HYBRIDOMA Volume 11, Number 6, 1992 Mary Ann Liebert, Inc., Publishers

Generation of Monoclonal Antibodies Against Human T Cell Receptor ß Chain Expressed in Transgenic Mice



imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, U.K. 2Department of Immunology, St. Mary's Hospital Medical School, Imperial College of Science, Technology and Medicine, Norfolk Place, London


1PG, U.K.


The generation of a panel of monoclonal antibodies specific for different variable (V) regions of human T cell receptors will be of great importance in the study of T cell-mediated diseases. However, relatively few such reagents exist, due in part to the poor immunogenicity of TcRs on the surface of human T cells. We have employed a strategy in which T cells from a transgenic

line expressing a human Vß3Cßl TcR were used to immunise syngeneic conventional mice to generate two monoclonal antibodies specific for human T cell receptors. Binding of antibody JOVI-3, which stained approximately 5% of human peripheral blood CD3 positive T cells, correlated with the expression of the human TcR Vß3 gene segment. Antibody JOVI-1 recognised a determinant on the majority of TcRs, staining 50-75% of peripheral blood T cells and T cell lines expressing different Vß regions. Some TcRs, however, failed to react with this antibody. Both antibodies immunoprecipitated detergent-solubilised TcR molecules and were capable of inducing proliferation of peripheral blood T cells. mouse

INTRODUCTION The results of a number of independent studies have demonstrated a for role T cells in the pathogenesis of autoimmunity, allergy and cancer. Two approaches have in particular been used to examine TcR usage in T cells

found at disease sites (see, for example, refs 1-6). One approach involves the generation of T cell clones from these sites and the analysis of the sequences of TcR a and ß chains predicted from cloned cDNA sequences. Another approach has been to expand T cell populations with CD3-specific antibodies and to analyse TcRs using the Polymerase Chain Reaction. Although these 701

much useful information, they are indirect, and give no information on the in situ location and phenotype of the T cells. The optimal strategy would be to use a serological approach, but this has been hampered by the paucity of available antibodies specific for TcR V regions. The lack of human TcR V region-specific reagents is due in part to the poor immunogenicity of TcRs expressed on the surface of T cells compared with other surface molecules, such as CD3 and CD45. Various approaches have been used to overcome this problem. Purified soluble TcRs have been used to produce several new Va- and Vß-specific reagents (7, 8). Alternatively, murine T cell transfectants expressing chimaeric TcRs comprising a specific

approaches yield

human a or ß chain have been used as immunogens (9). In this report we describe the use of T cells from transgenic mice expressing a human TcRß chain to immunise syngeneic conventional mice. Two novel monoclonal antibodies (mAbs) resulting from this fusion were characterised in detail. One antibody recognises a determinant apparently specific for the Vß3 region. The other antibody recognises the majority of aß TcRs on peripheral blood T cells and may define a novel epitope on the Cß, or






Transgenic mice. The transgenic mouse line used in this study was generated as part of a project to study the immune response to a viral antigen and will be the subject of a future publication (manuscript in preparation). Briefly, a genomic clone containing a productively rearranged TcRß gene was obtained by screening a cosmid library prepared using DNA from the human T helper clone, HA 1.7, with a Cß probe (10). HA 1.7 recognises the C-terminal peptide from influenza haemagglutinin in the context of HLA-DR1 (11) and expresses the Vß3 Dßl Jßl.2 Cßl TcRß chain (12). A 9kb EcoRl fragment containing the rearranged HA1.7 TcRß gene from the cosmid clone was subcloned into a pBS vector and the CD2 enhancer, as a 3kb Hindlll fragment (13), was subcloned into the Hindlll site of the TcRß fragment. The resulting 12kb fragment was excised from the plasmid and used to establish a transgenic mouse line. Northern and serological analyses established that the human TcRß construct allelically excluded rearrangement of the murine TcRß locus and showed a T cell-specific pattern of expression at the cell surface (manuscript in


Production of mAb. Antibodies to human TcR were raised by immunisation of conventional syngeneic mice with cells prepared from thymus, spleen and mesenteric lymph nodes from the HA1.7 TcRß chain transgenic mouse line. 107 cells in 2 00 pi PBS were injected intraperitoneally on day 0, and booster immunisations were given intravenously after 14 and 28 days. Sera from test bleeds were analysed for reactivity with Vß3 gene products by cytofluorometry. After resting the mice for 200 days, an additional booster 702

to termination. The with the NS1 myeloma cell spleen was removed and the isolated cells fused line using 1ml of 50% PEG 4000 added over 30 seconds. The fused cells were subsequently resuspended in serum free medium before culturing in RPMI 1640 supplemented with 20% FCS, penicillin/streptomycin, L-glutamine, ßME(ß-mercaptoethanol), OPI (oxalacetic acid, pyruvic acid, bovine insulin) and HAT (hypoxanthine, aminopterin, thymidine) in 96 well U-bottomed tissue culture plates. Screening of the hybridomas was carried out by cytofluorometry using the Vß3Cßl TcR expressing CH7C17 cell line (a Jurkat mutant lacking endogenous TcRß chain and transfected with the ß chain of HA 1.7; ref 12), the Vß8Cßl TcR expressing Jurkat cell line and the TcR negative CEM cell line. Cells were stained for surface TcR expression by standard methods using a fluorescein conjugated goat-anti-mouse Ig second



given intraperitoneally


days prior

and analysed on a FACScan (Becton Dickinson). subcloned three times by limiting dilution to 0.5 cells/well Hybridomas into HAT free medium supplemented with 20% FCS, penicillin/streptomycin, L-glutamine and ßME. Stable hybridomas were subsequently routinely cultured in complete RPMI-1640 medium supplemented with 10% FCS, pencillin/streptomycin, L-glutamine and ßME and culture supernatants stored at 4°C or -20°C as a source of monoclonal antibodies.

layer antibody (Sigma) were

Cvtofluorometric analysis. T cell lines, T cell clones and PBL from healthy volunteers were incubated with hybridoma culture supernatants and reactivity was detected with fluorescein- or phycoerythrin conjugated goat anti-mouse Ig second layer antibodies (Sigma). Briefly, cells were incubated with culture supernatant for 20 min at 4°C, washed x3 in PBS containing 0.1% sodium azide and 1% BSA, incubated with second layer antibody for 20 min at 4°C and washed x3 before being analysed by flow cytometry. For 2 colour analysis PE-labelled PBL were further incubated with a range of fluorescein conjugated antibodies specific for T cell surface antigens (anti-CD3 FITC, antiCD4 FITC, anti-CD8 FITC, anti IL2R FITC and anti-HLA-DR FITC; all purchased from Becton Dickinson) for 20 min at 4°C in the presence of 10% normal mouse serum and washed x3 before analysis by flow cytometry. Immun ohistnch cm istrv.

Fresh human tonsil was snap frozen in liquid nitrogen and stored at Cryostat sections of frozen tissue were cut at 6pm, air dried, fixed in fresh acetone and stained using the indirect immunoperoxidase technique. Sections were labelled with primary antibody followed by peroxidase conjugated goat-anti-mouse Ig (Sigma), and incubated for 10 min with hydrogen peroxide and diaminobenzadine (DAB) before being counterstained with haematoxylin. Positive cells were identified by the presence of a brown reaction product.


Surface labelling and immunoprecipitation. Protein A-Sepharose CL4B beads (Pharmacia) were incubated for 1 hour at 4°C with normal mouse serum (nms), JOVI-1 hybridoma supernatant, JOVI-3 hybridoma supernatant or W6.32 (anti-MHC Class I) hybridoma 703

supernatant, then washed x3 in lysis buffer (0.75% Triton-X-100, lOmM iodoacetamide, 2mM phenylmethyl sulfonyl fluoride, ImM each of leupeptin, antipain, pepstatin and chymostatin) before use. 2 x 107 cells were washed twice in PBS and labelled with 0.5mCi 125I (IMS30, Amersham) using the

lactoperoxidase technique (14). Labelled cells were lysed, centrifuged for 30 min in a microfuge at 4°C and the resulting cell lysate precleared with Protein A-Sepharose beads followed by nms coated Sepharose beads. 125I labelled surface molecules were subsequently immunoprecipitated with JOVI-1-, JOVI-3- or W6.32-coated Sepharose beads and analysed by SDS-PAGE using a 10% polyacrylamide gel followed by autoradiography. activation. Freshly isolated PBL from healthy volunteers were cultured at a density of 105 cells per well in 96 well U-bottomed tissue culture plates in a total volume of 200pl in complete RPMI 1640 medium supplemented with 10% FCS at 37°C in a humidified 5% CO2 incubator. All cultures were performed in triplicate. Anti-CD3 mAb 50pl/ml (UCHT1, culture supernatant), Staphylococcus aureus enterotoxin B 10pg/ml (SEB; Sigma), JOVI-1 mAb 50pl/ml (culture supernatant), JOVI-3 mAb 50pl/ml (culture supernatant) and IL-2 100pl/ml (lymphocult, Biotest UK Ltd) were diluted in RPMI 1640 medium and used at the concentrations indicated. Cultures were incubated for 48 hours, lOOpl of supernatant was removed from each well and stored at -70°C until used for lymphokine assay. lpCi [3H] methyl-thymidine (3H-TdR; Amersham) was then added to each well, and cultures incubated for a further 6 hours before harvesting onto paper filters using a titertek automatic cell harvester (Flow, Herts, U.K.). Filters were dried at room temperature, transferred to plastic scintillation vials containing 3ml EcoSint (National Diagnostics) and the amount of radioactivity incorporated into DNA was determined using an automated liquid scintillation beta counter. Data are presented as mean counts per minute (cpm) of triplicate cultures ±- one standard deviation. T cell

RESULTS characterisation of .IOVI-3 mAb. The JOVI-3 hybridoma secreted IgG2a heavy chain. The JOVI-3 mAb was initially screened for binding to the HA1.7 TcR transfected CH7C17 cell line (12) and to a panel of T lineage tumour lines (not shown). JOVI-3 also stained the HA1.7 T helper clone and two allergen specific T helper clones (Fig 1) that were shown by anchor PCR analysis to express the Vß3 region (L. Wedderburn, unpublished observation). However, it failed to stain Jurkat (Fig 1) or any other T cell line or clone tested. The JOVI-3 mAb was used to stain PBLs from several individuals. As shown in Figure 2, about 5% of CD3-positive PBLs were recognised by JOVI-3, although the precise percentage varied in different individuals (range 0.8%7.2%, n=ll). Taken together with the pattern of staining of T cell lines and clones, these results suggest that JOVI-3 recognises an epitope on the Vß3 region of the TcRß chain. Serological


Jurkat 4> w


a> JÛ




3 c



Fluorescence FACS Analysis of JOVI-3 mAb Expression. Cells expressing either Vß3Vß8-containing TcRs were stained with JOVI-3 and analysed by flow cytometric analysis.




anti-CD3 FIGURE 2



m Abs.

Dual Parameter FACS Analysis of PBL Stained with Anti-CD3 and Cells, labelled with JOVI-3 followed by a PE-conjugated goat anti-mouse Ig second

layer, were further incubated with FITC-conjugated anti-CD3. Labelled cells were analysed by flow cytometric analysis. The chart shown is a representative experiment. Similar results were obtained in 10 other individuals.

Serological characterisation of JOVM mAh. The JOVI-1 hybridoma also secreted IgG2a heavy chain. JOVI-1 was similarly screened for binding to the panel of cells described above. As shown in Table 1, this mAb showed a much broader pattern of reactivity than JOVI3. Thus, JOVI-1 reacted with the Cßl TcR-expressing Jurkat, HA 1.7 and CH7C17 cell lines with high levels of staining. A panel of T cell clones expressing Cßl TcR also stained well (data not shown). In contrast, JOVI-1 failed to react with the Cß2 TcR-expressing HPB-ALL cell line, the yô TcRexpressing ICRFT1 cell line or with a Vß3Cß2 TcR expressing clone (L. Wedderburn, personal communication), which were all strongly CD3 positive. 705


low levels of JOVI-1 staining were observed on a Vß7 Cß2 TcR transfected cell line (Paul Bowness, personal communication) and on a Vß6 cell TcR T clone Cß2 (L. Wedderburn; personal communication), expressing although the intensity of staining was greatly reduced compared to the level of CD3 staining (data not shown). ,


Reactivity of JOVI.l mAb. T cell lines and clones were stained with JOVI.l mAb and analysed by flow cytometry.

T Cell Line



stainingb) I

T Cell Clone









+ +



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+ +




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+ -




+ -







JOVI-1 high staining JOVI-1 low staining No staining

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a) Chinese hamster overy cells expressing



high staining;

+-, low









The JOVI-1 antibody reacted with 50-75% of CD3-positive PBL from different individuals (range 51.3%-74.2%, n=12). When the percentage of JOVI-1-positive T cells in the CD8 PBL T cell subset was analysed, CD8positive T cells were subdivided by the JOVI-1 antibody into two populations, JOVI-1 negative and JOVI-1 high (Fig 3A). The JOVI-1 negative subset failed to stain with JOVI-1 but expressed high levels of CD3 antigen, whereas the JOVI-1 high subset stained strongly with both anti-CD3 and JOVI-1 antibodies (data not shown). In contrast, dual parameter FACS analysis with JOVI-1 and anti-CD4 mAbs revealed a spread of JOVI-1 staining on CD4 positive cells from JOVI-1 high to JOVI-1 low, in addition to the JOVI-1 negative population (Figure 3b). Notably, high levels of CD3 staining were evident on all of the CD4 positive cells (not shown), including the JOVI-1 low subset. Staining with anti-




s> 19-5


%# anti-CD8


FIGURE 3 Dual Parameter FACS Analysis of PBL Stained with JOVI-1 and AntiCD8 or Anti-CD4 mAbs. Cells were labelled and analysed as described in the legend to Figure 2. FITC-conjugated anti-CD8 was used in Figure 3a and FITC conjugated anti-CD4 in Figure 3b. -

IL-2R and anti-HLA-DR antibodies failed to show concordance with JOVI-1

reactivity (data



and JOVI-3. stained by the immunoperoxidase histochemistry technique with JOVI-1, JOVI-3 and MX9 (and Vß8 TcR; ref 15) mAbs. JOVI-1 mAb stained large numbers of cells in the T dependent areas of the tonsil (Fig 4a). In contrast JOVI-3 mAb (Fig 4b) and MX9 (anti-Vß8TcR) mAb (Fig 4c) stained scattered cells, consistent with the pattern of reactivity of antibodies specific for Vß subfamilies.

Immunohistochemical_analysis with JOVII Frozen sections of human tonsil


analysis with JOVM and JOVI-3 antibodies. The results of immunoprecipitation analysis carried out following surface iodination of T cell lines are shown in Figure 5. Two bands corresponding to proteins of about 40kDa and 45kDa were detected in immunoprecipitates from the Vß3 Cßl TcR transfectant CH7C17 using either JOVI-1 or JOVI-3. The JOVI-1 antibody also immunoprecipitated a heterodimer from Vß8 Cßl TcR-expressing Jurkat cells. However, no specific bands were immunoprecipitated from Jurkat cells using JOVI-3. No labelled bands were observed in autoradiographs of immunoprecipitates from CEM or HPB-ALL cell lines with either antibody. These data are consistent with the immunofluorescence results presented above and demonstrate that both antibodies recognise TcRs when expressed at the cell surface or in detergent solubilised form. Class I molecules were precipitated from all cell lines with antibody W6.32.



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FIGURE 4 Immunoperoxidase Histochemistry with JOVM and JOVI-3 mAb. Tonsils were snap frozen, sectioned and stained with JOVM (Figure 4a), JOVI-3 (Figure 4b) and MX9 (anti-Vß8 TcR) (Figure 4c) mAbs. JOVI-1 mAb stained large numbers of cells in T dependent areas of the tonsil, whereas JOVI-3 and MX9 mAb stained scattered cells, consistent with the pattern of reactivity of antibodies specific for Vß subfamilies. -








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O O -3 -3



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30 —

FIGURE 5 Immunoprecipitation Analysis with JOVM and JOVI-3 Antibodies. Cells surface labelled with 125I by the lactoperoxidase technique, lysed and immunoprecipitated with JOVI-1, JOVI-3, W6.32 (anti-HLA-A, -B, -C) or normal mouse serum (nms) coupled to Protein ASepharose beads. The bound antigens were analysed by SDS-PAGE under reducing conditions followed by autoradiography. -












IL-2 IL-2





FIGURE 6 Mitogenic Activity of JOVM and JOVI- 3. PBL were cultured at a density of 105 cells per well and stimulated with anti-CD3 mAb, SEB, JOVI-1 or JOVI-3 in the absence or presence of IL-2. Proliferation was determined by measuring [3H]-TdR incorporation. Results are expressed as mean c.p.m. ± SD from triplicate wells. -


Mitogenic action of JOVM and JOVI3. The ability of JOVI-1 and JOVI-3 to activate T cells was shown by culturing PBL with these antibodies. PBL were cultured at a density of 105 cells per well and stimulated with anti-CD3 mAb, SEB, JOVI-1 and JOVI-3 in the absence or presence of IL-2. As shown in Figure 6, both JOVI-1 and JOVI-3 were mitogenic, JOVI-1 eliciting about five-fold greater stimulation than JOVI-3. This result is consistent with the observation that JOVI-1 recognises a substantially greater proportion of T cells than JOVI-3. In each case proliferation was enhanced by inclusion of IL-2 in the culture medium. Consistent with the proliferative data, supernatants harvested from cultures at 48 hours contained lymphokines capable of supporting factor dependent cell lines (data not shown). Effect of SEB on JOVM and JOVI-3 reactivity with PBL. The Staphylococcus aureus enterotoxin SEB induces vigorous responses in human T cells expressing Vß3, 12, 14, 15, 17 and 20 regions (16). When PBL were cultured for nine days with SEB, the proportion of CD3 positive cells reacting with JOVI-3 increased by at least three-fold from 3% to 10% (Figure 7). In contrast, the proportion of JOVI-1 expressing CD3-positive cells failed to increase significantly after SEB stimulation (74% at the onset of culture compared to 78% at 9 days).

day 9

day O





3mA ...Í./M-1.-


FIGURE 7 JOVI-3 Reactivity on PBL Cultured with SEB. PBL were cultured for 9 days with SEB. JOVI-3 reactivity was assessed by FACS analysis before and after incubation with the -

superan tigen.

DISCUSSION We describe here the generation of human TcR-specific monoclonal antibodies using T cells from a transgenic mouse line expressing a hybrid receptor comprising a murine TcRa chain and human TcRß chain as immunogen. Two antibodies resulting from the fusion were extensively characterised. Both antibodies clearly recognised determinants on the TcR as evidenced by their ability to immunoprecipitate TcR a and ß chains from labelled T cells. 710

The antibody JOVI-3, was specific for the human Vß3 TcR gene product, evidenced by the following criteria. Firstly, it reacted with the T cell clone HA1.7 and with a Jurkat transfectant CH7C17, both of which express Vß3, but not with Vß5- or Vß8-expressing cell lines. This antibody also reacts with two additional Vß3-expressing T cell clones but not with Vß6 or Vß9 expressing T cell clones. Secondly, JOVI-3 reacts with a small percentage of PBL T cells, typically about 1-10% in different individuals. This level is in good agreement with Vß3 TcR usage determined using the anchor polymerase chain reaction technique (17). Thirdly, the percentage of PBLs reactive with JOVI-3 increases after incubation with the bacterial superantigen SEB that has been shown to stimulate T cells expressing Vß3 TcRs. In contrast, JOVI-1 reacts with T cells expressing several different TcRß V regions and with all T cell lines expressing Cßl TcR. Interestingly, it fails to react with the Vß5Cß2 expressing T cell line HPB-ALL. However, a transfected T cell line and a T cell clone expressing Cß2 TcR showed reactivity with JOVI-1, although the level of staining was greatly reduced. Furthermore, JOVI-1 reacts with 50-75% of CD3+ PBL, although the staining profile of this mAb is significantly different in the CD4 and CD8 subpopulations. Thus, the CD8-positive PBL population was divided into two groups by JOVI-1 mAb (JOVI-1-high and JOVM-negative), whereas the CD4-positive PBL population exhibited a spread of JOVM staining. The variation in the level of staining with JOVM is not a consequence of downregulation of TcR levels on a proportion of CD4+ cells because this population expresses uniformly high CD3 levels. It is, therefore, unclear what epitope on the TcR is recognised by JOVI-1. It is possible that it recognises a supertypic determinant on Vß domains or a Vß epitope that is influenced by the particular Va that is associated with the Vß domain. Alternatively, JOVM may recognise a Cß determinant that is masked on some T cells by interactions of the TcR with other proteins. In this context, it is clear that the TcR forms dynamic associations at the cell surface with a number of other surface structures (18-22). This explanation is, however, not consistent with the observation that JOVM failed to immunoprecipitate the TcR from detergent extracts of HPB-ALL cells when weak protein-protein associations would be expected to be disrupted. Further work, particularly with JOVM-positive and -negative PBL will be required to establish the molecular basis for JOVM recognition of the TcR and whether the antibody defines a distinct functional subset of T cells. In summary, we have generated two antibodies that react with native cell This report (i.e. surface) and detergent solubilised human TcRs. constitutes the first description of a Vß3-specific mAb which will be of use in studies of T cell mediated diseases such as autoimmunity and allergy. The JOVI-1 mAb, which defines a novel epitope on a large number of TcRs, will be especially useful in purification of human TcRs for biochemical and structural studies and in the determination of the tissue architecture of T cells by immunohistology. JOVM may also be of use clinically in purging a large component of the T cell repertoire. as


ACKNOWLEDGEMENTS This work was funded by the Imperial Cancer Research Fund, the Medical Research Council and the Wellcome Trust. We thank Drs L. Wedderburn, R. O'Hehir and P. Bowness for communicating unpublished data, and Drs P. Lionetti and T. MacDonald for performing the immunohistology

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correspondence and reprint requests Owen,

Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, U.K.


Received for publication: 8/4/92 Accepted: 9/3/92



Generation of monoclonal antibodies against a human T cell receptor beta chain expressed in transgenic mice.

The generation of a panel of monoclonal antibodies specific for different variable (V) regions of human T cell receptors will be of great importance i...
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