidium bromide-stained gels.

RESULTS AND DISCUSSION

Production of mAb lCSVc( specific for a chimeric protein containing a T cell antigen receptor Vu region

In a previous report, a chimeric Va T cell receptor- Ck immunoglobulin genes construction was used to stably transfect the myeloma cell line J558L (Mariuzza and Winter, 1989). The Vu region sequence was initially obtained from a cDNA clone derived from a DR7-restricted human T cell clone (P28) specific for the CBl peptide of the B chain of diphtheria toxoid (DT) (Triebel et al., 1988). This sequence was shown to belong to the Vu 4.3 family, the Ja gene segment corresponding to the JclF germline sequence (Caccia et al., 1988). The transfectant efficiently synthetized and also secreted the Va Ck chimeric protein as a non-covalent homodimer of 65 kDa. BALB/c mice immunized i.p. with the VuCk transfectant developed a serological response against immunoglobulin k light chains as well as against clone P28 (data not shown). In order to raise specific mAb to the Va region of the T-cell antigen receptor, we therefore

FLUORESCENCE INTENSITY

Fig. 1. Immunofluorescence analysis of mAb 1 CSVa. P28 T cells were incubated at 4°C with pure culture supematant from the various secreting hybridomas. Ascitic fluid of anti-CD3 mAb X35 (l/400 dilution) was used as a positive control. The cells were then stained with FITC-conjugated goat anti-mouse IgG antibodies. (A) a negative supematant;

(B) 1CSVa; (C) CD3-specific mAb X35.

fused mouse myeloma Sp2/0 with the immunized mouse spleenocytes. Secreting hybridomas were screened by indirect immunofluorescence on P28 T lymphocytes and one positive clone termed lC5Vcr (IgGl kappa) was isolated (Fig. 1).

Specificity of mAb lC5Va was first analyzed on the VcrCk chimeric protein secreted by the transfected J558L cells. 35S-methionine labeled culture super- natants from the transfected cells were immunoprecipitated with either an anti-k human light chain antiserum or mAb lC5Va and analyzed by SDS PAGE under reducing conditions. As shown in Fig. 2, both mAb lC5Vcr (lane c) and anti-k antiserum (lane d) immunoprecipitated the same family of 32-35 kDa bands. The predicted size of the VuCk chimeric protein was 25 kDa but we reported that its secreted form was modified by the myeloma host by post- translational and terminal glycosylations to give this family of bands (Mariuzza and Winter, 1989). Lane a shows the SDS-PAGE analysis of the whole 35S- labeled proteins present in the culture supernatant of the VcrCk transfectant and lane b the result obtained with a control BALB/c ascitic fluid. The 25 kDa band in lane b and c represents the mouse lambda chain constitutively secreted by J558L (see lane a) and precipitated by the rabbit anti-mouse IgG antiserum used in this experiment with mAb lC5Vcr and control ascitic fluid (see material and methods). The small amount of lambda chain immunoprecipitated in lane d with anti-k antiserum is likely due to k/lambda cross-reactivity as previously suggested (Mariuzza and Winter, 1989).

mAb 1CSVu recognizes P28 T ceil antigen receptor

Since mAb lC5Vcr bound P28 T cells, the above results strongly suggested a specificity of the selected hybridoma for the Va part of the chimeric protein. In order to demonstrate that mAb lC5Va is directed against the a chain variable region of clone P28, both

1130 GEORFES BISMUTH et al.

Fig. 2. SDS-PAGE analysis of the supernatant of VaCk-transfected J558L cells immunoprecipitated by mAb ICSVa. 106/ml VaCk transfectants were labeled in uiuo with 35S methionine (100 pCi/ml) for 16 h at 37°C and the labeled supernatant immunoprecipitated with either 1CSVa mAb or anti-human k light chain sheep antiserum coupled to protein A-sepharose beads. A normal BALB/c ascitic fluid was used as a negative control. The immune complexes were analysed under reducing conditions by SDS-PAGE (12.5%). Lane a: unprecipitated supernatant; lane b: control ascitic fluid; lane c: lC5Va mAb; lane d: anti-k antiserum. In lane b and c protein A-sepharose beads were preincubated with rabbit anti-mouse

IgG (see material and methods).

comodulation experiments of the epitope recognized by mAb 1CSVa together with the CD3 molecular complex and biochemical studies were carried out.

The physical association between TCR and CD3 is actually well established. It was first demonstrated in comodulation studies of the CD3 molecular complex with the m//j’ heterodimer usually achieved by incubation of T cells with antibodies directed against either structure (Meuer et al., 1983; Reinherz et al.,

1982). As shown in Fig. 3a, preincubation of clone P28 with mAb lC5Va during 18 hr at 37°C led to a clear decrease of the cell surface expression of the CD3 molecule recognized by anti-CD3 mAb X35 in a direct immunofluorescence assay. Reciprocally, a reduced expression of the epitope recognized by mAb lC5Va was also observed by incubating the cells with the anti-CD3 mAb X35 (data not shown). Moreover, incubation of T cell clone P28 with 10 ng/ml PMA, known to down-regulate the CD3/TCR complex (Cantrell et al.. 1985) was found to reduce to the same extent 1CSVsr and CD3 expression (Fig. 3b). Taken together these data provide evidence that the cell surface molecule recognized by mAb lCSVc( is physically linked to CD3.

In order to further assess the specificity of mAb lC5Va, P28 T cells were cell surface-labeled with lz51 and immunoprecipitates from the solubilized membranes subjected to SDS-PAGE. Figure 4, panel A, shows that in Triton X-100 extracts, mAb lC5Vcr immunoprecipitates under non-reducing conditions (lane a) a 90 kDa protein and in reducing conditions (lane b) one band of 43/44 kDa. In digitonin extracts (panel B) and under reducing conditions, anti-CD3 mAb X35 (lane d) coprecipitated the same 43/44-kD band together with the different CD3 molecules ranging from 16 to 28 kDa. mAb lC5Vcr (lane c) also precipitated in digitonin extracts the CD3 complex but weakly as compared to mAb X35. All the present biochemical results are therefore consistent with the notion that mAb lC5Vm is directed at the Vet region of P28 TCR. It is to be noticed that when analyzed in reducing conditions the a chain and the fi chain of clone P28 are indistinguishable.

We cannot conclude from our results whether mAb lC5Vcr recognizes a Va4.3 gene segment product or a Vu4.3/JuF-encoded determinant. As shown in Table 1, among a panel of nine different DT-specific T cell clones from the same individual, two clones P28

mAb to human TCR VU region-containing chimeric protein 1131

a

FLUORESCENCE INTENSITY

Fig. 3. Comodulation of the epitope recognized by mAb ICSVa with the CD3 molecular complex. (a) P28 T cells (5 x IOs/ml) were incubated in complete culture medium either with mAb 1CSVa (A) or with normal BALB/c ascitic fluid as a control (B) (l/400 dilution for each) for 18 hr at 37°C. The cells were then washed and labeled with FITC-conjugated anti-CD3 mAb X35. CD3 expression of clone P28 at the beginning of the experiment is also shown (C). (b) P28 T cell (5 x lO’/ml) were incubated in complete culture medium with (closed curve) or without (open curve) 10 ng/ml of PMA for 1 hr at 37°C. The cells were then washed and labeled either with mAb lC5Va (upper panel) or mAb X35 (anti-CD3) (lower panel)

followed by FITC-conjugated anti-mouse IgG.

and P26 were lC5Vsl positive. Nucleic acid sequence of the a chain expressed by clone P26 has not been determined. However, it is clear from recent studies (F. Triebel, personnal communication) that reactivity of a mAb against different V/J associated gene segment products sharing the same V gene does not imply a specificity of this mAb to the V gene-encoded determinant. PBL from different donors were also studied for the expression of lC5Va. No positive cells could be detectable by FACS analysis (data not shown). Note also from Table I that there is appar- ently no correlation between the class II restriction

Table I. ICSVcz expression and DRor : DR7fl I restriction of different DT-specific human T cell clones”.

ICSVU T cell clones Index of proliferation expression”

Antigen presenting cells PBL DRz : DR7P I

P28 133.78 14.88 + ScA 157.05 I .08 _

P22 4.67 1.00 _

Pll 78.86 0.44 _

P26 32.30 28.28 + P30 26.25 8.32 _

P25 25.33 I .30 _

PI3 21.90 13.63 _

P29 14.77 I .04 -

“5 x 10JT cells from the different clones were cultured with 2 x IO’ autologous PBL or 2.5 x IO4 transfected L cells bearing DRa : DR7B I class II molecules, in the presence of IO pg/ml of DT or IO fig/ml of Varidase as a control irrelevant antigen. The proliferation was measured on day 4 after a 16 hr pulse with I PCi of (‘Hjthymidine. The results are expressed as the ratio between DT-specific and Varidase-control cpm values (212 to II36 cpm with Varidase depending from the tested clone). All assays were done in triplicate.

‘lC5Va expression was followed by indirect immunofluorescence as described in the material and methods section.

of the clones and the expression of ICSVa since two additional DR7/l l-restricted clones, PI3 and P30, (others were DRp4;DRw53_restricted, Bismuth et al., 1990) were lC5Va negative (Table I).

mAb lC5 Va triggers the intracellular events typical of human T lymphocyte activation

The CD3/TCR molecular complex plays a key role to initiate events that culminate in T cell activation by foreign antigens. It is actually believed that its two major components have separate functions: the a//? heterodimer mediates T cell recognition of antigen and MHC while the CD3 multimetric component would convert the recognition event into a transmembrane signal initiating T cell activation (Manger et al., 1988). The activating properties of mAb lC5Va were therefore investigated both at the biochemical and functional level to indicate that an alteration of the Va region of the a chain itself was sufficient to deliver a signal to the T cell.

In the T cell activation process, recent attention has focused upon a signalling pathway involving the hydrolysis by phospholipase C (PLC) of inositol phospholipids as one of the earliest biochemical events triggered by specific antigen (Imboden et al., 1987; Pate1 et al., 1987) or mAb against cell surface activation molecules like the CD3/TCR complex (Imboden and Stobo, 1985) or the CD2 (Bismuth et al., 19886; Pantaleo et al., 1987). As a result of this enhanced activity of PLC, both an accelerated turn-over of phosphoinositides (Bismuth et d., 1988~) and an increased production of IPs (Bismuth et al., 19886; Pantaleo et al., 1987) is usually noticed. As

1132 GEORGES BISMUTH et al.

Fig. 4, SDS-PAGE analysis of cell surface molecules immunoprecipitat~ from the “I’ cell clone P28 by lC5Vu and anti-CR3 mAbs. P28 T cells were surface labeled with I mCi tz51 using the lactoperoxvdase technique. The labeled cells were Iyzed in buffer containing either 0.5% Triton X-100 (A) or 1% &v/v) digitonin (B). Imm~nopre~ipitation of the lysates were performed either with ICSVc( or X35 (anti-CD3) mkbs coupled to pro&t A-sepharose beads. A normal BALB/c ascitic fluid was used as a negative control. The precipitated proteins were analyzed by SDS-FACE (12.5%) under reducing (R) or non reducing (NR) conditions followed by autoradiography. a: ICSVc((NR); b and c: ICSVafR); d: X35(R);

e: normal ascitic fluid(R).

shown in Fig. 5, upper panel, 1 hr incubation of P28 T cells with mAb ICWE induced a substantial increase in both PI and phosphatidic acid (PA) 32P-iabe1ing compared to control. A similar treatment with either mAb Xl I+ D&6, a mitogenic pair of anti-CD2 mAb (Huet et al., 1986), or anti-CD3 mAb X35 also ted to enhanced labeling of the two phosphohpids. Addition- ally, mAb 1 CSVcr was also found to induce phosphoinositides hydrolysis leading to an important generation of IFS (Fig. 5, lower panel). The results obtained with mAbs specific for the CD2 and the CD3 molecules, and with antigen, presented by DRc( : DR7@ 1 class II expressing transfectants pulsed overnight with DT, are also shown. It is to be noticed here that, whatever the activating signal delivered at

the level of the CD3jTCR molecular complex, almost identical responses, in terms of both intensity (see Fig. 5) and kinetics (data not shown), were usually observed. By comparison PLC activation triggered via the CD2 molecule was markedly higher (Fig. 5). Identical results were also obtained with the other ICSVor positive T cell clone (data not shown). Taken together, the rest&s obtained here demonstrate that a direct alteration of the Vo! structure of the T cel1 antigen receptor triggers the PI cycle-related activating pathway,

These biochemical results suggested that the TCR- related activation pathway may be initiated through the specific binding of a mAb to the Va part of the human TCR s( chain. In order to assess the functional


nl. as. CD2 CD3 1 C5Va

nl. as. CD2 CD3 lC5Va DT

Fig. 5. mAb 1CSVa induces an increased turnover of PA and PI and an increased production of IPs in P28 T lymphocytes. Upper panel: P28 T cells (10 x lob/ml) were preincubated during 5 min at 37°C with 20 pCi/ml of ‘*PO, in phosphate-free medium. Aliquots (100 ~1) of the cell suspension were then mixed either with the combination of Xl 1 plus D66 CDZ-specific mAbs, or the anti-CD3 mAb X35, or the mAb 1CSVa (l/400 dilution of an ascitic fluid for each). A l/400 dilution of a normal BALB/c ascitic fluid was used as a control. The reaction was stopped after an additional 60 min incubation period at 37°C and the “‘P-labeled phospholipids extracted and quantified as described in material and methods. The results are expressed as the mean cpm for lo6 cells of triplicate determination. Lower panel: P28 T cells (1.5 to 2 x lo6 cells/ml) were labeled to isotopic equilibrium with 10 pCi/ml of myo-[2-3Hlinositol during 18 hr in inositol free medium. The labeled cells were then washed extensively and incubated at lO’/ml in Hank’s buffer containing 10 mM LiCl for 20 min at 37°C. Aliquots (100 ~1) of the cell suspension were then mixed with the different mAbs (l/400 dilution of an ascitic fluid for each) or with 5 x lo5 DR7B 1 transfectants pre-pulsed 18 hr with 50 pg/ml of antigen DT. A l/400 dilution of a normal BALB/c ascitic fluid was used as control. The reaction was stopped after an additional 30 min incubation period and the IPs quantified as described in the Material and Methods Section. The results were expressed as the ratio between these IPs values and the value found just after the 20 min LiCl incubation period (1324 & 74 dpm). Each

determination was performed in triplicate.

events related to that activation process, the ability of mAb lC5Va to increase the expression of specific mRNA, and in parallel to induce a T cell proliferative response was investigated. In a recent report, we have studied IL2 receptor (IL2R), IL2 and y interferon mRNA expression during the stimulation of human T cells, including clone P28, through the CD3 molecular complex. A coordinate up-regulation of these messengers was observed (Paillard er al., 1988). As shown in Fig. 6 (left panel), binding of mAb

MIMM 27111-F

1CSVcr to clone P28 weakly increased levels of IL2 and IFN y mRNA transcripts, while a marked rise of IL2R mRNA transcripts was observed. However, as previously reported for CD3-mediated stimulation (Weiss et al., 1987), a cooperative effect, mainly for lymphokine production, appeared when the clone was stimulated with mAb 1CSVcl together with PMA which role is presumably to mediate PKC activation (Nishizuka, 1984). As expected from these results mAb lCSVc( was not or very weakly mitogenic when


lCSVa/PMA hours

IL2

hours 0 2 4 18 2 4

.

113200 111600 l/600 1 400

lC5Va dilutions

Fig. 6. Time course analysis of mRNA expression and cell proliferation induced by mAb 1CSVa. Left panel: P28 T cells were incubated in complete culture medium with mAb 1CSVa (l/400 dilution of an ascitic fluid) in the presence or not of PMA (1 ng/ml). Total cellular RNA was extracted at the indicated times and analyzed after Northern blot transfer by a sequential hybridization of the filter with the indicated riboprobes. mRNA sizes are also indicated. Right panel; P28 T cells (5 x 104) were cultured with various dilutions of mAb 1CSVu in the presence (closed symbols) or not (open symbols) of 1 ng/ml of PMA. The proliferation was quantified on day 3 after a 16 hr pulse with [3H]thymidine (1 pCi/well). Values represent

the mean cpm of triplicate culture.

used in a soluble form but triggered a high proliferative response of clone P28 in the presence of 1 ng/ml of PMA (Fig. 6, right panel). This suggests that, as reported with CD3 mAbs (Manger et al., 1988), PKC activation by soluble lCSVc( is too transient to induce a sufficient lymphokine production. These functional studies ultimately demonstrate that a signal delivered at the level of the Vu region of the human T cell antigen receptor may trigger most of the intracellular events usually observed in the human T cell activation process.

CONCLUSION

Several types of mAbs have been raised against the cc/p TCR, including anti-clonotypic antibodies that are able to recognize the unique combination of an individual TCR LX/B heterodimer (Allison et al., 1982; Haskins et al., 1983; Meuer et al., 1983) antibodies that are specific for a single TCR protein chain (Saito et al., 1987) or mAbs that are specific for individual TCR variable regions, mainly certain V/I families in the mouse system (Goverman et al., 1985; Haskins et al., 1984; Kappler ef al., 1987; Staerz et al., 1985). In this work, the use as immunogen of an original chimeric protein containing the TCR Vu region of a human DT-specific T cell clone, gave us the opportunity to raise a mAb evidently directed

against a human TCR Va region epitope. This re- agent allowed us to study the possible involvement of the human TCR Vu region as an independent target for a putative activating signal. Indeed, the structural basis for ligand recognition by the cl//? heterodimer remains controversial. Although the consensus, based upon gene sequences analysis of different TCR (Dem- bit et al., 1986) and upon gene transfer experiments (Saito and Germain, 1987; Winoto et al., 1986), is that the contribution of the two chains cannot be separated, a predominant interaction of one chain with antigen or MHC was recently suggested. Thus, concerning the a chain, a major role was indicated for it in cytochrome C and arsonate-specific murine T cell clones (Sorger et al., 1987; Tan et al., 1988). From these results, it has been proposed that T cell antigen receptor a chains may be involved in making the major contacts with antigen epitopes (Tan et al., 1988). Similar observations have not been yet established in the human system. However, the finding that mAb lC5Va triggers most of the intracellular events usually observed in T cell activation, possibly reflects an independent role for the Vcc region in the natural process of the human T cell stimulating pathway by foreign antigens.

Acknowledgement-We thank Dr Robert W. Karr for HLA class II transfectants.


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A human TCR-Ig chimeric protein used to generate a TCR α chain variable region-specific mAb

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