Generation of mAb against soluble human TcR polypeptides
Eur. J. Immunol. 1991. 21: 2111-2119
Brigitte DevauxoAaO, Pamela J. Bjorkmanovo, Christine Stevenson*, William G r e f l , John F. Elliottom, Charles Sagerstrod, Carol ClaybergerO, Alan M. K r e n s k e + and Mark M. DavisAo Howard Hughes Medical InstituteA and Department of Microbiology and Immunologyo, Department of Pediatrics*, Department of Cardiothoracic Surgeryo, Stanford University School of Medicine, Stanford
Generation of monoclonal antibodies against soluble human T cell receptor polypeptides* One approach to the diagnosis and therapy of Tcell-mediated diseases is to develop reagents specific for Tcell receptor (TcR) variable (V) regions. To date, however, TcR expressed on the surface of antigen-specific T lymphocytes have proven to be poorly immunogenic. As a result, few monoclonal antibodies (mAb) recognizing human variable regions are available. In this report, we have used the “phosphatidylinositol linkage” strategy to generate soluble forms of two human allogeneicTcR derived from human cytotoxicT lymphocytes (CTL) known to be specific for HLA-A2 and HLA-Aw68/HLA-Aw69, respectively. Monomeric TcRu and p chains from the HLA-A2-specific CTL were purified in large quantities from CHO cells and each was used to immunize mice to generate mAb. In particular, the anti$ chain mAb, denoted anti-Vgl3, stain a significant (- 5%) fraction of human peripheral blood u/p T lymphocytes, immunoprecipitate native anti-A2 TcR molecules, and activateT cells transfected with the relevant a and p chain cDNA. Anti-a chain mAb were also obtained against a constant region determinant which can immunoprecipitate detergent-solubilized polypeptides. In general, we find that immunizations with soluble protein are far superior to those with cells bearing TcR chimeras or in combination with the purified protein.
1 Introduction Antibodies to specific TcR V region families have had a number of important uses in mouse models, particularly in following specific receptor-bearing cells [l-51, monitoring responses to internal or external superantigens [6,7], and in ablating autoimmune Tcells [8,9]. Applications to human diseases have been hampered, however, by the paucity of Vg-specific mAb, which currently cover only 4 [ 10-151 of the 25 or more distinct Vg families (, J. Bell et al., manuscript submitted). TcR V region-specific mAb have been exceedingly difficult to generate and there are almost as many different protocols in the literature as there are antibodies. Even in the mouse, specific mAb have been developed which cover about half of the 21 Vg sequence groups. There are even fewer antibodies recognizing V,
[I 94981 ~~
sequences. A more complete set of monoclonal reagents would be extremely useful for identifying specific subsets of autoimmune T cells in tissue infiltrates, assessing the clonality of Tcell tumors, or characterizing the response to specific pathogens. Such antibodies are also an important starting point for the development of reagents that could remove or inactivate harmful populations of Tcells, confering a great deal of specificity with fewer of the side effects associated with nonspecific T cell depletion. In this report, we describe the isolation of a number of human TcRu and fl cDNA and their expression on the surface of fibroblast cells via a glycan-phosphatidylinositol (PI) anchor [17-201. Specific cleavage of this linkage with the enzyme PI-phospholipase C (PLC) , and capture of the TcR proteins on antibody affinity columns results in large quantities of pure protein (> 1mg). Immunizations with these soluble proteins proved very efficient for the generation of specific mAb. We present data concerning a novel anti-Vgl3 mAb which stains approximately 5% of human peripheral a@ T lymphocytes.
* This work was supported by HHMI and NIH. EMBO Scholar and an HHMI Research Associate. Established Investigator of the American Heart Association. Fellow of the American Cancer Society. 0 Present address: Brigitte Devaux, Immunologic Pharmaceutical Corporation, 855 California Avenue, Palo Alto, CA 94304, USA. Present address: Pamela J. Bjorkman, Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. Present address: John F. Elliott, Department of Medicai Microbiology and Infectious Diseases, University of Alberta, Edmonton, Canada T6G 2H7. A
Correspondence: Mark M. Davis, Howard Hughes Medical Institute, Beckman Center, Unit in Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford CA 94305-5425, USA
Abbreviations: PI: Phosphatidylinositol GPI: Glycosyl PI 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
2 Materials and methods 2.1 Construction of cDNA libraries cDNA libraries were constructed from two previously described human allogeneic Tcell clones, ALI1.1 (HLAA2 specific) and AL8.1 (HLA-Aw68/Aw69 specific) . Cytoplasmic RNA was isolated by the method of Favoloro et al.  from 5 x lo7cells. About 25 pg of total RNA was used to make double-stranded cDNA . Xho I adapters were ligated to the cDNA, which was then cloned into the XhoI site of AZAP (Stratagene, Inc., San Diego, CA).The ligations were packaged in vitro and the resultant phage screened with probes from murine a and p TcR C region genes. cDNA clones of length 1500 bp (a)and 1300 bp (p) were isolated, sequenced, and found to encode full-length OO14-2980/91/O909-2111$3.50 + .25/0
B. Devaux, I? J. Bjorkman, C. Stevenson et al.
Eur. J. Immunol. 1991. 21: 2111-2119
TcRa and p chains that were joined in frame to D andor J gene segments. A second full-length TcR a gene was also isolated from the ALI1.l library, but was joined outof-frame to the J gene segment.
MA) previously bound to Staphylococcus aureus or (b) with uncoupled fl monomers only. The TcR immune complex and the (3 protein were resuspended in PBS before immunization. Approximately 60 pg of purified proteins was injected into each mouse (6 injections in 3 weeks; 10 pghnjection). After the last immunization, LN were 2.2 Construction of expression vectors removed and the isolated cells fused with the myeloma cell pBJl-NeolAdalP-HPAP and pBJ2lA2alP line Sp2/0 using 1 ml of 60% PEG 4000 over 1 min. The fused cells were resuspended in medium supplemented The TcR-human placental alkaline phosphatase (€PAP) with 15% FCS, 2 mM L-glutamine, hypoxanthine azaserine chimeras were constructed as described in Lin et al. . and plated into six microtiter plates. The first screening of Briefly, the A2fiMPAP-S’ cDNA was cloned into pBJ1- the hybridomas was done by solid-phase ELISA. Briefly, Neo  between the unique Xho I and Not I sites of the CHO transfectants (anti-A2 TcR a , p and alp positive polylinker located 3’ of the SRa promoter. The clones) were dried onto Dynatech (Alexandria,VA) ImmuA2a/HPAP-S‘ was first introduced into pBJ1. A SalI Ion I1 plates. After blocking of nonspecific binding with fragment containing the A2 a-€PAP cDNA as well as SRa 0.5% powdered milk/PBS, SN from the hybridomas were and SV40 poly(A) was then introduced into the unique added and incubated for 1 h at room temperature. HorserSal I site of pBJl-Neo/A2 (3-HPAP, generating pBJ1- adish peroxidase (HRP) conjugated to goat anti-mouse Ig Neo/A2a/fi-€PAP The a and (3 anti-A2 TcR complete was added and incubated for 45 min. The peroxidase cDNA were introduced into pBJl (vector designated substrate was then added and positive SN were screened by pBJ2/A2 a(3) using the same strategy. The plasmid color development. These SN were then tested for binding pBJ2/A2a(3 does not contain a neomycin gene and was to CHO transfectants using FCM. FITC-conjugated goat therefore co-transfected with pRSV gpt for the establish- anti-mouse Ig was used as secondary antibody. Untransfected CHO cells were used as negative control. ment of stable lines. 2.3 Site-directed mutagenesis and transfection of eukaryotic cells Site-directed mutagenesis was performed as described from single-strand DNA templates prepared in CJ236, using the M13 helper phage R408 . All eukaryotic cells were grown in RPMI supplemented either with 5% FCS (Jurkat) or 10% calf serum (CHO, L, CV1, HeLa), 50 pM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin G., and 100 pg/ml streptomycin. Hypoxanthine (15 yg/ml), mycophenolic acid (1 pg/ml), and xanthine (200 pg/ml), were added to make HMX-selective media. The expression vectors were introduced into the cell lines by electroporation . Cells ( lo7) were washed once in ice-cold 1 x HBS , and 10 pg of plasmid DNA was added. The mixture (1 ml) was then placed in a sterile prechilled Bio-Rad (Richmond, CA) electroporation cuvette (0.4 cm) and pulsed at 230 V and 940 pF capacitance. After pulsing, the cells were immediately placed on ice, diluted with medium and plated into either 10cm round plates (for attached cells) or 24-well plates (for cells growing in suspension). Selective medium (G418 800 pg/ml for CHO cells or HMX 1 x for Jurkat cells) was added 36 h after transfection. Resistant transfectants, obtained after 15-20 days of culture were then stained with specific mAb ( 5 pg/ml) and analyzed by FCM using a FACStar Plus (Becton Dickinson, Mountain View, CA). High expressing cells were sorted twice, collected and analyzed again after 2 weeks of culture to verify the stability of surface expression. 2.4 Production of mAb Six BALB/c mice (6 weeks old) were immunized into the rear footpads with either (a) anti-A2a and fi PI-linked monomers purified from CHO transfectants coupled to aFl and (3F1 mAb (kindly provided by T Cell Sciences, Boston,
SN positive after both screenings were used to stain Jurkat anti-A2 a/(3(whole cDNA) positive transfectants, as well as T cell tumor lines and HLA-A2-specific T cell lines generated from PBL. Hybridomas were subcloned by limiting dilution to 0.3 celYwell and then injected into pristaneprimed BALBlc mice for production of immune ascites. The antibodies were purified from ascites using a protein A-Sepharose column, eluted at pH 6.0 and dialyzed against 1 x PBS.
2.5 Calcium flux measurements Calcium ion concentration [Ca2+]was measured using the calcium-sensitive fluorescent dye indo-1 . Transfected Jurkat cells (Jurkat 2.10, 2.15, 34 and W T 2 x 10’) were washed in MEM supplemented with 10% FCS (MEM-10) and loaded with indo-1 AM (acetoxy-methyl ester of indo-1, Molecular Probes, Eugene, OR). The cells were incubated at 37°C (S% CO2) in 2 ml of MEM-10 with 3 p~ indo-1 AM for 30 min, washed extensively and resuspended in MEM-10 at 2 x lo6 cells/ml. The hydrophobic acetoxymethyl ester groups on indo-1 enable it to pass readily into the cell. Once in the cell, these residues are cleaved and the dye remains trapped inside. For analysis of [Ca2+], 2 x 10’ indo-1-loaded cells were pelleted at 2000 rpm for 45 s, and incubated at 37°C for 3 min. [Ca”] responses to single antibodies were determined by incubating 2 x 10’ indo-1 AM-loaded cells with 30 pl of mAb (250 pg/ml; OKT3, BAM13 or KJ25) for 3 min at room temperature. [Ca2+] responses to double antibody treatment were determined by first incubating 2 x lo5 indo-1 AM-loaded cells with 30 pl of mAb (BAM13 or KJ25) for 6 min on ice, then pelleting them at 2000 rpm for 45 s and incubating them for 3 min at room temperature with 30 p1 of goat anti-mouse Ig (250 kg/ml, Cappel, Downington, PA).The cells were resuspended in 300 pl of a Ca2+ buffer (140 mM NaCl, 4 mM KCI, 1 mM Na2HP04, 0.8 mM MgS03, 1.8 mM CaC12, 25 mM Hepes buffer, pH
Generation of mAb against soluble human TcR polypeptides
Eur. J. Immunol. 1991.21: 2111-2119
adjusted to 7.4). Fluorescence intensity was analyzed on an SLM 8000 Spectrofluorometer in which the dye was activated by an excitation wavelength of 350 nm and emissions at 405 nm (violet) and 485 nm (blue) were measured. Calibration of indo-1 was performed as previously described .When Ca2+complexes with the dye, the color shifts from blue (Ca2+free) to violet (Ca2+bound). A rise in [Ca2+]corresponds to a relative shift from blue to violet fluorescence and thus precise concentration of dye is not a critical factor in the measurement of [Ca2+]levels.
3.1 Sequences of anti-HLA-A2 and anti-AwW69a and p cDNA clones The TcR cDNA were cloned from the allogeneic cytotoxic ALI1.l and AL8.1 T lymphocyte clones . These TcR, specific for HLA-A2 and HLA-Aw6WAw69, were sequenced using primers from murine a and 0 C regions. The sequence of both ci and 0 chain cDNA is shown in Fig. 1. The V, from the AL1.l clone was identical to a V gene segment from V,8.1 family (the HAP41 sequence reported by Yoshikai et al. ).TheVp gene segment from ALI1.l was 94.4% homologous with the Vp13.2 sequence described by Kimura et al.  and 100% identical to a partical Vp13 related sequence (aa68-113) described by Bregado et al. . Accordingly, we propose designating this gene segment Vp13.3.
2.6 T cell stimulation assay Protein A-purified OKT3 and BAM13 or goat anti-mouse (GaM) IgG were used at a concentration of 200 pglml to coat 96-well microtiter plates overnight at 4°C. Excess antibody was removed by extensive washing with PBS. BAM13 and KJ25 mAb were then added to the wells coated with GaM IgG and left an additional 2 h at 37°C. After washing, 2 x lo5 cells from Jurkat 2.10 and 2.15 as well as 1 ng of PMA (5 ng/ml) were incubated with the bound antibodies in a final volume of 200 pl for 20-24 h at 37 "C.
The V, sequence from the HLA-Aw68/Aw69-specificTcell clone AL8.1 represents aV, gene partially characterized by Mengle-Gaw et al. , while its Vp gene segment is a member of the Vp15 family and is identical to the ATL21 (Vp15.1) sequence reported by Kimura et al. . The J region of the anti-A2cr cDNA has not been previously described, while the anti-A2 0 gene uses Jp2.3 . The anti-Aw68/69 J, and Jp are homologous to J,T described by Yoshikai et al.  and Jp2.7 , respectively. The 3' cDNA sequences of bothTcR studied correspond to the constant region Cp2. A summary of theTcR anti-A2 and anti-Aw68/69 V, J and C region usage is given in Table 1.
2.7 IL2 assays and data analysis
HT2 cells  were washed three times in RPMI supplemented with 10 mM Hepes, pH 7.4, and 4 X lo3 cells were added to duplicate, twofold dilutions of SN in a final volume of 100 p1. After 17-20 h at 37 "C,wells were pulsed with 1 pCi = 37 kBq of [3H]dThd for 4 h, harvested and counted. One unit/ml of IL2 was defined as the concentration at which half-maximal [3H]dThd incorporation occurred. A four-parameter logistic curve using the ARCUS RADIMM program (adapted from Dr. D. J. Finney by Tony Kyne) was used to fit the resulting data and standards.
A soluble form of human TcR would be extremely useful: (a) to study the interaction between theTcR and the MHC
ICL G GT +ICY GACCTCGAGCTACGTCAGGGCCTGGAGCACCTGCCATGAGCAT~~GCCTCCTGTGCTGTGCAGCCTTTCCTCTCCTGTGGGCAGGTCCAGT~IU\~G~~=~~~T~ACTCAGACCCCM S I S L L C C A A F P L L W A G P V N A G V T Q T P K Id1 ILI I S 1
Figure 1. Nucleotide and protein sequences of the anti-A2 and antiAw68169 TcR c( and p chain gene. The anti-A2 and anti-Aw68169 TcR were obtained from the HLA-A2 (ALI1.1)-
and HLA-Aw68IAw69 (AL8.1)-specific T cell clones, respectively. The figure shows only the sequences of the +It D -It J +I+ c TTCTGTGCCAGCAGTTACGCGGGGGCCTTACTTAACACAGATACGCAGTATTTTGGCCCAGGCACCCGG~T=ACA~T~=~C=~~G~~~~~RAR V regions which have not been previously described or which have only partial homology with other TcR V I t L v A G ~ C A R T G R R G A C A T T T G C T G G A T T T ~ C G T T C C T G T T T T T G T G G C ~ G C A G C T G G A C T G T ~ ~ G A G T ~ G A G G f f i ~ G G A ~ G ~ G G A G C ~ G A G T C T ~ T T C C T G A G T G T C C G A G A G Gregions. GAGACA~ The nucleotide and amino M K T F A G F S F L F L W L Q L D C H S R G E D V E P S L F L S V R E G D S acid residues from the Vp13.2 sequence described by Kimura et al.  different from those of the anti-A2 R R Vp are shown above and below (in parenthesis), respectively the anti-A2 TcR Vp sequence. The dashed line above the anti-A2 TcR Vp and the anti-Aw68169TcR V, genes shows the portion of the genes previously described ( and , respectively). F
3.2 Expression of the human anti-A2 TcR in a lipid-linked form
4 t J
Eur. J. Immunol. 1991. 21: 2111-2119
B. Devaux, F! J. Bjorkman, C. Stevenson et al.
Table 1. Summary of the anti-A2 and anti-Aw68169 TcR V and C region usage
TcR anti-A2 u TcR a n t i 4 2 f3 TcR anti-AwW69a TcR a n t i - A M 6 9 f3
V"8.1 vp13.3 V, homologous to HUMCRATI vg15.1
class I molecule (also available in a soluble form), and/or (b) to generate mAb against specific regions of the TcR (particularly V regions). The strategy used to generate a lipid-linked form of a murine TcR has been described by Lin et al. . Employing this same approach for the anti-A2 TcR sequences, the last 35 amino acids of human placental alkaline phosphatase [33, 341 (HPAP-S') were used to replace the transmembrane and cytoplasmic portions of the anti-A2a and p chains. In the chimeric molecule, the HPM-S'signal was fused to the fifth amino acid residue of theTcR a or p chain located 3' of the last cysteine before the transmembrane domain. After in v i m mutagenesis, both A2 a/HPAP-S' and A2 b/HPAP-S' cDNA were inserted into an expression vector, pBJ1-Neo , generating the plasmid pBJl-Neo/A2 dp-HPAF? To verify that the cDNA were properly expressed after transfection of pBJ1Neo/A2a@-HPAP in eukaryotic cells, and the chimeric CI and p chains transported to the cell surface, the expression vector was introduced into CHO cells by electroporation. Stable transformants expressing the neomycin gene were selected in presence of the antibiotic G418 (800 pg/ml). 100
Jp2.3 JrsT J9.7
Cp2 CU Cp2
Resistant clones were then pooled and sorted twice for the 5% brightest double-positive cells after staining with a F l and pF1 mAb, specific for all human TcR a and p chains, respectively. Although the a F l and pF1 mAb detected both PI-linked anti-A2a and p chains on the surface of the live CHO transformants (Fig. 2C and D), they do not stain IiveTcells (,T Cell Sciences, Inc., unpublished results), suggesting that these epitopes are obscured by CD3 molecules on Tcells. Untransfected CHO cells were also stained with the two mAb (Fig. 2A and B) and used as a negative control to confirm the specificityof the staining.While the integration of the p chain cDNA was very stable and the level of expression did not change after several weeks of culture, the amount of a chain progressively decreased on the cell surface even when the antibiotic was maintained in the culture medium. This phenomenon can be observed in Fig. 2C where the population of brightly stained cells obtained after sorting of the cells (data not shown) is slowly replaced by a population of dull cells. were transfected with pBJl-Neo/A:! a@-HPAP to determine if the a chain would be expressed on the surface in these cell lines and if the expression would remain high after several passages of the cells.The result of the staining of the various transfectants with a F l mAb indicated that the a chain of the anti-A2 TcR was absent from the surface of all the human, monkey and mouse cell lines tested. In
I I < / l / " ' 2.
Jurkat clone 34
Jurkat clone 2.1 0
was detected in Jurkat 2.10,2.15 and Jurkat WT but not in Jurkat 34, proving the presence of TcR molecules on the surface of Jurkat 2.10,2.15 and Jurkat,WT.When BAM13 was tested on the same four Jurkat lines, only Jurkat 2.10 and 2.15 showed a raise in [Ca2+], indicating that BAM13 was able to specifically activate T cells bearing the anti-A2 TcR. None of the clones are sensitive to KJ25, an antibody specific for the mouse Vp8.1 family.
3.6.2 IL 2 production 43
Figure 7. Immunoprecipitation of the anti-A2 TcR from Jurkat transfectants with BAM13 mAb. Cells (lo7)from Jurkat 2.10 and 2.15 (A2 a@+)as well as Jurkat 34 (CD8+) were surface iodinated using lactoperoxidase and glucose oxidase. Lysates from these cells were incubated with 5pg/ml of BAM13 mAb (lanes 2-4 and 6-8) for 1 h at 4°C. Lysate from Jurkat 2.10 was also immunoprecipitated with aF1 mAb (1 pg/ml) in the same conditions. Protein A beads, first incubated with goat anti-mouse IgG (5 pglimmunoprecipitation) for 1 h at 4°C were then added to each lysate and the samples were rotated another hour at 4°C. After boiling, protein samples, either nonreduced (lanes 1-4) or reduced (lanes 5-S), were loaded on a 10% polyacrylamide gel.The gel was then stained with Coomassie blue, dried and exposed for autoradiography. The number on the left side indicate the position of the molecular mass markers.
To determine whether the increase in [Ca2+]would result in an induction of IL2 production by the Jurkat transfectants, we coated plates with OKT3, BAM13 and KJ25 either directly (OKT3, BAM13) or/and indirectly using goat anti-mouse IgG (BAM13 and KJ25). The amount of IL2 produced by Jurkat 2.10 and 2.15 was increased approximately 100-fold in response to BAM13 stimulation (Fig. 8B).The background level of IL2, determined using
BAM13 (lanes 3 and 4). As expected, this band was absent in Jurkat 34 (lane 2). Reduction of the samples (lanes 5-8) resulted in the appearance of two bands corresponding to anti-A2a and chain monomers (50 kDa and 39 kDa molecular mass, respectively) only in samples where heterodimers had been detected. Thus, BAM13 was able to immunoprecipitate anti-A2 TcR heterodimers.
GaM + BAMl3
Two of the mAb raised against the anti-A2 u chain were also able to immunoprecipitate the anti-A2 TcR present in the Jurkat transfectants as well as in Jurkat WTand HPB-ALL cells (data not shown) This result indicates that these antibodies recognize an epitope located on the C region of the u chain (as the cell lines tested have only the C regionin common), and that this epitope is shared between the native and PI-linked forms of TcR a. 3.6 Activation of Jurkat transfectants expressing the anti-A2 alp TcR with BAM13
Figure 8. (A) Calcium flux induction in Jurkat transfectants in presence of BAM13 mAb. The level of intracellular calcium in Jurkat transfectants (Jurkat 2.10,2.15 and 34) as well as in Jurkat Jurkat transfectants (Jurkat 34, 2.10 and 2.15) as well as WTwas measured after incubation of the cells with OKT3. BAM13 Jurkat WT cells were loaded with the fluorescent dye and KJ25 mAb (see Sect. 2.5). (B) Induction of Jurkat 2.10 and 2.15 proliferation by BAM13 mAb.The lymphokine assay was set indo-1, incubated for 3 min with either BAM13, OKT3 or up as described in Sect. 2.6. The graph represents the amount of KJ25 (hamster anti-mouse 2B4 TcR p chain) antibodies, IL2 produced by each Jurkat clone after stimulation with the and the concentration of intracellular calcium bound to the different mAb. The black and hatched bars reflect the results dye was measured as described in Sect. 2.5 (Fig. 8A). After obtained in two separate experiments. The background in each incubation with OKT3 mAb, a significant increase in [Ca2+] experiment was less than 0.01 U/ml of IL2.
3.6.1 Intracellular calcium flux
Eur. J. Immunol. 1991. 21: 2111-2119
B. Devaux, F! J. Bjorkman, C. Stevenson et al.
either no antibody or KJ25 to coat the plate, or Jurkat 34 stimulated with BAM13,was < 0.01 U/ml.This experiment shows that the BAM13 mAb is efficient inducing activation of Tcells bearing the anti-A2 TcR (though not quite as efficient as OKT3). Moreover, the IL 2 production consequential to the activation can be directly correlated to an increase in [Ca2+]i.
4 Discussion We describe here a useful new approach to generating mAb against specific TcR V regions by immunizing with soluble TcRa and p chains derived from GPI-linked chimeras. A large number of hybridomas producing anti-TcR a antibodies (43/80) were obtained with the solubleTcR protein while none were produced after immunizations with CHO or M12 (B cells) transfectants. Similarly, we obtained 28 hybrids specific for the TcR p-PI transfectants, of which four were able to stain native Vp13.3 transfected Jurkat cells. These results demonstrate that the efficiency of raising TcRspecific antibodies was much better with soluble proteins than with the same immunogens expressed on cells. One of the antibodies, BAM13 (anti$), stains a significant fraction of peripheral a@T cells ( 5 % ) and the anti-a reagent is able to immunoprecipitate native TcR a polypeptides. Jameson et al.  have also been sucessful in making an mAb to soluble TcR protein that reacts with normal V,11-expressing murine T cells, but the Ig fusion vector that they used only works with V, gene segments . Surprisingly, the GPI-linked anti-A2 TcR molecules found on the cell surface were exclusively monomeric with no heterodimers being formed. In contrast, a murine PI-linked TcR (2B4) was expressed as a heterodimer on the surface of CHO transfectants . This difference may reflect a decreased efficiency of transport of the human PI-linked a chain (but not p chain) in most of the cell types transfected. Although we were able to detect a chain on the surface of CHO cell transfectants, expression was not stable and disappeared slowly after several weeks of culture. Recently, Slanetz and Bothwell  reported that CHO cells expressed a PI-linked TcR p but not an d B determinant, whereas a mouse Tcell lymphoma (BW5147) did. In our case, however, we obtained the same results whether the TcR chimeras were transfected into a human T cell line (Jurkat) or into a variety of other mouse and human cell lines, indicating that is not a CHO-specific problem. Comparison of the two human a chains to the mouse 2B4 a chain reveals only one obvious difference, which is an additional glycosylation site in the human C, region at amino acid 165. It is possible that this site is exposed to glycosylation during processing of the PI-linked chimera and not in the native form (possibly due to CD3 polypeptide association). This might then prevent a@heterodimer assembly. It is also apparent from this study that many epitopes on TcR C, and Cg domains are inaccessible when TcR are expressed on Tcells but are exposed when GPI linkage is employed or when Tcells are detergent solubilized [the T Cell Sciences anti-cx antibody, aF1, the new anti-a antibody we describe here (AC1) as well as pF1 do not react with TcR expressed by unfixed Tcells]. One likely explanation is that.the epitopes recognized by the different
antibodies are areas of close contact with the CD3 polypeptides or are obscured by the presence of CD3 in their vicinity. The fact that so many of the antigenic portions of C, and Cg are masked helps to explain why attempts to make pan-TcR specific antibodies often failed in earlier investigations. We show that BAM13, an anti-Vp13.3 mAb can immunoprecipitate the anti-A2 TcR from Jurkat cells expressing the anti-A2 TcR and can stimulate the same Jurkat cells to produce IL2. This latter result demonstrates that the anti-A2 TcR present on the surface of Jurkat cells is functional. We also tested the activation of the Jurkat TcR transfectants using EBV transformed HLA-A2 expressing B lymphoblasts as the alloantigen (data not shown). Neither transfectant (Jurkat 2.10 and 2.15) produced significant amounts of IL2 when co-cultured with the B lymphoblasts. Since Gabert et al. have shown that MHC class I specificity could be reconstituted in a mouse MHC class 11-restrictedT cell hybridoma only after transfer of both TcR and Ly-2 genes into the recipient cell line, the lack of I L 2 production in our study could be due to the absence of CD8 molecules on the surface of the cells.To test this hypothesis, we transfected the CD8 a chain cDNA in Jurkat 2.15 and selected cells expressing both anti-A2 TcR and CD8 a chain. Although CD8 was then present on the surface, Jurkat 2.15 still did not produce detectable quantities of I L 2 when incubated with the HLA-A2-expressing B cells. Since the Jurkat cells used in these experiments do not produce large amounts of IL2 even after stimulation with OKT3 it is possible that the level of IL2 produced by the cells after allostimulation is too low to be detected in our assay or that the affinity between anti-A2 TcR and A2 molecules is weak and thus not sufficient to induce activation of the Jurkat cells.
In summary, we have produced and characterized two new
anti-TcR antibodies using PI-linked TcRa and p chains as immunogens. This strategy should be useful to generate other mAb specific for different TcR a and chains variable regions and could be generalized to the production of mAb against any protein not available in sufficiently large quantities or poorly immunogenic when on the surface of the cell. We wish to thank A . Lin for useful discussions, G . Albrecht for critical reading of the manuscript and B. Robertson for secretarial assistance.
Received April 29, 1991.
5 References 1 Roehm, N., Herron, L., Cambier, J., DiGuisto, D . , Haskins, K., Kappler, J. W. and Marrack, F!, Cell 1984. 38: 577. 2 Kappler, J. W., Roehm, N . and Marrack, F!, Cell 1987. 49: 273. 3 Sha, W. C., Nelson, C. A., Newberry, R. D., Kranz, D. M., Russell, J. H. and Loh, D.Y., Nature 1988. 336: 73. 4 Kisielow, F!, Bliithmann, H., Staerz, U. D., Steinmetz, M. and Von Boehmer, H., Nature 1988. 333: 742. 5 Berg, L. J., Pullen, A. M . , Fazekas de St. Groth, B., Mathis, D., Benoist, C. and Davis, M. M., Cell 1989. 58: 1035. 6 Pullen, A. M., Marrack,F! and Kappler, J.W.,Nature 1988.335: 796.
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Generation of mAb against soluble human TcR polypeptides
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