The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Annenkov, A. E.
Right arrow Articles by Chernajovsky, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Annenkov, A. E.
Right arrow Articles by Chernajovsky, Y.
Right arrowPubmed/NCBI databases
*Substance via MeSH
The Journal of Immunology, 1998, 161: 6604-6613.
Copyright © 1998 by The American Association of Immunologists

Loss of Original Antigenic Specificity in T Cell Hybridomas Transduced with a Chimeric Receptor Containing Single-Chain Fv of an Anti-Collagen Antibody and Fc{epsilon}RI-Signaling {gamma} Subunit1

Alexander E. Annenkov*, Sasha P. Moyes*, Zelig Eshhar{dagger}, Rizgar A. Mageed* and Yuti Chernajovsky2,*

* Kennedy Institute of Rheumatology, London, United Kingdom; and {dagger} Department of Immunology, Weizmann Institute of Science, Rehovot, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell hybridomas HCQ6 and MD.45 acquired Ab-type specificity to collagen type II, when engrafted with a chimeric cell surface receptor, scC2Fv/{gamma}, which includes the single-chain Fv domain (scFv) of the anti-collagen type II mAb C2 and the signaling {gamma} subunit of the Fc{epsilon}RI. When transduced into MD.45 cells, scC2Fv/{gamma} or its mutated form lacking immunoreceptor tyrosine-based activation motif (ITAM), scC2Fv/{gamma}IC-, formed mainly homodimers. A small proportion of these molecules formed heterodimers with endogenous CD3{zeta} in these hybridoma cells. By contrast, in HCQ6 cells, the majority of scC2Fv/{gamma} and scC2Fv/{gamma}IC- molecules formed heterodimers with CD3{zeta}, and only a small proportion of them was expressed as homodimers. Stimulation with plastic-immobilized collagen induced IL-2 production in scC2Fv/{gamma}-transduced MD.45 cells, but not in MD.45 cells transduced with the ITAM-less chimera scC2Fv/{gamma}IC-. HCQ6 cells transduced with scC2Fv/{gamma} responded to plastic-bound collagen. Due to the high content of CD3{zeta}-associated chimeras, HCQ6 cells transduced with the ITAM-less scC2Fv/{gamma}IC- chimera were also responsive to plastic-bound collagen. When cells were stimulated with collagen in solution, MD.45 cells transduced with scC2Fv/{gamma} produced IL-2, whereas transduced HCQ6 cells were unresponsive, hence suggesting that the ability of cells transduced with scC2Fv chimeras to respond to soluble collagen correlated with predominant expression of divalent scC2Fv/{gamma} homodimers, but not monovalent scC2Fv/{gamma}-CD3{zeta} or scC2Fv/{gamma}IC--CD3{zeta} heterodimers. Of interest, expression of CD3 subunits in hybridomas transduced with scC2Fv chimeras was reduced, resulting in decreased response to cognate Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Type II collagen is expressed predominantly in articular cartilage and highly conserved across species. Mice immunized with bovine type II collagen develop autoimmune arthritis (1), which bears similarity with rheumatoid arthritis. Collagen-induced arthritis in mice is partially mediated by type II collagen-reactive CD4+ lymphocytes producing Th1 cytokines (2). These cells can transfer arthritis to normal animals. If these collagen-specific T lymphocytes are genetically modified ex vivo to produce constitutively an antiinflammatory protein, such as soluble TNF receptor (3), or TGF-ß (4), their pathogenic potential can be outbalanced by this antiinflammatory protein, because these cells reduce inflammatory reaction in the joints when injected into arthritic animals. Adoptive transfer of pathogenic self-reactive Th1 lymphocytes genetically modified to produce IL-10 (5), or transfer of self Ag-specific Th2 lymphocytes (6), has been shown to exert a therapeutic effect in other models of autoimmune diseases dominated by Th1 response.

Significant amounts of autologous self-reactive lymphocytes to use as delivery vehicles for therapeutic antiinflammatory genes are unlikely to be available in patients. However, it is perhaps possible to achieve tissue-specific homing of autologous lymphocytes by transducing them with a fusion receptor capable of coupling autoantigen recognition with TCR-mediated intracellular signaling. Such fusion receptors representing scFv of a tumor Ag-specific Ab fused with a signaling subunit CD3{zeta} or Fc{epsilon}RI{gamma} have been used for targeting CTL to tumor cells (7, 8). These signaling subunits contain ITAM,3 the consensus motif whose tyrosine phosphorylation is one of the major membrane-proximal events in the activation of TCR-signaling pathway (9, 10).

To engraft T cells with MHC-nonrestricted specificity to collagen type II, which might be sufficient to make them home to the joints, we cloned a chimeric receptor representing collagen type II-binding ectodomain in the form of scFv of the anti-collagen mAb C2 (11) fused with the signaling {gamma} subunit of Fc{epsilon}RI. To characterize this chimeric receptor, scC2Fv/{gamma}, biochemically and functionally, it was transduced into T cell hybridomas, where it is expressed as a homodimer as well as a heterodimer with endogenous CD3{zeta} subunit. The transduced hybridomas, MD.45 (12) and HCQ6 (13), responded to collagen type II by production of IL-2. Dependent on predominant expression of scC2Fv/{gamma} homodimers or scC2Fv/{gamma}-CD3{zeta} heterodimers, transduced hybridoma cells demonstrated different cross-linking requirements for stimulation with collagen type II. Of interest, the chimeric receptor interferes with expression of endogenous CD3 subunits in transduced T cell hybridomas. As a result of this interference, their response to cognate Ags was considerably lower than in wild-type cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue culture and Ab production

MD.45 is a H-2d-restricted CTL hybridoma generated from a BALB/c mouse (12). HCQ6 is a type II collagen-specific H-2q-restricted CD4+ hybridoma generated from a DBA/1 mouse (13), generously donated by R. Holmdahl (Lund, Sweden). Hybridoma-producing type II collagen-specific mAb C2 was a gift from L. Klareskog (Stockholm, Sweden). All hybridoma cells were cultured in DMEM supplemented with 10% FCS (Life Technologies, Renfrewshire, U.K.). The mAb C2 was purified from ascitic fluid of mice injected with the C2 hybridoma cells. Polyclonal anti-mAb C2 antiserum was raised in a rabbit by immunization with purified mAb C2. GP+E86 cells, a National Institutes of Health 3T3-based ecotropic murine-packaging cell line (14), were cultured in DMEM supplemented with 10% newborn calf serum (Life Technologies).

Cloning of the rearranged H and L chain V region genes of mAb C2

Total RNA was extracted from C2 hybridoma cells using guanidinium isothiocyanate (15). cDNA for the H and L chains were synthesized from this RNA preparation using SuperScript II RNase H-Reverse Transcriptase (Life Technologies), and primers complementary to mouse IgG C region (mC{gamma}1, 5'-AGAGTTCCAKGTCAAGGTCACT; K = G or T), or {kappa}-chain C region (mC{kappa}1, 5'-GTAGAAGTTGTTCAAGAAGCACAC-3'). The cDNA was poly(G) tailed at the 3' end using the enzyme terminal deoxynucleotidyl transferase (Pharmacia, Hertfordshire, U.K.), and amplified using anchored PCR. A primer complementary to the poly(G) tail (anch2pc, 5'-ACGAATTCTAGAGTCGACCCCCCCCCCCCCC) was used together with a primer complementary to mouse IgG C region upstream of the mC{gamma}1 site (nested primer mC{gamma}2; 5'-GAAATARCCCTTGACCAGGC; R = G or A), or a {kappa} L chain region upstream of the constant mC{kappa}1 site (mC{kappa}2, 5'-AGATGTTAACTGCTCACTGGA). All primers were from Oswell DNA Service Laboratory (Southampton, U.K.). The anchor primer was allowed to extend for 15 min at 70°C, before the addition of the second primers mC{gamma}2 or mC{kappa}2. The PCR was conducted as follows: 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min in a Hybaid Omnigene programmable thermocycler (Hybaid, Middlesex, U.K.). The PCR products were cloned using the Invitrogen TA cloning vector (pCRTMII; Invitrogen BV, Leek, The Netherlands). Plasmid DNA was sequenced using Sequenase T7 DNA polymerase (United States Biochemical, Amersham International, Buckinghamshire, U.K.) with mC{gamma}2 or mC{kappa}2.

Construction of chimeric genes in retroviral vector

The L chain V region of the collagen type II-specific mAb C2 (C2VL) was amplified by PCR from the plasmid pCRII using oligonucleotide primers 5'-AAGATCTGGACATTGTGCTGACACAG and 5'-CCTGTTTCAACCTTTATTTTGGCCAGTGGA, which contain BglII and BstEII sites, respectively. The PCR product was cut with the same enzymes.

A DNA sequence encoding the transmembrane and cytoplasmic domain of the human Fc{epsilon}RI{gamma} subunit with BstEII (upstream) and XhoI (downstream) sites was cut out of pRSVscFvR{gamma} (7) by digestion with XhoI, blunting with Klenow fragment of DNA polymerase (Boehringer Mannheim, East Sussex, U.K.), and digestion with BstEII. The plasmid pIg16 (16) contains a DNA sequence encoding a polyglycine-serine linker (GGGGS)3 bounded with XbaI site at the 5' end and BglII site at the 3' end. In this plasmid, (GGGGS)3 is followed by a DNA sequence encoding the 3' portion of the insert with Asp718 restriction site at its 3' end. To prepare pIg16 for subcloning of C2VL and {gamma} subunit into it, its DNA was cut with Asp718, filled in with Klenow fragment to form blunt ends, and digested with BglII. The C2VL and {gamma} subunit were subcloned at one time into the Asp718-blunted/BglII-cleaved pIg16 immediately downstream of (GGGGS)3, C2VL in front of the {gamma} subunit. The product contained a DNA fragment coding for (GGGGS)3-C2VL-{gamma} subunit. This fragment containing XbaI (upstream) and restored XhoI (downstream) sites was excised with the same enzymes.

A DNA fragment encoding the H chain V region of the mAb C2 (C2VH) was amplified by PCR from the plasmid pCRII using oligonucleotide primers 5'-TGGATCCTGAACACACATCCCTTACCATGG and 5'-GTCTAGATGAGGAGACTGTGAGAGT, the latter containing XbaI site. The PCR product was filled in with Klenow fragment to form blunt ends, and digested with XbaI. The C2VH was subcloned at one time with the (GGGGS)3-C2VL-{gamma} subunit into SnaB1/SalI-cleaved retroviral vector pBabeNeo (17), C2VH in front of the (GGGGS)3-C2VL-{gamma} subunit, to yield pBabeNeo+scC2Fv/{gamma} containing the final insert C2VH-(GGGGS)3-C2VL-{gamma} subunit (EMBL database accession number X9676).

A nonsignaling version of scC2Fv/{gamma} was produced by truncation of its intracellular domain. DNA from pBabeNeo+scC2Fv/{gamma} was cut with SalI, filled in with Klenow fragment, and recircularized with T4 DNA-ligase to introduce a frame shift and stop codon. As a result of this modification, the entire intracellular domain apart from its first arginine was replaced with an irrelevant sequence of 14 amino acids yielding scC2Fv/{gamma}IC-.

Vector production and transduction of lymphocytes

The GP+E86 cells were transfected using calcium phosphate precipitation and glycerol shock, as described previously (4). The cells were transfected with 20 µg of plasmid DNA from pBabeNeo+scC2Fv/{gamma} or pBabeNeo+scC2Fv/{gamma}IC-. Transfected cells were selected in 1 mg/ml G418. T cell hybridomas were infected with viruses containing genes of the chimeric receptors by coculture overnight with the transfected GP+E86 cells. Hybridoma cells transduced with scC2Fv/{gamma} or scC2Fv/{gamma}IC- were selected with 3 mg/ml of G418. This resulted in four transduced cell lines, including MD.45 cells expressing scC2Fv/{gamma} or scC2Fv/{gamma}IC-, and HCQ6 cells expressing scC2Fv/{gamma} or scC2Fv/{gamma}IC-, called, respectively, MD.45(C2{gamma}), MD.45(C2{gamma}IC-), HCQ6(C2{gamma}), and HCQ6(C2{gamma}IC-). Hybridoma cells transduced with an empty retroviral vector pBabeNeo only, MD.45(BabeNeo) and HCQ6(BabeNeo), were used as a control.

Biotinylation of cell surface proteins

To label cell surface proteins with biotin, viable cells were washed five times in PBS with 1 mM CaCl2 and 1 mM MgCl2 and resuspended in the same buffer (5 x 106 cells/ml). Sulfosuccinimidyl 6-(biotin-amido) hexanoate (Pierce, Rockford, IL) was added from stock solution in DMSO to a final concentration of 0.1 mg/ml. The cells were incubated for 1 h on ice and washed five times in PBS.

Immunoprecipitation

Surface-biotinylated or nonlabeled cells were solubilized by incubating for 30 min on ice in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40 (BDH, Poole, U.K.), 1 µg/ml leupeptin, 1 µg/ml chymostatin, 1 µg/ml pepstatin A, 1 mM PMSF, 1 mM aprotinin, and 10 mM iodoacetamide (all from Sigma, St. Louis, MO). IgG from rabbit anti-C2 antiserum was covalently bound to protein A-Sepharose beads (Pharmacia) by incubating the beads with the rabbit anti-C2 antiserum and 20 mM dimethylpimelimidate (Pierce) in 0.2 M borate buffer, pH 9, for 45 min at room temperature. The reaction was stopped with 0.2 M ethanolamine, pH 8, and the beads were washed five times in PBS. The protein A-Sepharose beads with covalently bound anti-C2 IgG were used for immunoprecipitation of nuclear-free supernatants of lysed surface-biotinylated cells. The immunoprecipitation was performed for 4 h at 4°C. For immunoprecipitation with the anti-CD3{zeta} mAb 6B10.2 (Santa Cruz Biotechnology, Santa Cruz, CA), nuclear-free supernatants of nonlabeled cells were incubated with 1 µg/ml of the mAb for 1 h on ice, and the immune complexes were precipitated with protein A-Sepharose beads for 1 h at 4°C. After immunoprecipitation, protein A-Sepharose beads were washed five times with lysis buffer.

Immunoblotting

Nuclear-free cell lysates, or immunoprecipitated proteins, were separated by SDS-PAGE and transferred onto nitrocellulose membrane, which was then blocked with nonfat milk. Biotinylated scC2Fv chimeras were detected by blotting with streptavidin-biotinylated horseradish peroxidase complex (Amersham). Nonlabeled scC2Fv chimeras were detected by immunoblotting with 1/1000 dilution of rabbit anti-C2 antiserum, followed by F(ab')2 of donkey anti-rabbit IgG conjugated with horseradish peroxidase (Amersham). For detection of CD3{zeta}, blots were incubated with 1 µg/ml of the anti-CD3{zeta} mAb 6B10.2, followed by horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham International). All blots were developed using a chemoluminescence kit (ECL, Amersham) and exposed to an x-ray film (Kodak-Pathe, Paris, France).

Quantitation of cellular proteins

The x-ray films were semiquantitatively analyzed with Bio-Rad GS-670 Imaging Densitometer (Bio-Rad, Hertfordshire, U.K.). In transduced hybridoma cells, scC2Fv chimeras form homodimers as well as heterodimers with endogenous CD3{zeta}. The following formula was used to calculate the molar ratio of the homodimers to the heterodimers:

where Mhmd is the intensity of the band representing homodimer of scC2Fv/{gamma} or scC2Fv/{gamma}IC-; and Mhtrd is the intensity of the band representing heterodimer of the scC2Fv chimera and CD3{zeta}.

Flow cytometry

For detection of cell surface-expressed scC2Fv/{gamma} and scC2Fv/{gamma}IC-, cells were stained with 1/200 dilution of the rabbit anti-C2 antiserum, followed by FITC-labeled F(ab')2 fragment of goat anti-rabbit IgG Ab (Sigma). The samples were analyzed with a FACS (Becton Dickinson, Mountain View, CA) using Consort 30 software. Cell surface-expressed CD3 was detected by incubating cells with 10 µg/ml of anti-mouse CD3 mAb 145-2C11, followed by incubation with FITC-conjugated affinity-purified goat anti-Armenian hamster IgG (Stratech Scientific, Luton Beds, U.K.). Samples were analyzed with a FACS (Becton Dickinson) using Cell Quest software.

Cell stimulation

Collagen type II was purified from bovine articular cartilage (18). T cell hybridomas were stimulated with collagen attached to the wells of a flat-bottom 96-well plate, or added to culture medium. Rabbit IgG enriched with Abs specific to Fv domain of the mAb C2 (further referred to as anti-mAb C2 IgG) was purified from the antiserum by a two-step affinity chromatography. First, anti-mouse IgG Fc Abs were depleted from the antiserum by passing through the column of mouse IgG coupled to Sepharose (Pharmacia). Anti-mAb C2 IgG was then isolated from this antiserum by affinity chromatography on mAb C2 bound to Sepharose (Pharmacia). Anti-mAb C2 IgG was also used for cell stimulation either attached to the microtiter wells or added to culture medium. In some experiments, the hybridomas were stimulated with the anti-CD3{epsilon} mAb 145-2C11 attached to the microtiter wells. For stimulation with their cognate Ags, MD.45 cells were cocultured with their target EL-4 cells, and HCQ6 cells were cultured with collagen added to culture medium in the presence of freshly isolated DBA/1 splenocytes. Concentration of murine IL-2 in culture supernatants was measured for quantitation of cell activation. It was determined by ELISA using mAb JES6-1A12 (PharMingen, San Diego, CA) as coating Ab and biotinylated mAb JES6-5H4 (PharMingen) for detection. The lower detection limit of this ELISA was 15 pg/ml.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of scC2Fv chimeras on the surface of transduced T cell hybridomas

T cell hybridomas MD.45 and HCQ6 transduced with scC2Fv/{gamma} or scC2Fv/{gamma}IC- expressed these chimeric receptors on the cell surface, as demonstrated by FACS analysis of cells stained with the polyclonal anti-mAb C2 antiserum (Fig. 1Go). Levels of scC2Fv/{gamma} and scC2Fv/{gamma}IC- expression were equivalent in transduced HCQ6 cells, and comparable in transduced MD.45 cells.



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 1. Expression of scC2Fv/{gamma} and scC2Fv/{gamma}IC- on the surface of T cell hybridomas. MD.45, MD.45(C2{gamma}IC-), MD.45(C2{gamma}), HCQ6, HCQ6(C2{gamma}IC-), and HCQ6(C2{gamma}) cells were stained with 1/200 dilution of normal rabbit serum (solid line), or 1/200 dilution of rabbit anti-mAb C2 antiserum (dotted line), followed by FITC-labeled secondary goat anti-rabbit IgG, and analyzed by FACS.

 
Subunit organization of scC2Fv/{gamma} in T lymphoid cells

To determine the m.w. of scC2Fv/{gamma}, pBabeNeo+scC2Fv/{gamma}-transfected GP+E86 packaging fibroblasts were surface biotinylated, and their lysates were immunoprecipitated with rabbit anti-C2 antiserum. Fig. 2Go demonstrates that transfected GP+E86 cells express scC2Fv/{gamma} in the form of a disulfide-linked homodimer. As averaged from seven independent experiments (immunoprecipitation and immunoblotting), its molecular mass is 74 ± 5 kDa (mean ± SD). Under reducing conditions, scC2Fv/{gamma} migrated as a single band of 36.7 kDa (Fig. 2Go), which corresponds to the predicted molecular mass of the monomeric protein.



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 2. Formation of homodimers by scC2Fv/{gamma} molecules. Lysates of surface-biotinylated wild-type (WT) and BabeNeo+scC2Fv/{gamma}-transfected GP+E86 cells (4 x 106 and 2.5 x 106 cells, respectively) were immunoprecipitated with rabbit anti-mAb C2 IgG covalently coupled to protein A-Sepharose, and analyzed by SDS-PAGE. Gel (7.5 and 12%) was used for separation under nonreducing conditions or in the presence of 2-ME, respectively.

 
We analyzed the subunit structure of scC2Fv/{gamma} chimera in transduced lymphoid cells, bearing in mind that these molecules contain the transmembrane and intracellular domains of the Fc{epsilon}RI-signaling {gamma} subunit that has been shown to associate with the CD3{zeta} subunit (19, 20). To this end, cell lysates were separated by SDS-PAGE under nonreducing conditions, transferred onto nitrocellulose membrane, and immunoblotted with rabbit anti-C2 antiserum. Fig. 3Go demonstrates that in both T cell hybridomas, in addition to homodimerization, scC2Fv/{gamma} forms a heterodimer of 54 kDa with an endogenous protein. Analogous homo- and heterodimers were present in cells transduced with scC2Fv/{gamma}IC-, suggesting that the cytoplasmic domain of scC2Fv/{gamma} was not involved in the intermolecular association (Fig. 3Go). Based on the difference in the m.w. between monomeric scC2Fv/{gamma} and the heterodimer, the latter could represent scC2Fv/{gamma} associated with endogenous CD3{zeta}, which has been directly demonstrated by immunoprecipitation (see below). The four types of homodimeric and CD3{zeta}-associated scC2Fv chimeras expressed in the T cell hybridomas are presented schematically in Fig. 4Go.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 3. Subunit structure of scC2Fv/{gamma} and scC2Fv/{gamma}IC-transduced into T cell hybridomas. Nuclear-free lysates of wild-type (WT) and scC2Fv/{gamma}IC-- or scC2Fv/{gamma}-transduced HCQ6 or MD.45 cells were resolved by SDS-PAGE under nonreducing conditions and immunoblotted with rabbit anti-mAb C2 antiserum, followed by peroxidase-labeled goat anti-rabbit IgG F(ab')2. Film was exposed for 5 min. The arrows point at the homodimers of scC2Fv chimeras (hmd), or heterodimer of scC2Fv chimeras and CD3{zeta} (htrd).

 


View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 4. Schematic representation of homodimeric and CD3{zeta}-associated scC2Fv/{gamma} and scC2Fv/{gamma}IC-. (scC2Fv/{gamma})2 and (scC2Fv/{gamma}IC-)2 are homodimers of scC2Fv chimeras; scC2Fv/{gamma}-CD3{zeta} and scC2Fv/{gamma}IC--CD3{zeta} are heterodimers of scC2Fv chimeras and endogenous CD3{zeta}. (GGGGS)3 is a peptide linker between VH and VL in scC2Fv chimeras.

 
When transduced into MD.45, scC2Fv chimeras were expressed predominantly as homodimers, whereas heterodimers of chimeric molecules and CD3{zeta} were more abundant in HCQ6 cells. As determined by densitometric analysis of immunoblots presented in Fig. 3Go, the molar ratio of homodimers to heterodimers in MD.45(C2{gamma}) and MD.45(C2{gamma}IC-) cells was 2.57 and 2.7, respectively. In HCQ6 cells transduced with scC2Fv/{gamma} or scC2Fv/{gamma}IC-, this ratio was 0.19 and 0.22, respectively. In other words, 1 of 3.5 chimeric receptors in transduced MD.45 cells is a heterodimer, and 1 of 6 receptors in transduced HCQ6 cells is a homodimer. The higher proportion of CD3{zeta}-associated scC2Fv chimeras in HCQ6 cells may be accounted for by a fivefold higher CD3{zeta} content in wild-type HCQ6 cells than in wild-type MD.45 cells (not shown data of immunoblotting).

Fig. 5Go demonstrates that the heterodimers found in transduced T cell hybridomas represent the CD3{zeta}-associated scC2Fv chimeras, because they could be immunoprecipitated with an anti-CD3{zeta} mAb from Nonidet P-40-lysed HCQ6(C2{gamma}) and HCQ6(C2{gamma}IC-) cells. No band of this m.w. was precipitated from lysates of wild-type HCQ6 cells by the anti-CD3{zeta} mAb, or from lysates of HCQ6(C2{gamma}) cells by an isotype-matched control IgG1 (Fig. 5Go). Although proteins were separated under nonreducing conditions in this experiment, slight degradation of Igs used for immunoprecipitation did occur. As a result of this degradation, immunoblotting with rabbit anti-mouse mAb C2 antiserum visualized the heavy chains of the mouse anti-CD3{zeta} mAb 6B10.2, or control mouse IgG1. This signal is significantly weaker than the band corresponding to undegraded IgG on this film (not shown).



View larger version (50K):
[in this window]
[in a new window]
  		FIGURE 5. Association of scC2Fv/{gamma} and scC2Fv/{gamma}IC- with CD3{zeta} in T cell hybridomas. Nuclear-free lysates of wild-type (WT) or transduced HCQ6 cell (108 cells per immunoprecipitation) were immunoprecipitated with the anti-CD3{zeta} mAb 6B10.2 or control-purified mouse IgG1, resolved by SDS-PAGE under nonreducing conditions, and immunoblotted with rabbit anti-mAb C2 antiserum, followed by peroxidase-labeled F(ab')2 of donkey anti-rabbit IgG. Film was exposed for 20 s. The arrow points at the heterodimer of scC2Fv chimeras and CD3{zeta}.

 
IL-2 production by transduced hybridomas stimulated with collagen type II

To assess the ability of scC2Fv chimeras to function as cell surface receptors, transduced and nontransduced MD.45 and HCQ6 cells were stimulated with type II collagen, and IL-2 levels in their culture supernatants were determined. Collagen was either added to culture medium or attached to plastic. It is reasonable to assume that, compared with plastic-immobilized collagen, collagen in solution has lower capacity to cross-link scC2Fv chimeric receptors. In the absence of collagen, none of the wild-type cell lines or transduced cells produced detectable levels of IL-2. Collagen in solution, or immobilized to plastic, did not stimulate IL-2 secretion in wild-type MD.45 or HCQ6 cells. At the same time, as expected, wild-type hybridoma cells produced high levels of IL-2 in response to stimulation with the anti-CD3 mAb 145-2C11 (Fig. 10Go).



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 10. Reduced response to anti-CD3 stimulation in T cell hybridomas transduced with scC2Fv/{gamma} or scC2Fv/{gamma}IC-. HCQ6 (A) or MD.45 (B) cells, wild-type (WT, squares), or transduced with an empty retroviral vector (BabeNeo, triangles), or with scC2Fv/{gamma} (closed circles), or with scC2Fv/{gamma}IC- (open circles), were stimulated overnight with anti-CD3{epsilon} mAb 145-2C11 attached to microtiter wells at indicated concentrations. Cell concentration during stimulation was 2.5 x 105/ml. IL-2 content in culture supernatants was measured using murine IL-2 ELISA. Results of three independent experiments are demonstrated as mean ± SEM. SEM bars are not shown where they are smaller than the symbol.

 
In response to stimulation with plastic-immobilized collagen, MD.45(C2{gamma}) and HCQ6(C2{gamma}) cells secreted IL-2, demonstrating biphasic dose-response relationship (Fig. 6Go, A and D). Optimal concentration of collagen for coating microtiter wells was within the range of 1–10 µg/ml. IL-2 production progressively declined, as the concentration rose further. scC2Fv/{gamma} binds to collagen through the antigenic epitope recognized by native mAb C2, because preliminary treatment of collagen-coated wells with the mAb C2 inhibited IL-2 production by MD.45(C2{gamma}) cells (Fig. 6GoC). This did not result from a nonspecific loss of responsiveness or viability in the hybridoma cells in the presense of the mAb C2, because IL-2 response of wild-type MD.45 cells to the anti-CD3 mAb 145-2C11 in control experiments was unaffected by the mAb C2. Similarly, if microtiter wells coated with 1 µg/ml collagen were pretreated with 50 µg/ml mAb C2 before adding cells, IL-2 production by HCQ6(C2{gamma}) and HCQ6(C2{gamma}IC-) was inhibited by 93 and 97%, respectively (not shown).



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 6. Collagen-induced IL-2 secretion of scC2Fv/{gamma} or scC2Fv/{gamma}IC--transduced T cell hybridomas. MD.45 cells (A to C), wild-type (WT, closed squares), or transduced with scC2Fv/{gamma} (closed circles), or scC2Fv/{gamma}IC- (closed diamonds), were stimulated overnight with indicated concentrations of collagen type II attached to plastic (A) or with collagen in culture medium (B). In some cases, collagen attached to plastic at 10 µg/ml was blocked with indicated concentrations of mAb C2 before addition of cells (C). HCQ6 cells (D and E), wild-type (WT, closed squares), or transduced with scC2Fv/{gamma} (closed circles), or scC2Fv/{gamma}IC- (closed diamonds), were stimulated overnight with indicated concentrations of collagen type II attached to plastic (D), or with plastic-bound anti-mAb C2 IgG (E). Some microtiter wells coated with anti-mAb C2 IgG were blocked with 2 µg/ml of mAb C2 (E, open circles). Cell concentration during stimulation was 106 ml-1. IL-2 content in culture supernatants was measured using murine IL-2 ELISA. Results of representative experiments are demonstrated as mean ± SEM of triplicates. SEM bars are not shown where they are smaller than the symbol.

 
As expected, MD.45(C2{gamma}IC-) cells did not produce significant levels of IL-2 in response to stimulation with plastic-immobilized collagen (Fig. 6GoA). However, HCQ6(C2{gamma}IC-) cells stimulated with plastic-immobilized collagen secreted substantial levels of IL-2, which were about 70% of IL-2 levels secreted by HCQ6(C2{gamma}) cells (Fig. 6GoC and Table IGo). The high level of responsiveness to plastic-immobilized collagen in HCQ6(C2{gamma}IC-) cells finds explanation in a relatively high expression of scC2Fv/{gamma}IC--CD3{zeta} heterodimers in these hybridoma cells (Fig. 3Go).


View this table:
[in this window]
[in a new window]
  		Table I. IL-2 production by infected hybridomas1

 
MD.45(C2{gamma}) cells were responsive to soluble collagen (Fig. 6GoB). As summarized in Table IGo, their IL-2 production induced by optimal concentration of soluble collagen was 81% of the IL-2 production induced by stimulation with optimal concentration of plastic-immobilized collagen. By contrast, HCQ6(C2{gamma}) cells stimulated with soluble collagen produced only 2.4% of the amount of IL-2 that they produced in response to stimulation with plastic-immobilized collagen (Table IGo). When transduced HCQ6 cells were stimulated with polyclonal anti-mAb C2 IgG instead of collagen type II, they demonstrated similar cross-linking requirements for activation. Thus, anti-mAb C2 IgG attached to microtiter wells was stimulatory for these cells (Fig. 6GoE), whereas IgG added to culture medium (the highest concentration tested was 20 µg/ml) did not induce detectable levels of IL-2 in them (not shown). In similarity to response to plastic-immobilized collagen, response to plastic-immobilized anti-mAb C2 IgG was totally inhibited by pretreatment of the microtiter wells with mAb C2 (Fig. 6GoE). MD.45(C2{gamma}IC-) and HCQ6(C2{gamma}IC-) cells were unresponsive to collagen in solution (Fig. 6GoB and Table IGo).

Subunit structure of the scC2Fv chimeras in transduced cells is perhaps an important factor determining cross-linking requirements for activation of these cells by collagen. The capacity to respond to collagen in solution appears to correlate with the presence of scC2Fv/{gamma} homodimers in transduced cells, because MD.45(C2{gamma}) cells, the only cells that responded to soluble collagen, contained the highest level of these homodimers. Meanwhile, cells that were unable to respond to soluble collagen either did not contain them, like MD.45 and HCQ6 cells transduced with scC2Fv/{gamma}IC-, or, like HCQ6(C2{gamma}) cells, expressed lower level of the homodimers than MD.45(C2{gamma}) cells (Fig. 3Go). This allows speculation that heterodimers of endogenous CD3{zeta} and scC2Fv chimeras do not initiate intracellular signaling events in response to stimulation with soluble collagen. To determine whether or not the heterodimers were capable of binding collagen from solution, we studied the effect of preincubation with soluble ligand on subsequent cell activation with plastic-immobilized collagen. Results of such experiments performed using HCQ6(C2{gamma}IC-) cells in which CD3{zeta}-scC2Fv/{gamma}IC- heterodimer is the only ITAM-containing receptor for unprocessed collagen are shown in Fig. 7Go. Soluble collagen inhibited IL-2 production induced by plastic-immobilized collagen, which suggests that it blocks binding of the CD3{zeta}-associated scC2Fv/{gamma}IC- to plastic-immobilized collagen by occupying the receptor or causing its internalization without initiation of downstream signaling events leading to IL-2 production. Another possible explanation is delivery of a negative regulatory signal by binding of soluble collagen to the heterodimer. IL-2 production by wild-type HCQ6 cells stimulated with plastic-immobilized anti-CD3 mAb 145-2C11 was unaffected by soluble collagen (not shown).



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 7. Blockade of CD3{zeta}-associated scC2Fv/{gamma}IC- in HCQ6(C2{gamma}IC-) cells with soluble collagen type II. HCQ6(C2{gamma}IC-) cells were incubated at 106 ml-1 with indicated concentrations of collagen in culture medium for 1 h and then transferred to microtiter wells coated with collagen at 2 µg/ml and cultured overnight. IL-2 content in culture supernatants was measured using murine IL-2 ELISA. Results of a representative experiment are demonstrated as mean ± SEM of triplicates. SEM bars are not shown where they are smaller than the symbol.

 
Loss of TCR/CD3-mediated response in transduced T cell hybridomas

We asked whether or not response to cognate Ags was altered in T cell hybridomas transduced with scC2Fv chimeras. TCR-mediated activation of HCQ6 cells can be quantitatively estimated by IL-2 release in response to stimulation with type II collagen processed and presented by syngeneic APC. As scC2Fv chimeras expressed in HCQ6(C2{gamma}) and HCQ6(C2{gamma}IC-) cells were unresponsive to soluble collagen, IL-2 production induced in these cells by soluble collagen in the presence of APC could be fully attributed to TCR-mediated stimulation. In the experiment presented in Fig. 8Go, wild-type or scC2Fv chimera-transduced HCQ6 cells stimulated with soluble collagen in the absence of APC did not secrete detectable levels of IL-2 (not shown). In response to stimulation with soluble collagen presented by DBA/1 splenocytes (Fig. 8GoA), HCQ6(C2{gamma}) and HCQ6(C2{gamma}IC-) cells produced much lower levels of IL-2 than nontransduced HCQ6 cells. The overall ability of HCQ6(C2{gamma}) and HCQ6(C2{gamma}IC-) cells to produce IL-2, however, was not impaired, because under optimal conditions their response to unprocessed plastic-immobilized collagen (Fig. 8GoB) was equivalent to the response of wild-type HCQ6 cells stimulated with APC-presented collagen at 100 µg/ml (Fig. 8GoA). Preliminary experiments demonstrated that this concentration of collagen was optimal for the splenocyte-dependent activation of HCQ6 cells (not shown). To study the effect of scC2Fv chimeras on TCR-mediated functions in MD.45 cells, wild-type and transduced MD.45 cells were stimulated with their specific target EL-4 cells (12) (Fig. 8GoC). Profound decrease in IL-2 response was observed in transduced cells. Response of hybridomas transduced with an empty retroviral vector was not different from that of wild-type cells (not shown).



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 8. Loss of the TCR-mediated response in scC2Fv/{gamma} or scC2Fv/{gamma}IC--transduced hybridomas. A total of 5 x 105 ml-1 of HCQ6 cells (A and B), wild-type (WT, squares), or transduced with scC2Fv/{gamma} (circles), or scC2Fv/{gamma}IC- (diamonds), was stimulated overnight with indicated concentrations of type II collagen in culture medium in the presence of 5 x 106 ml-1 of syngeneic splenocytes as APC (A), or with indicated concentrations of plastic-immobilized collagen without splenocytes (B). A total of 106 ml-1 of MD.45 cells (C), wild-type (WT, squares), or transduced with scC2Fv/{gamma} (circles), or scC2Fv/{gamma}IC- (diamonds), was stimulated overnight with indicated concentrations of EL-4 cells. IL-2 content in culture supernatants was measured using murine IL-2 ELISA. Results of representative experiments are demonstrated as mean ± SEM of triplicates. SEM bars are not shown where they are smaller than the symbol.

 
Trying to elucidate the underlying mechanism of the reduced responsiveness to cognate Ags in T cell hybridomas expressing the chimeric receptors, we compared expression of endogenous CD3{zeta} and CD3{epsilon} in transduced and wild-type hybridoma cells. Immunoblot analysis presented in Fig. 9GoA demonstrates that, compared with wild-type cells, scC2Fv chimera-transduced cells contained reduced amount of CD3{zeta}. Even in transduced HCQ6 cells, in which heterodimers of scC2Fv chimeras and CD3{zeta} were readily detectable by immunoblotting with the anti-mAb C2 antiserum (Fig. 3Go), the chimera-associated CD3{zeta} could not be revealed by immunoblotting with the anti-CD3{zeta} mAb 6B10.2, presumably due to an insufficient sensitivity of this staining. Therefore, overall decrease in CD3{zeta} content in the Nonidet P-40-soluble fraction of the cell lysates, rather than its redistribution in favor of the scC2Fv chimera-bound form, occurred in the transduced hybridomas. Results of FACS analysis (Fig. 9GoB) demonstrated that CD3{epsilon} expression was also decreased on the surface of hybridoma cells transduced with scC2Fv chimeras, but not in control cells transduced with an empty retroviral vector. Data presented in Fig. 9GoB are from one of five independent experiments. The decrease in CD3{epsilon} expression was highly reproducible, but its degree varied between these experiments from slight reduction compared with control to nearly undetectable levels. Such variation of CD3{epsilon} expression in scC2Fv chimera-transduced cells is compatible with functional down-modulation of endogenous CD3 subunits in the presence of the chimeric receptors, rather than with selection of clones expressing low CD3{epsilon} levels during generation of transduced cells. Fig. 10Go demonstrates that reduced expression of the CD3{epsilon} subunit in hybridomas expressing scC2Fv chimeras was accompanied by a decreased response to stimulation with anti-CD3{epsilon} mAb 145-2C11. The reduction in responsiveness to anti-CD3{epsilon} was perhaps greater than one would expect, considering only partial decrease in the expression of CD3{epsilon} in scFv chimera-transduced cells. The response of cells, transduced with an empty retroviral vector, however, remained unaltered (Fig. 10Go).



View larger version (54K):
[in this window]
[in a new window]
  		FIGURE 9. Reduced CD3 expression in scC2Fv/{gamma} or scC2Fv/{gamma}IC--transduced T cell hybridomas. Nuclear-free lysates of wild-type (WT) or scC2Fv chimera-transduced cells were resolved by SDS-PAGE under nonreducing conditions and immunoblotted with the anti-CD3{zeta} mAb 6B10.2, followed by sheep anti-mouse IgG. Film was exposed for 30 min (A). MD.45, MD.45(BabeNeo), MD.45(C2{gamma}IC-), MD.45(C2{gamma}), HCQ6, HCQ6(BabeNeo), HCQ6(C2{gamma}IC-), and HCQ6(C2{gamma}) cells were stained with 10 µg/ml normal hamster IgG (dotted line), or 10 µg/ml anti-CD3{epsilon} mAb 145-2C11 (solid line), followed by FITC-labeled goat anti-hamster IgG, and analyzed by FACS (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that transduction with the gene encoding scC2Fv/{gamma}, a chimeric receptor containing scFv domain of the anti-collagen mAb C2 and the Fc{epsilon}RI{gamma} subunit, confers Ab-type specificity to collagen on T cell hybridomas. In addition, expression of endogenous CD3 subunits and response to the cognate Ag in T cell hybridomas expressing scC2Fv/{gamma} are reduced. This receptor forms homodimers as well as heterodimers with endogenous CD3{zeta} subunit. The homodimerization of scC2Fv/{gamma} and its association with CD3{zeta} do not require integrity of its cytoplasmic domain, as this ability is fully maintained in its mutein, scC2Fv/{gamma}IC-, lacking the Fc{epsilon}RI{gamma}-signaling domain. Presumably, homodimerization of scC2Fv chimeras and their covalent association with CD3{zeta} are mediated by their Fc{epsilon}RI{gamma}-derived transmembrane domains, because a cysteine residue in this domain is known to mediate homodimerization of native Fc{epsilon}RI{gamma}, as well as its association with CD3{zeta} (19). Decreased expression of endogenous CD3{zeta} subunit in T cell hybridomas transduced with scC2Fv chimeras is a major reason for decreased TCR-mediated responsiveness because this subunit is a limiting factor for expression of TCR/CD3 complex (21). The exact mechanism of reduction in CD3{zeta} expression in T cells transduced with Fc{epsilon}RI{gamma}-containing chimeras remains unclear. However, it does not appear to result from sequestration of CD3{zeta} with the engrafted chimera, as overall decrease in CD3{zeta} expression was observed in scC2Fv chimera-transduced hybridomas. One could hypothesize that for an unknown reason, production of CD3{zeta} is suppressed in the presence of scC2Fv chimeras, or CD3{zeta}-scC2Fv/{gamma} and CD3{zeta}-scC2Fv/{gamma}IC- heterodimers have higher turnover rate, compared with homodimeric CD3{zeta}, thus leading to a rapid depletion of a newly synthesized CD3{zeta}. Increased association of CD3{zeta} with the cytoskeleton and its disappearance from the Nonidet P-40-soluble fraction (22) in transduced hybridomas is also a possibility. With regard to decreased CD3{zeta} expression, the T cell hybridomas transduced with scC2Fv chimeras are reminiscent of unresponsive T cells in conditions such as rheumatoid arthritis, HIV infection, or cancer (23, 24, 25). The decrease in the TCR-mediated response seems to be specific for chimeras containing Fc{epsilon}RI{gamma} subunit, since MD.45 cells expressing a chimera containing scFv and TCR {alpha} or ß subunit apparently remained fully responsive to stimulation with EL-4 cells (26).

Taking into account that dependence on CD3{zeta} is a ubiquitous regulatory mechanism of TCR/CD3 expression (27), TCR-dependent response of primary lymphocytes transduced with Fc{epsilon}RI{gamma} subunit-containing chimeras would perhaps also be reduced. Mitogenic stimulation of T lymphocytes in vitro, to facilitate their infection with scC2Fv/{gamma}-containing retrovirus, might lead to expansion of autoimmune T cell clones. Loss of sensitivity to the original Ag in the transduced cells, however, would prevent such clones from revealing their harmful potential.

The Fc{epsilon}RI{gamma} and CD3{zeta} belong to the same molecular family, and their transmembrane domains have 58% amino acid identity (28). These domains mediate noncovalent interaction of Fc{epsilon}RI{gamma} and CD3{zeta} with other components of Ig FcR or the TCR/CD3 complex (27, 29, 30). In addition, the ectodomain of CD3{zeta} is also involved in association with other CD3 subunits (31). This suggests a possibility that apart from CD3{zeta}, other endogenous CD3 subunits are noncovalently associated with homo- and heterodimers of the chimeric receptors and contribute to intracellular signal transduction upon oligomerization of these receptors. As far as the homodimers of scC2Fv chimeras are concerned, the observation that MD.45(C2{gamma}IC-) cells that predominantly express homodimers of the ITAM-less chimera scC2Fv/{gamma}IC- do not respond to collagen (Fig. 6Go, A and B) argues against this possibility. A number of published reports suggest that Fc{epsilon}RI{gamma} and CD3{zeta} are interchangeable signaling subunits. Thus, Fc{epsilon}RI{gamma} can substitute CD3{zeta} in supporting assembly and function of the TCR/CD3 complex in CD3{zeta} knockout mice (32). Another line of evidence suggesting functional similarity between the two signaling molecules is the observation that in basophils, the Fc{epsilon}RI{gamma} subunit can activate Syk kinase (33), the tyrosine kinase playing an important role in TCR-mediated signaling (34, 35). Therefore, scC2Fv/{gamma} homodimers and heterodimers of scC2Fv chimeras with CD3{zeta} may initiate qualitatively similar downstream signaling events in T cells.

Apparently, scC2Fv/{gamma} homodimers and heterodimers of scC2Fv chimeras and CD3{zeta} have different cross-linking requirements for activation, because the capacity of transduced hybridoma cells to respond to collagen in solution correlated with predominant expression of the homodimers (Figs. 3Go and 6Go). The reason that heterodimers of scC2Fv chimeras and CD3{zeta} need plastic-attached ligand for efficient oligomerization is probably the fact that they have only one ligand-binding site per receptor, hence, lower avidity to collagen. Soluble collagen, however, binds to the monovalent heterodimers, as it inhibits cell activation by plastic-immobilized collagen (Fig. 7Go). It remains unclear whether soluble collagen is unable to induce optimal oligomerization of the heterodimers, or its interaction with the receptor results in the generation of a negative regulatory signal. Such negative regulatory signal delivered through heterodimers could play a role in reduced response of chimera-transduced HCQ6 cells to collagen presented by splenocytes (Fig. 8GoA). This, however, seems to be unlikely because in the absence of collagen, transduced hybridomas also exhibited decreased TCR/CD3-mediated responses (Figs. 8GoC and 10).

Our recent observation on T cell hybridomas transduced with scC2Fv/CD8/{zeta}, a chimera representing scC2Fv ectodomain fused with CD8{alpha} hinge region and CD3{zeta} transmembrane and cytoplasmic domains (manuscript in preparation), lends support to the notion that expression of divalent receptors is a requirement for responsiveness to soluble collagen. Thus, scC2Fv/CD8/{zeta} is expressed only as a homodimer in both MD.45 and HCQ6 cells, without association with endogenous CD3{zeta} in either of them, and both hybridomas transduced with scC2Fv/CD8/{zeta} respond to soluble collagen by IL-2 production. Collagen in solution can activate cells expressing scC2Fv/{gamma} homodimers because it is a multivalent Ag. Multivalent soluble Ags, however, may differ in the ability to induce oligomerization of scFv-containing chimeras optimal for generating intracellular signals in T cells. For instance, MD.45 cells transduced with the chimera consisting of the trinitrophenol-specific scFv and Fc{epsilon}RI{gamma} were unresponsive to fowl {gamma}-globulin-coupled trinitrophenol in solution (7). The ratio between scC2Fv/{gamma} homodimers and scC2Fv/{gamma}-CD3{zeta} heterodimers in transduced primary lymphocytes would determine their cross-linking requirements for activation with collagen. Therefore, had such genetically modified cells been used for gene therapy of arthritis, this ratio would be an important factor that would affect their behavior in vivo.

Does the difference between the homo- and heterodimers in the composition of their cytoplasmic domains contribute to their differential cross-linking requirements? With this regard, of interest is the observation of Sunder-Plassmann et al. (36), who have shown that T cells expressing a chimeric receptor containing all three ITAM of CD3{zeta} could be activated by cross-linking with a mAb specific to the receptor ectodomain, whereas the chimera with single ITAM required additional cross-linking with a secondary reagent for activation. However, no such correlation has been found in the present study, because the scC2Fv/{gamma} homodimers, receptors that are more readily cross-linked with collagen, contain fewer ITAM per receptor than the heterodimers of CD3{zeta} and scC2Fv chimeras. Apparently, in this case, the structure of the ectodomain is more important in determining cross-linking requirements for receptor activation than the number of ITAM in the cytoplasmic domain.

In summary, transduction with the gene of the chimeric receptor scC2Fv/{gamma} composed of a single-chain collagen-specific Ab fused with Fc{epsilon}RI{gamma} confers Ab-type specificity to collagen on T cell hybridomas. This receptor is expressed as a homodimer as well as a heterodimer with endogenous CD3{zeta}. The two forms of the receptor differ in their cross-linking requirements for oligomerization. The original TCR-mediated reactivity of the transduced cells is reduced profoundly, as a result of which they become specific predominantly to collagen type II.


   Acknowledgments
 
We thank Prof. M. Feldmann and Dr. B. Foxwell for critically reading the manuscript.


   Footnotes
 
1 This work was supported by the Nuffield Foundation, London, and Arthritis Research Campaign, Chesterfield, United Kingdom. Back

2 Address correspondence and reprint requests to Dr. Yuti Chernajovsky, Molecular Biology Laboratory, Kennedy Institute of Rheumatology, 1 Aspenlea Road, London, W6 8LH, U.K. E-mail address: Back

3 Abbreviations used in this paper: scFv, single-chain Fv domain; H chain, heavy chain; ITAM, immunoreceptor tyrosine-based activation motif; L chain, light chain. Back

Received for publication January 2, 1998. Accepted for publication August 28, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Holmdahl, R., J. M. C. Nordling, P. Larsson, L. Jansson, T. Goldschmidt, M. Andersson, L. Klareskog. 1989. Collagen induced arthritis: an experimental model for rheumatoid arthritis with involvement of both DTH and immune complex mediated mechanism. Clin. Exp. Rheumatol. 3:51.
  2. Mauri, C., R. O. Williams, M. Walmsley, M. Feldmann. 1996. Relationship between Th1/Th2 cytokine patterns and arthritogenic response in collagen-induced arthritis. Eur. J. Immunol. 26:1911.[Medline]
  3. Chernajovsky, Y., G. Adams, O. L. Podhajcer, G. M. Mueller, P. D. Robbins, M. Feldmann. 1995. Inhibition of transfer of collagen-induced arthritis into SCID mice by ex vivo infection of spleen cells with retroviruses expressing soluble tumor necrosis factor receptor. Gene Ther. 2:731.[Medline]
  4. Chernajovsky, Y., G. Adams, K. Triantaphylopoulos, M. F. Ledda, O. L. Podhajcer. 1997. Pathogenic lymphoid cells engineered to express TGF ß1 ameliorate desease in a collagen-induced arthritis model. Gene Ther. 4:553.[Medline]
  5. Moritani, M., K. Yoshimoto, S. Li, M. Kondo, H. Iwahana, T. Yamaoka, T. Sano, N. Nakano, H. Kikutani, M. Itakura. 1996. Prevention of adoptively transferred diabetes in nonobese diabetic mice with IL-10-transduced islet-specific Th1 lymphocytes. J. Clin. Invest. 98:1851.[Medline]
  6. Gua, D. J., D. R. Hinton, S. A. Stohlman. 1995. Self-antigen-induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice. J. Immunol. 155:4052.[Abstract]
  7. Eshhar, Z., T. Waks, G. Gross, D. G. Schindler. 1993. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody binding domains and the {gamma} or {zeta} subunits of the immunoglobulin and T-cell receptors. Proc. Natl. Acad. Sci. USA 90:720.[Abstract/Free Full Text]
  8. Weijtens, M. E. M., R. A. Willemsen, D. Valerio, K. Stam, R. L. H. Bolhius. 1996. Single chain Ig/{gamma} gene-redirected human T lymphocytes produce cytokines, specifically lyse tumor cells, and recycle lytic capacity. J. Immunol. 157:836.[Abstract]
  9. Samelson, L. E., R. D. Klausner. 1992. Tyrosine kinases and tyrosine-based activation motifs. J. Biol. Chem. 267:24913.[Free Full Text]
  10. Romeo, C., M. Amiot, B. Seed. 1992. Sequence requirements for induction of cytolysis by the T cell antigen/Fc receptor {zeta} chain. Cell 68:889.[Medline]
  11. Holmdahl, R., K. Rubin, L. Klareskog, E. Larsson, H. Wigzell. 1986. Characterization of the antibody response in mice with type II collagen-induced arthritis, using monoclonal anti-type II collagen antibodies. Arthritis Rheum. 29:400.[Medline]
  12. Kaufman, Y., G. Berke, Z. Eshhar. 1981. Cytotoxic T lymphocyte hybridomas that mediate specific tumor-cell lysis in vivo. Proc. Natl. Acad. Sci. USA 78:2502.[Abstract/Free Full Text]
  13. Brunsberg, U., K. Gustafsson, L. Jansson, E. Michaelsson, L. Ahrlund-Richter, S. Pettersson, R. Mattson, R. Holmdahl. 1994. Expression of transgenic class II Ab gene confers susceptibility to collagen-induced arthritis. Eur. J. Immunol. 24:1698.[Medline]
  14. Markowitz, D., S. Goff, A. Bank. 1988. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J. Virol. 62:1120.[Abstract/Free Full Text]
  15. Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156.[Medline]
  16. Brigido, M. M., M. Polymenis, B. D. Stollar. 1993. Role of mouse VH10 and VL gene segments in the specific binding of antibody to Z-DNA, analyzed with recombinant single chain Fv molecule. J. Immunol. 150:469.[Abstract]
  17. Morgenstern, J. P., H. Land. 1990. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18:3587.[Abstract/Free Full Text]
  18. Miller, E. J.. 1972. Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry 11:4903.[Medline]
  19. Anderson, P., M. Caligiuri, C. O’Brien, T. Manley, J. Ritz, S. F. Schlossman. 1990. Fc{gamma} receptor type II (CD16) is included in {zeta} NK receptor complex expressed by human natural killer cells. Proc. Natl. Acad. Sci. USA 87:2274.[Abstract/Free Full Text]
  20. Orloff, D. G., C. Ra, S. J. Frank, R. D. Klausner, J.-P. Kinet. 1990. Family of disulfide-linked dimers containing the {zeta} and {eta} chains of the T-cell receptor and the {gamma} chain of Fc receptors. Nature 347:189.[Medline]
  21. Weissman, A. M., S. J. Frank, D. G. Orloff, M. Mercep, J. D. Ashwell, R. D. Klausner. 1989. Role of the {zeta} chain in the expression of the T cell antigen receptor: genetic reconstitution studies. EMBO J. 8:3651.[Medline]
  22. Caplan, S., M. Baniyash. 1996. Normal T cells express two T cell antigen receptor populations, one of which is linked to the cytoskeleton via {zeta} chain and displays a unique activation-dependent phosphorylation pattern. J. Biol. Chem. 271:20705.[Abstract/Free Full Text]
  23. Maurice, M. M., A. C. Lankester, A. C. Bezemer, M. F. Geertsma, P.-P. Tak, F. C. Breedveld, R. A. W. van Lier, C. L. Verweij. 1997. Defective TCR-mediated signaling in synovial T cells in rheumatoid arthritis. J. Immunol. 159:2973.[Abstract]
  24. Salvadori, S., B. Gansbacher, A. M. Pizzimenti, K. S. Zier. 1994. Abnormal signal transduction by T cells in mice with wild type tumors is not seen in mice bearing IL-2 secreting tumors. J. Immunol. 153:5176.[Abstract]
  25. Stefanova, I., M. W. Saville, C. Peters, F. R. Cleghorn, D. Schwartz, D. J. Venzon, K. J. Weinhold, N. Jack, C. Bartholomew, W. A. Blattner, R. Yarchoan, J. B. Bolen. 1996. HIV infection-induced posttranslational modification of T cell signaling molecules associated with disease progression. J. Clin. Invest. 98:1290.[Medline]
  26. Gross, G., T. Waks, Z. Eshhar. 1989. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc. Natl. Acad. Sci. USA 86:10024.[Abstract/Free Full Text]
  27. Weissman, A. M., M. Baniyash, D. Hou, L. E. Samelson, W. H. Burgess, R. D. Klausner. 1988. Molecular cloning of the {zeta} chain of the T cell antigen receptor. Science 239:1018.[Abstract/Free Full Text]
  28. Kuster, H., H. Thompson, J.-P. Kinet. 1990. Characterization and expression of the gene for the human Fc receptor {gamma} subunit. J. Biol. Chem. 265:6448.[Abstract/Free Full Text]
  29. Kurosaki, T., I. Gander, J. V. Ravetch. 1991. A subunit common to an IgG Fc receptor and the T-cell receptor mediates assembly through different interactions. Proc. Natl. Acad. Sci. USA 88:3837.[Abstract/Free Full Text]
  30. Lanier, L. L., G. Yu, J. H. Fillips. 1991. Analysis of Fc{gamma}RIII (CD16) membrane expression and association with CD3{zeta} and Fc{epsilon}RI-{gamma} by site-directed mutation. J. Immunol. 146:1571.[Abstract]
  31. Bolliger, L., B. Johansson, E. Palmer. 1997. The short extracellular domain of the T cell receptor {zeta} chain is involved in assembly and signal transduction. Mol. Immunol. 34:819.[Medline]
  32. Liu, C.-P., V.-J. Lin, M. Huang, J. W. Kappler, P. Marrack. 1997. Development and function of T cells in T cell antigen receptor/CD3 {zeta} knockout mice reconstituted with Fc{epsilon}RI{gamma}. Proc. Natl. Acad. Sci. USA 94:616.[Abstract/Free Full Text]
  33. Kimura, T., H. Kihara, S. Bhattacharyya, H. Sakamoto, E. Appella, R. P. Siraganian. 1996. Downstream signaling molecules bind to different phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) peptides of the high affinity IgE receptor. J. Biol. Chem. 271:27962.[Abstract/Free Full Text]
  34. Farber, D. L., O. Acuto, K. Bottomly. 1997. Differential T cell receptor-mediated signaling in naive and memory CD4 T cells. Eur. J. Immunol. 27:2094.[Medline]
  35. Kolanus, W., C. Romeo, B. Seed. 1993. T cell activation by clustered tyrosine kinases. Cell 74:171.[Medline]
  36. Sunder-Plassmann, R., F. Lialios, M. Madsen, S. Koyasu, E. L. Reinherz. 1997. Functional analysis of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal transduction: the two YxxL segments within a single CD3{zeta}-ITAM are functionally distinct. Eur. J. Immunol. 27:2001.[Medline]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
J. M. Clark, A. E. Annenkov, M. Panesar, P. Isomaki, Y. Chernajovsky, and A. P. Cope
T cell receptor {zeta} reconstitution fails to restore responses of T cells rendered hyporesponsive by tumor necrosis factor {alpha}
PNAS, February 10, 2004; 101(6): 1696 - 1701.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
A. E. Annenkov, G. M. Daly, T. Brocker, and Y. Chernajovsky
Clustering of immunoreceptor tyrosine-based activation motif-containing signalling subunits in CD4+ T cells is an optimal signal for IFN-{gamma} production, but not for the production of IL-4
Int. Immunol., May 1, 2003; 15(5): 665 - 677.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Isomaki, M. Panesar, A. Annenkov, J. M. Clark, B. M. J. Foxwell, Y. Chernajovsky, and A. P. Cope
Prolonged Exposure of T Cells to TNF Down-Regulates TCR{{zeta}} and Expression of the TCR/CD3 Complex at the Cell Surface
J. Immunol., May 1, 2001; 166(9): 5495 - 5507.
[Abstract] [Full Text] [PDF]

Home page
 Rheumatology (Oxford)Home page
Y. Chernajovsky
Gene transfer as therapy for rheumatoid arthritis: why, what and how?
Rheumatology, September 1, 1999; 38(9): 804 - 806.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Annenkov, A. E.
Right arrow Articles by Chernajovsky, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Annenkov, A. E.
Right arrow Articles by Chernajovsky, Y.
Right arrowPubmed/NCBI databases
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS