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

Alexander E. Annenkov1,3, Gordon M. Daly1, Thomas Brocker2 and Yuti Chernajovsky1

1 Bone and Joint Research Unit, St Bartholomew’s and Royal London School of Medicine and Dentistry, Queen Mary, University of London, London EC1M 6BQ, UK 2 Max Planck Institute for Immunobiology, Freiburg D-79011, Germany 3 Present address: Biological Sciences Department, Imperial College London, London SW7 2AZ, UK

Correspondence to: Y. Chernajovsky; E-mail: y.chernajovsky{at}qmul.ac.uk
Transmitting editor: D. Wallach


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD4+ T cells with pre-defined MHC-unrestricted specificity to type II collagen (CII) were engineered for cell-based anti-inflammatory gene therapy of autoimmune arthritis. To this end, recombinant chimeric immunoreceptors, C2{gamma} or C2{zeta}, were expressed in primary mouse keyhole limpet hemocyanin (KLH)-specific Th1 and Th2 cells using retrovirus vector-based somatic cell gene transfer. The ectodomain of these tyrosine-based activation motif (ITAM)-containing immunoreceptors is a single-chain IgG variable domain of an anti-CII mAb. The engineered cells might arrest migration when they encounter CII in articular cartilage. Up to 19 and 55% of transduced CD4+ T cells expressed respectively C2{gamma} and C2{zeta}. The expression of C2{gamma} or C2{zeta} on the surface of CD4+ T cells was down-regulated upon binding CII, and cells activated in such a way proliferated, up-regulated CD25 expression and produced cytokines. Comparison of cytokine levels normalized by the number of producer cells revealed that C2{gamma} and C2{zeta} were as potent as TCR in the induction of IFN-{gamma}, but induced lower levels of IL-4. It appears that the reason why CD4+ T cells stimulated through C2{gamma} and C2{zeta} produce low levels of IL-4 is a lack of integration between co-stimulatory signals required for the optimal production of this cytokine and the ITAM-dependent signals generated by the immunoreceptors. The significance of these data for the development of anti-inflammatory gene therapy based on CD4+ T cells targeted to a tissue-specific protein is discussed.

Keywords: cytokine, gene therapy, mouse, TCR, Th1, Th2


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tissue-specific autoimmune diseases are thought to be caused by self-reactive Th1 cells, a CD4+ T cell subset producing IFN-{gamma} upon antigenic stimulation, whereas CD4+ T cells producing IL-4 (Th2 cells), transforming growth factor (TGF)-ß1 or IL-10 have regulatory properties (1,2). Antigenic stimulation, among other effects, causes migration arrest of self-reactive pathogenic CD4+ T cells and their accumulation at sites of disease. Regulatory subsets of CD4+ T lymphocytes may also exert their protective effects in an antigen-specific manner (37). As demonstrated in mouse collagen-induced arthritis (812) and other models of autoimmunity (1316), pathogenic self-reactive CD4+ T cells may acquire properties of regulatory cells after expression in them of an anti-inflammatory protein, such as TGF-ß1, IL-10, soluble CD35 or soluble p75 tumor necrosis factor receptor. However, targeting regulatory CD4+ T cells to tissue-specific antigens by expressing in them recombinant immunoreceptors of appropriate specificity might be more feasible for cell-based gene therapy of human autoimmune diseases.

The activation of CD4+ T cells is induced when the clonotypic subunits of TCR, {alpha} and ß TCR chains, bind to their cognate MHC class II loaded with an antigenic peptide. These subunits, which are unable to transduce intracellular signals on their own, are non-covalently associated with the signalling subunits, the tetrameric complex CD3{gamma}{epsilon}{delta}{epsilon} and TCR{zeta}{zeta} homodimer. A consensus amino acid sequence of these signalling subunits, immunoreceptor tyrosine-based activation motif (ITAM) (17), is indispensable for the function of TCR and other immunoreceptors.

In order to target keyhole limpet hemocyanin (KLH)-specific CD4+ T cells isolated from lymph nodes of KLH-immunized mice to CII (an extracellular matrix protein that is mainly confined to articular cartilage) these cells were transduced with recombinant genes encoding the chimeric immunoreceptor C2{gamma} (termed scC2Fv/{gamma} in previous reports) (18) or C2{zeta} (termed scC2Fv/CD8/{zeta} in previous reports) (19) using retrovirus vector-based somatic cell gene transfer protocol (20). The CII-recognizing ectodomain of these ITAM-containing immunoreceptors is a single-chain IgG variable domain (scFv) of the anti-type II collagen (CII) antibody C2 (21). As expected, CD4+ effector T cells expressing these chimeric immunoreceptors acquired MHC-unrestricted responsiveness to CII. This ITAM-dependent signal was sufficient to induce the optimal production of IFN-{gamma}, but IL-4 production was low.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Retroviral constructs and packaging cell lines
Packaging cells producing replication-defective retrovirus vector with a recombinant gene of C2{gamma} or C2{zeta}, or the reporter enhanced green fluorescent protein (eGFP), were generated as described (18,19).

Gene transfer into primary mouse lymphocytes
To obtain KLH-specific lymphocytes, DBA/1 mice were anaesthetized with Hypnorm and immunized with 200 µg KLH (Sigma, Poole, UK) in 100 µl of a 1:1 emulsion of PBS and complete Freund’s adjuvant (1 µg/ml Mycobacterium tuberculosis; Becton Dickinson, Oxford, UK) at the base of the tail. Cells from inguinal lymph nodes of these mice (KLH-LNC) were isolated 1 week post-immunization and cultured in vitro in DMEM supplemented with 10% FCS (Gibco/BRL, Life Technologies, Paisley, UK) and 50 µM 2-mercaptoethanol.

During the first 3 days in culture KLH-LNC were stimulated with 100 µg/ml KLH (5 x 106 cells/ml). Viable cells were then separated by centrifugation on a Lymphoprep (Robin Scientific, Solihull, UK) density gradient. In some experiments, CD4+ T cells were purified from these cells via magnetic cell sorting by negative selection using Murine CD4+ T Cells Enrichment Cocktail (StemCell Technologies, Meylan, France), which contained antibodies against CD8, CD11b, CD45R, Gr-1 and TER 119, and MACS separation columns (Miltenyi Biotec, Bisley, UK) according to the manufacturer’s instructions.

KLH-LNC or purified CD4+ T cells (96% CD4+) were then stimulated with 20–50 ng/ml human recombinant IL-2 (PeproTech, London, UK; 107 U/mg) for 1 day (106 cells/ml). This was followed by centrifugation-assisted transduction (20). The transduction was performed using retrovirus vector packaged in PT67 cells, except for the experiment presented in Fig. 6, where the retroviral stock was produced in GP+E86 packaging cells. After transduction, cells were further expanded by stimulation with 50 ng/ml IL-2 at a cell density of 2.5 x 105/ml. They were used in functional studies after 2 days post-transduction, unless otherwise noted.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6. Proliferation of KLH-LNC stimulated through TCR or C2{zeta} is independent of MCS. (A) Proliferation of KLH-LNC in response to KLH. KLH-LNC were re-activated in vitro as if for transduction, plated at 1.5 x 106/ml and re-stimulated with the indicated concentrations of KLH with or without MCS (4 x 106/ml) for 1 day. [3H]Thymidine was added to culture medium (1 µCi/well) for the last 5 h of stimulation. Cells were harvested and [3H]thymidine incorporation was measured with a scintillation ß counter. Results are shown as mean ± SEM of triplicates. SEM bars are not shown where they are smaller than the symbol. (B) Proliferation of C2{zeta}-transduced KLH-LNC in response to CII. KLH-LNC were transduced with C2{zeta} (C2{zeta}) or with the control retroviral vector pBabe neo (control) using replication-defective retrovirus vector packaged by GP+E86 cells. They were stimulated for 1 day at a cell density of 1.5 x 106/ml with CII, which was immobilized on plastic or added to culture medium. [3H]Thymidine (1 µCi/well) was added to culture medium for the last 5 h of stimulation. Results are shown as mean ± SEM of triplicates. SEM bars are not shown where they are smaller than the symbol. For KLH-LNC transduced with the control vector pBabe neo, stimulated samples were not significantly different from non-stimulated ones at all concentrations of CII. All values of CII-stimulated samples of C2{zeta}-transduced KLH-LNC, except that labelled with an asterisk, were significantly higher than non-stimulated ones (two-tailed Student’s t-test; P < 0.05).

 
Flow cytometry
The expression of cell-surface proteins in KLH-LNC or purified CD4+ T cells was analysed by two- or three-colour flow cytometry after staining with various combinations of the following fluorochrome-conjugated mAb: FITC-labeled GK1.5 (anti-CD4–FITC; Becton Dickinson), phycoerythrin (PE)-labeled H129.19 (anti-CD4–PE; Becton Dickinson), CyChrome-labeled H129.19 (anti-CD4–CyChrome; Becton Dickinson), CyChrome-labeled 17A2 (anti-CD3–CyChrome; Becton Dickinson), FITC-labeled 7D4 (anti-CD25–FITC; Becton Dickinson) and PE-labeled PC61.5.3 (anti-CD25–PE; Serotec, Oxford, UK). The percentages of B and CD8+ T cells were determined by staining with rat anti-{kappa} chain mAb OX20 (Becton Dickinson) or rat anti-mouse CD8 mAb 53.6.72-14 (22), using the phycoerythrin-conjugated F(ab)2 fragment of goat anti-rat IgG (Serotec) as a second-stage stain. Fluorochrome- and isotype-matched rat IgG (ImmunoKontakt, Abingdon, UK) was used as a negative control. All specific and control antibodies were used at a saturating concentration (5–10 µg/ml).

To obtain antibodies recognizing the ectodomains of C2{gamma} and C2{zeta}, polyclonal antiserum against the CII-specific mouse mAb C2 was raised by immunizing a rabbit with the whole molecule of this mAb. IgG was purified from this anti-serum using Protein G–Sepharose (Pharmacia, St Albans, UK) and digested with papain (Boehringer Mannheim, Lewes, UK). Anti-mAb C2 Fab was separated from Fc by affinity chromatography on Protein G–Sepharose and biotinylated. Double- and triple-staining of KLH-LNC was carried out using subset-specific mAb together with this biotinylated anti-mAb C2 Fab or with biotinylated normal rabbit Fab (Stratech Scientific, Luton, UK) as a negative control. Both Fab were used at a concentration of 25 µg/ml. Streptavidin–PE (SAPE; Serotec) was used as the second-stage stain. The samples were pretreated with 5% normal rat and rabbit serum (Stratech Scientific) prior to incubation with Fab and mAb.

The Th1/Th2 composition of CD4+ T cells was analyzed by flow cytometry. Cells were stimulated with a combination of 20 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma) and 1 µM ionomycin (Sigma) or with an anti-CD3{epsilon} mAb (145-2C11; Becton Dickinson) immobilized on plastic at an optimal concentration of 10 µg/ml determined by titration. The duration of stimulation was 4 and 6 h respectively with the addition of 10 µg/ml Brefeldin A (Sigma) for the last 2 and 4 h respectively. Cells were washed in PBS, fixed for 20 min at room temperature in 4% paraformaldehyde (Sigma) in PBS, and washed in PBS and 0.1% BSA. Cells were then permeabilized with 0.5% saponin (Sigma) in PBS, 0.1% BSA and 5% normal rat serum. After 5 min, the combination of PE-conjugated anti-IL-4 (11B11; Becton Dickinson) and allophycocyanin-conjugated anti-IFN-{gamma} (XMG1.2; Becton Dickinson) diluted in PBS, 0.1% BSA, 0.5% saponin and 5% normal rat serum was added, so that the final concentration of each mAb was 10 µg/ml. Following incubation for 20 min at room temperature, cells were washed in PBS, fixed for 15 min in 1% paraformaldehyde in PBS, and suspended in PBS and 0.01% sodium azide.

Flow cytometry was performed on a FACSCalibur using CellQuest software (Becton Dickinson).

Functional assays
KLH-LNC were stimulated with KLH, CII or plastic-immobilized anti-CD3 for 24 h, or as indicated. CII was purified from bovine articular cartilage (23) and used attached to the wells of a flat-bottom plate or added to culture medium. Stimulation of cells with KLH or CII was carried out in the presence or in the absence of mitomycin C-treated splenocytes (MCS). MCS were DBA/1 splenocytes treated with 50 µg/ml of mitomycin C (BDH, Loughborough, UK) for 30 min at 37°C and washed 5 times in 50 ml of culture medium at 4°C. In some experiments, KLH-LNC were treated with inhibitors of intracellular signalling molecules, including cyclosporin, SB203580 and bisindolylmaleimide I hydrochloride (Calbiochem, Nottingham, UK) or with mCTLA-4–Ig (R & D, Abingdon, UK) that were added to the cells 30 min prior to stimulation with KLH.

Changes in cellular expression of recombinant proteins and CD25 in response to stimulation were determined by flow cytometry. The concentration of murine cytokines IL-4 and IFN-{gamma} in supernatants of stimulated cells was determined by ELISA using BVD4-1D11 and R4-6A2 mAb respectively for coating, and biotinylated BVD6-2492 and XMG1.2 mAb respectively for detection (all from Becton Dickinson). Serial dilutions of recombinant mouse IL-4 and IFN-{gamma} (both from PeproTech) were used as standards. The detection limit of these assays was 30 pg/ml. To determine DNA synthesis, 1 µCi [3H]thymidine (Amersham International, Little Chalfont, UK)/well of a 96-well plate was added and its uptake was measured 5 h later.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of CD4+ T cells with MHC-unrestricted specificity to CII
The composition of KLH-LNC was determined after transduction by flow cytometry using subset-specific mAb. These cells contained 60–80% of activated, presumably KLH-specific, CD4+ T cells (dot-plots in Fig. 1), 10–30% of B cells (CD3CD4IgG {kappa} chain+), 5–10% of CD8+ T cells (CD3+CD4CD8+) and 5–10% of quiescent CD4+ T cells (FSClowSSClowCD4low) without specificity to KLH. As retrovirus vector was used for gene transfer, the highest frequency of transduced cells was among activated CD4+ T and B cells, subsets with the highest level of cell division. As T and B cells have similar susceptibility to retrovirus vector-mediated gene transfer (20), their ratio among transduced cells reflected the T:B cell ratio in the KLH-LNC samples, with CD4+ T cells constituting ~70% of all transduced cells, and remaining cells being mostly B cells. CD4low and CD8+ T cells expressing recombinant genes constituted <2% of all engineered cells.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1. Expression of retrovirally transduced genes in CD4+ T cells. (A) C2{gamma} and C2{zeta} expression. KLH-LNC were transduced with the genes of C2{gamma} or C2{zeta}. On the third day post-transduction, they were triple-stained with anti-CD4–FITC, anti-CD3–CyChrome and biotinylated anti-C2 IgG Fab followed by SAPE, and analysed by flow cytometry. Activated CD4+ T cells were electronically gated as shown on the dot-blots on the basis of their light scatter characteristics (R1), and expression of CD4 and CD3 (CD4+ T cells). The histograms show C2{gamma} or C2{zeta} expression in activated CD4+ T cells. The markers on the histograms were set so that M1 included at least 95% of cells in the samples of C2-transduced KLH-LNC incubated with normal rabbit IgG Fab (transduced cells + control Fab). The background staining, which is the percentage of cells in M2 in the samples of non-transduced KLH-LNC incubated with rabbit anti-C2 IgG Fab (non-transduced cells + anti-C2 Fab), was 17%. The numbers on the histograms represent the percentage of cells in M2 in the samples of KLH-LNC transduced with C2{gamma} or C2{zeta} and incubated with rabbit anti-C2 IgG Fab (transduced cells + anti-C2 Fab). (B) The increase in the percentage of apparently C2+ cells in KLH-LNC samples transduced with C2{gamma} or C2{zeta}, or non-transduced when these samples were incubated with increasing concentrations of anti-C2 IgG Fab. The values plotted on the graph were obtained by flow cytometry analysis of non-transduced (control) or C2{gamma}- or C2{zeta}-transduced KLH-LNC double-stained with anti-CD4–CyChrome and biotinylated anti-C2 IgG Fab followed by SAPE. (C) The expression of the control gene eGFP. On the third day after transduction with the eGFP gene, KLH-LNC were incubated with anti-CD3–CyChrome mAb and anti-CD4–PE. The histogram shows eGFP expression in activated CD4+ T cells electronically gated on the basis of their light scatter characteristics and expression of CD4 and CD3.

 
The monovalent IgG Fab used for detection of C2 chimera expression produced non-specific staining of non-transduced KLH-LNC that could not be completely eliminated by titration without significantly reducing a specific signal (graph in Fig. 1A). The concentration of 25 µg/ml was chosen as the working concentration for our experiments because it gave optimal separation between specific and non-specific signals. As determined by flow cytometry using this concentration of anti-mAb C2 Fab in seven independent experiments, the expression levels of C2{gamma} and C2{zeta} in CD4+ T cells were respectively 14–19 and 27–55% after subtraction of the background staining (9–17%).

Ligand-induced down-regulation of C2{zeta} in genetically modified CD4+ T cells
Cell-surface expression of the chimeric immunoreceptor C2{zeta} in transduced CD4+ T cells decreased upon simulation with its ligand CII (Fig. 2). The decrease resulted from C2{zeta} ligation, but not from cell activation as such, because it did not occur when the cells were stimulated with their cognate antigen KLH in the presence of MCS. The expression of the control protein eGFP was not changed by stimulation with CII, arguing against an unlikely possibility that CII inhibited recombinant gene expression (Fig. 2). C2{zeta} expression was completely inhibited in wells coated with 10 µg/ml CII (Fig. 2). C2{gamma} also appeared to be down-regulated, but this was difficult to verify due to a low level of its expression.



View larger version (68K):
[in this window]
[in a new window]
 
Fig. 2. Decrease in the expression of C2{zeta} in CD4+ T cells upon binding to CII. KLH-LNC transduced with C2{zeta} or eGFP were stimulated with CII immobilized on plastic at indicated concentrations or with 100 µg/ml KLH in culture medium, or cultured without stimulation. After 24 h, the eGFP-transduced cells were incubated with anti-CD4–CyChrome, and the C2{zeta}-transduced cells were double-stained with anti-CD4–CyChrome and biotinylated anti-C2 IgG Fab followed by SAPE. Expression levels of recombinant genes and CD4 were analysed by flow cytometry after electronically gating activated cells on the basis of their light scatter characteristics (analogous to R1 in Fig. 1A). The regions C2+ and C2 were set so that the C2+ region in the sample of control non-transduced KLH-LNC incubated with anti-C2 (not shown) included 5% of CD4+ T cells. Only some of the dot-plots are shown. The graphs demonstrate dependency of recombinant protein expression on CII concentration, as well as the inability of KLH to change this expression.

 
Since C2{zeta} is down-regulated following its cross-linking with CII, one would expect an increase in the expression of activation markers, such as the IL-2 receptor {alpha} subunit (CD25), in the C2{zeta} subset of CD4+ T cells after stimulation with plastic-immobilized CII. If C2{zeta}-transduced KLH-LNC were left for 4 days in the presence of IL-2, but without TCR- or C2{zeta}-dependent stimulation, CD25 was expressed in 58.7% of their C2{zeta} subpopulation of CD4+ T cells (Fig. 3, non-stimulated). Some of these samples were stimulated with plastic-immobilized CII or anti-CD3 during the last 24 h of the 4-day culture period. In response to CII, the expression of CD25 increased, in a dose-dependent manner, in the C2{zeta} subpopulation of CD4+ T cells in these samples, but not in the eGFP subpopulation of CD4+ T cells in KLH-LNC samples transduced with eGFP (Fig. 3). Stimulation with anti-CD3 induced CD25 expression in all C2{zeta} CD4+ T cells (Fig. 3, anti-CD3).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3. Up-regulation of IL-2R in C2{zeta}-transduced CD4+ T cells upon stimulation with CII. On the third day after transduction with eGFP or C2{zeta}, KLH-LNC were stimulated with plastic-immobilized anti-CD3 or CII for 1 day without MCS. The eGFP-transduced cells were double-stained with anti-CD4–CyChrome and anti-CD25–PE. The C2{zeta}-expressing cells were triple-stained with anti-CD4–CyChrome, anti-CD25–FITC and biotinylated anti-C2 IgG Fab followed by SAPE. The cells were analysed by flow cytometry. Transduced and non-transduced activated CD4+ T cells were electronically gated on the basis of their light scatter characteristics and expression of CD4 and recombinant gene (similar to gaiting in Fig. 2). The histograms show the expression of CD25 in activated CD4+ T cells not expressing eGFP or C2{zeta} (denoted as eGFP or C2{zeta} in Fig. 2). The markers were set so that M2 included 95% of cells that were stained with anti-CD25–PE or anti-CD25–FITC after stimulation with anti-CD3. The numbers show percentage of cells within the markers M2 after incubation with anti-CD25–PE or anti-CD25–FITC. Unfilled histograms in the top row show results of flow cytometry analysis of cells incubated with isotype control antibodies for anti-CD25–PE or anti-CD25–FITC. The graph shows dependency of CD25 expression in CD4+ T cells on the concentration of CII.

 
ITAM-dependent signals induced by cross-linking of chimeric immunoreceptors cannot integrate with co-stimulatory signals
Upon stimulation with KLH, KLH-LNC secreted both IFN-{gamma} and IL-4, the prototype cytokines of Th1 and Th2 cells respectively. We assume that KLH-specific CD4+ T cells are the only producers of IFN-{gamma} and IL-4 among KLH-LNC, because B cells are known to be strictly dependent on an exogenous source of these cytokines for antibody production (24); and CD8+ and CD4low T cells (25) in these samples are predominantly bystander cells without specificity to KLH (as suggested by their quiescent phenotype), resistance to retrovirus vector-mediated gene transfer and small numbers.

Producers of IL-4 and IFN-{gamma} among CD4+ T cells were highly segregated, as demonstrated by flow cytometry of purified CD4+ T cells after double staining with anti-IL-4 and anti-IFN-{gamma} mAb (Fig. 4). Thus, if these CD4+ T cells were stimulated with plastic-immobilized anti-CD3 2 days after transduction, single cytokine producers constituted 94.8% of all cells capable of producing IFN-{gamma} and/or IL-4 (Fig. 4, CD4+, day 2). The ratio of the number of cells producing only IFN-{gamma} to the number of cells producing only IL-4 (Th1:Th2 ratio) was 0.62 at this time point. As revealed using the same mode of stimulation (anti-CD3) on day 7 post-transduction, the percentage of single cytokine-producing cells remained unchanged (94%), but the Th1:Th2 ratio (3.1) became 5 times higher (Fig. 4, CD4+, day 7). If on day 7 after transduction, instead of anti-CD3, a combination of PMA + ionomycin was used for cell stimulation, a comparable percentage of polarized cells and Th1:Th2 ratio was observed (91.2% and 2.3).



View larger version (74K):
[in this window]
[in a new window]
 
Fig. 4. Polarization of Th1 and Th2 cytokine production in KLH-specific CD4+ T cells. CD4+ T cells were purified from KLH-LNC using magnetic beads after re-activation with KLH in vitro for 3 days. In order to determine their purity, these CD4+ T cells were analysed by flow cytometry after staining with anti-CD4–PE or a PE-conjugated isotype-control mAb (respectively solid and dotted lines on the histogram). The numbers on the histogram are the percentages of cells in M2 after staining with anti-CD4–PE. The cells were stimulated with IL-2 for 1 day, transduced with eGFP or left non-transduced and stimulated with IL-2 for an additional 2 or 7 days. They were then stimulated with plastic-immobilized anti-CD3 or PMA + ionomycin, double-stained with anti-IFN-{gamma}–allophycocyanin and anti-IL-4–PE, and analysed by flow cytometry. Dot-plots in the top row show electronic gaiting of eGFP-expressing CD4+ T cells. Other dot-plots show the expression of intracellular IFN-{gamma} and IL-4 in the whole-cell population (CD4+) and selectively in the transduced population of cells (eGFP+CD4+).

 
The percentage of Th1/Th2 polarized cells and Th1:Th2 ratio determined by the analysis of electronically gated eGFP-expressing CD4+ T cells (Fig. 4, eGFP+CD4+) on the second day after transduction was roughly the same as in the whole population of CD4+ T cells (94% and 0.46). However, the Th1:Th2 ratio in the subset of eGFP+CD4+ T cells did not increase as much as in the whole population of CD4+ T cells, constituting by day 7 post-transduction 0.85 and 0.79, as revealed by stimulation with anti-CD3 and PMA + ionomycin respectively. There was a slight decrease in the percentage of polarized cells among cytokine-producing cells in the transduced subset, which at this time point constituted 84.7 and 82.8% after stimulation with anti-CD3 and PMA + ionomycin respectively. Therefore, the Th1/Th2 composition of CD4+ T cells susceptible to retrovirus vector-mediated gene transfer remains more stable with time in culture than the composition of the CD4+ T cell population as a whole.

When KLH-LNC were stimulated with KLH in the absence of MCS, KLH-specific CD4+ T cells produced IFN-{gamma} and IL-4, presumably relying on the presentation of antigen by KLH-specific B cells. This stimulation, however, was suboptimal and the cytokine production further increased upon addition of MCS (Fig. 5A). In contrast, cell proliferation in response to KLH stimulation was independent of MCS (Fig. 6A). MCS may promote cytokine production of CD4+ T cells by increasing the density of cognate antigenic peptides. Additionally, the observation that cytokine production and cell division have different requirements for MCS (Fig. 5A and 6A) points towards a possibility that MCS may also provide unique co-stimulatory signals capable of selectively promoting production of effector cytokines. These signals are not provided by ‘endogenous’ B cells. Whatever co-stimulation is provided to CD4+ T cells by MCS, it is not mediated by CD28 because CTLA-4–Ig only marginally inhibited antigen-induced production of IL-4 and did not affect the production of IFN-{gamma} at all (Fig. 5 B).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 5. The effect of MCS on antigen-induced cytokine production in CD4+ T cells. (A) MCS promote cytokine production of CD4+ T cells. KLH-LNC were stimulated with KLH for 3 days and with IL-2 for 3 days, washed, and plated at indicated concentrations alone, or with MCS, or with KLH, or with MCS and KLH together. After 1 day, the concentration of IFN-{gamma} and IL-4 in their culture supernatants was measured by ELISA. (Top row) MCS were added at the constant concentration of 2 x 106/ml and KLH-LNC were titrated. Results are presented as mean ± SEM of triplicates. (Bottom row) KLH-LNC were plated at the constant concentration of 2.5 x 105/ml and MCS were titrated. Results are presented as mean of duplicates. (B) The effect of MCS is independent of CD28. KLH-LNC were stimulated with KLH in the presence of indicated concentrations of mCTLA-4–Ig, and the concentration of IFN-{gamma} and IL-4 in their culture supernatants was measured by ELISA. Results are presented as mean ± SEM of triplicates. Statistically significant difference (two-tailed Student’s t-test; P < 0.05) from samples not treated with mCTLA4-Ig is labelled with an asterisk.

 
Cross-linking C2{gamma} and C2{zeta} with plastic-immobilized CII initiates the cascade of ITAM-dependent signalling events. To elucidate whether or not these events could, in trans, integrate with co-stimulation provided by MCS, transduced KLH-LNC were stimulated with CII with or without MCS. In response to stimulation with CII, CD4+ T cells transduced with a chimeric immunoreceptor, but not control eGFP-expressing cells, secreted IFN-{gamma} and, inconsistently, IL-4 (Fig. 7). The inclusion of MCS during stimulation with CII only marginally increased the cytokine production of C2{zeta}-expressing CD4+ T cells and did not affect the cytokine production of C2{gamma}-expressing CD4+ T cells (Fig. 7, experiment 2). Thus, when CD4+ T cells receive an ITAM-dependent signal through the chimeric immunoreceptor, they cannot efficiently integrate this signal with co-stimulatory signals.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 7. Cytokine secretion of C2 chimera-expressing CD4+ T cells in response to CII. In experiment 1, KLH-LNC transduced with C2{gamma}, C2{zeta} or eGFP were stimulated with KLH or with CII bound to plastic at indicated concentrations at a cell density of 2 x 106/ml. Cytokine concentration in their culture supernatants was determined by ELISA after 1 day. Results are shown as mean of duplicates. In experiment 2, KLH-LNC transduced with C2{gamma}, C2{zeta} or eGFP were stimulated with KLH or with CII bound to plastic at indicated concentrations at a cell density of 2.5 x 106/ml. The stimulation was carried out in the presence or absence of MCS. Cytokine concentration in culture supernatants was determined by ELISA after 1 day. Results are shown as mean ± SEM of triplicates. SEM bars are not shown where they are smaller than the symbol. P values are shown for conditions where the difference between cells cultured with and without MCS was statistically significant (two-tailed Students t-test; P < 0.05).

 
The proliferative response of C2{zeta}-expressing cells stimulated with CII could not be further increased by inclusion of MCS (Fig. 6B), but antigen-induced proliferation of KLH-LNC was also independent of MCS (Fig. 6A), suggesting that ITAM-mediated signalling on its own may be sufficient for optimal cell proliferation.

A higher capacity of C2 chimeras to induce effector cytokine secretion in Th1 cells than in Th2 cells
Th1 and Th2 cells exhibited a remarkable similarity in the susceptibility to retrovirus vector-mediated gene transfer (Fig. 8). On the second day post-transduction, Th1 and Th2 subsets contained equivalent numbers of eGFP-expressing cells. Compared to the levels of gene expression in Th1 and Th2 subsets, the efficiency of gene transfer into subsets of non-Th1 and non-Th2 cells was slightly lower. The proportion of transduced cells in all the subsets remained relatively unchanged by day 7 post-transduction (Fig. 8).



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 8. Similar levels of recombinant gene expression in Th1 and Th2 cells. CD4+ T cells were purified from KLH-LNC (96% of cells were CD4+ after purification) and transduced with the gene of eGFP or left non-transduced. After 2 or 7 days post-transduction, the cells were stimulated with anti-CD3 absorbed on plastic at a concentration of 10 µg/ml or with a combination of 20 ng/ml PMA + 1 µM ionomycin. Intracellular cytokines were determined by flow cytometry after double staining cells with anti-IL-4–PE and anti-IFN-{gamma}–allophycocyanin. Only some of the dot-blots used for calculating the frequency of eGFP-transduced cells among Th1, Th2, non-Th1 and non-Th2 subsets are shown. Quadrants of the dot-plots are denoted as a, b, c and d, as shown on the diagram. The frequency of eGFP+ cells in Th1 or Th2 subset was calculated using the formula: [(cell number in quadrant c)/(cell number in quadrant a + cell number in quadrant c)] x 100. The frequency of eGFP+ cells in non-Th1 or non-Th2 subset was calculated using the formula: [(cell number in quadrant d)/(cell number in quadrant b + cell number in quadrant d)] x 100.

 
We next compared the capacity of the chimeric immunoreceptors to induce effector cytokine expression in Th1 and Th2 cells. As a point of reference for this comparison we used levels of IFN-{gamma} and IL-4 induced by antigenic stimulation. In order to completely exclude the contribution of cells other than CD4+ T cells to the cytokine production, KLH-specific CD4+ T cells were purified from KLH-LNC before transduction. Cytokine production was induced by stimulation with CII through a chimeric immunoreceptor or with KLH through the endogenous TCR under optimal conditions. The levels of cytokines measured in culture supernatants were normalized by the number of producer cells using formulas presented in Table 1. For the purpose of this comparison, all IFN-{gamma}-producing cells, including a small subset of cells producing both cytokines, are referred to as Th1 cells. Similarly, all IL-4 producers are denoted as Th2 cells. Normalized IFN-{gamma} levels induced by stimulation through C2{gamma} or C2{zeta} were similar to the levels induced by stimulation through the endogenous TCR. By contrast, IL-4 production of C2{gamma}- and C2{zeta}-expressing CD4+ T cells in response to CII stimulation was lower than IL-4 production of KLH-specific CD4+ T cells stimulated with KLH (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Production of cytokines by primary Th cells in response to stimulation through the recombinant immunoreceptor or endogenous TCR
 
Thus, the recombinant immunoreceptors C2{gamma} and C2{zeta} seem to have a greater capacity to induce production of effector cytokines when they are expressed in Th1 cells than when they are expressed in Th2 cells. This conclusion is in agreement with results of a series of previous experiments, including those presented in Fig. 7, where KLH-LNC were used instead of purified KLH-specific CD4+ T cells. In these experiments, antigen-induced and chimera-induced production of cytokines was normalized respectively by the number of CD4+ T cells or by the number of C2 chimera-expressing cells. The ratio between TCR- and C2{gamma}-dependent production of IFN-{gamma} was 1 ± 0.12 (mean ± SE, n = 3 experiments); the ratio between TCR- and C2{zeta}-dependent production of IFN-{gamma} was 1.06 ± 0.09 (mean ± SE, n = 5 experiments). The ratio between TCR- and C2{gamma}-dependent production of IL-4 was 2.41 in one experiment and in two other experiments no detectable IL-4 production was observed when C2{gamma}-expressing KLH-LNC were stimulated with CII. The ratio between TCR- and C2{zeta}-dependent production of IL-4 was 11.06 ± 7.51 (mean ± SE, n = 3) and in two other experiments no detectable IL-4 production was observed when C2{zeta}-expressing KLH-LNC were stimulated with CII. Comparable levels of IL-4 were produced by KLH-LNC in response to antigen in all these experiments.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TCR expression on the surface of T cells decreases during antigenic stimulation as a result of internalization. This decrease is a direct consequence of TCR ligation and does not require co-stimulation or intracellular signalling (26). The extent of this decrease positively correlates with the affinity of TCR binding to its cognate antigenic peptide–MHC complex (27) and functional responses of antigenically stimulated T cells, in turn, positively correlate with the extent of TCR down-regulation (28). In an early study, Valitutti et al. (29) measured TCR down-regulation to determine the proportion of TCR engaged by a ligand.

The expression of C2{zeta} on the surface of genetically modified CD4+ T cells decreased, in a concentration-dependent manner upon binding CII. Complete down-regulation of the immunoreceptor was observed when CII was attached to plastic at a concentration >=10 µg/ml, suggesting that at these concentrations all cell-surface-expressed C2{zeta} was engaged by CII. A submaximum engagement of the immunoreceptors, however, was sufficient to induce production of IFN-{gamma} at a level comparable to that induced by antigenic stimulation. In contrast, the level of IL-4 production mediated by C2{gamma} or C2{zeta} remained low, compared to the TCR-mediated production of this cytokine, even at those concentrations of CII which induced a complete down-regulation of chimeric immunoreceptor expression on the cell surface. Thus, it appears that effector functions of Th1 cells can be stimulated through the chimeric immunoreceptors more efficiently than effector functions of Th2 cells.

ITAM-containing fusion proteins, some of which were prototypes of the recombinant immunoreceptors C2{gamma} and C2{zeta}, were used to establish the non-redundant role of ITAM in TCR-mediated cell activation (3036). Clustering of ITAM-containing signalling subunits of the TCR complex induced by binding of TCR clonotypic subunits to a cognate antigenic peptide–MHC complex initiates a number of biochemical events ultimately leading to the assembly of a multiprotein complex, in which proteins with signalling domains are recruited directly or by virtue of adapter proteins. Together with this primary antigenic stimulus, T cells may also receive co-stimulatory signals that are transmitted through some of their cell-surface molecules, such as CD28, and may affect the outcome of antigenic stimulation. The two signalling pathways integrate by sharing some molecular components (37,38). It is unclear whether or not these co-stimulatory signals can be integrated with ITAM-dependent signalling in T cells stimulated through ITAM-containing chimeric immunoreceptors.

The failure of MCS to enhance cytokine production of CD4+ T cells stimulated through the chimeric immunoreceptors argues against a possibility that such integration of co-stimulatory and ITAM-dependent signalling occurs. This suggests a possible explanation of the decreased capability of chimeric immunoreceptors to induce IL-4 production in CD4+ T cells. Thus, the IL-4 response may be more stringently dependent on co-stimulation than the production of IFN-{gamma}. Therefore, the maximum attainable level of C2{zeta} cross-linking, which results in a complete internalization of the immunoreceptor, may still be suboptimal for IL-4 production, although the ITAM-dependent signal generated thereby is sufficient for a strong IFN-{gamma} response.

The nature of the MCS-dependent co-stimulatory signal that may be required for optimal production of effector cytokines, especially IL-4, remains unclear. We, however, excluded CD28 as a molecule mediating such signal by demonstrating that the blockade of the CD28-dependent pathway does not significantly inhibit IL-4 production. This confirms the earlier reports that CD28-mediated co-stimulation is not involved in IL-4 production of antigen-stimulated mouse effector CD4+ T cells (39). Some co-stimulatory receptors other than CD28 are known to have the capability to modulate the production of Th1 and Th2 effector cytokines (4045). They may be involved in the co-stimulation provided by MCS to KLH-specific CD4+ T cells. Additionally, CD4 co-receptor, which is thought to be important for Th2 responses (46), may not be recruited during the stimulation of CD4+ T cells through the chimeric immunoreceptor.

Secondary messengers and transcription factors have been implicated in controlling differential cytokine expression in Th1 and Th2 cells. Thus, Vav selectively activates the IL-4 promotor (47) and Itk plays a non-redundant role in modulating signals from the TCR–CD28 pathways that are specific for the establishment of stable IL-4, but not IFN-{gamma}, expression (48). Furthermore, Th1 and Th2 cells may differentially regulate the Ca2+ and K+ channel-dependent movement of Ca2+ ions (49,50), a biochemical event that plays an important role in the activation of calcineurin and protein kinase C (PKC) in antigen-activated CD4+ T cells (51).

We reason that identification of a signalling pathway that is involved in the production of IL-4, but not IFN-{gamma}, would suggest the experimentally testable hypothesis that this particular pathway is not activated during stimulation of engineered cells through a chimeric immunoreceptor. We used inhibitors of some well-studied signalling pathways in order to address the question of whether or not any of these pathways were differentially involved in the induction of Th1 and Th2 effector cytokines. To investigate whether or not Th1 and Th2 cells differed in their dependency on signalling mediated by calcineurin and PKC, these molecules were blocked with their respective inhibitors, cyclosporin A and bisindolylmaleimide I, in KLH-specific CD4+ T cells stimulated with KLH in the presence of MCS. The production of both IFN-{gamma} and IL-4 was inhibited by either treatment with similar IC50, which was 10 nM and 1 µM for cyclosporin A and bisindolylmaleimide respectively (not shown). SB203580, an inhibitor of another signalling molecule implicated in differential cytokine production in CD4+ T cells, p38 MAPK, tested over a range of concentrations from 0.1 to 10 µM, failed to inhibit the production of either cytokine by antigenically stimulated KLH-specific CD4+ T cells (not shown). As no difference between IFN-{gamma} and IL-4 production in the dependency on calcineurin, PKC or p38 MAPK was found in these experiments, a failure to engage any of these signalling pathways can be excluded as a possible explanation of the reduced capability of the chimeric immunoreceptors to mediate IL-4 production.

Several issues are to be considered regarding the development of anti-inflammatory gene therapy based on CD4+ T cells targeted to CII. First, expression levels of C2{zeta} were always higher than those of C2{gamma}. This cannot be explained by the different efficiency of transduction or staining, because a saturating concentration of the retroviral stock determined by preliminary titration was used, and binding of anti-C2 Fab, a monovalent reagent, could not be affected by the difference in valency between C2{gamma} and C2{zeta} (18,19). C2{zeta} may be more efficiently transported to the plasma membrane, because it contains the cytoplasmic and transmembrane domains of TCR{zeta} known to facilitate the transportation of other TCR–CD3 subunits to the cell surface (52). For this reason, C2{zeta} may be more suitable for therapeutic application. Secondly, it is unlikely that other leukocytes and resident cells expressing ligands for co-stimulatory receptors may enhance activation of C2 chimera-expressing CD4+ T cells when these cells encounter CII in the joints. Thirdly, the low levels of IL-4 secretion induced by stimulation of Th2 cells through C2{gamma} and C2{zeta} is a drawback because the production of this cytokine in inflamed joints is likely to be therapeutically beneficial. Perhaps, this could be overcome by combining an ITAM-containing immunoreceptor with an appropriate molecular module from co-stimulatory pathways by virtue of genetic engineering (53). Another approach to engineering therapeutic T cells that we are currently exploiting is a concomitant expression in T cells of two recombinant genes, of which one encodes for a tissue antigen-specific immunoreceptor and the other for IL-4 or another anti-inflammatory protein.


    Acknowledgements
 
This work was funded by the Arthritis Research Campaign, Multiple Sclerosis Society and Wellcome Trust, UK.


    Abbreviations
 
CII—type II collagen

eGFP—enhanced green fluorescent protein

ITAM—immunoreceptor tyrosine-based activation motif

KLH—keyhole limpet hemocyanin

KLH-LNC—lymph node cells from KLH-immunized mice

MCS—mitomycin C-treated splenocytes

PE—phycoerythrin

PKC—protein kinase C

PMA—phorbol 12-myristate 13-acetate

SAPE—streptavidin–phycoerythrin

scFv—single-chain IgG variable domain


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25). J. Immunol. 155:1151.[Abstract]
  2. Powrie, F., Correa-Oliveira, R., Mauze, S. and Coffman, R. L. 1994. Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity. J. Exp. Med. 179:589.[Abstract]
  3. Akhtar, I., Gold, J. P., Pan, L. Y., Ferrara, J. L., Yang, X. D., Kim, J. I. and Tan, K. N. 1995. CD4+ ß islet cell-reactive T cell clones that suppress autoimmune diabetes in nonobese diabetic mice. J. Exp. Med. 182:87.[Abstract]
  4. Chen, L. Z., Hochwald, G. M., Huang, C., Dakin, G., Tao, H., Cheng, C., Simmons, W. J., Dranoff, G. and Thorbecke, G. J. 1998. Gene therapy in allergic encephalomyelitis using myelin basic protein-specific T cells engineered to express latent transforming growth factor-ß1. Proc. Natl Acad. Sci. USA 95:12516.[Abstract/Free Full Text]
  5. Gua, D. J., Hinton, D. R. and Stohlman, S. A. 1995. Self-antigen-induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice. J. Immunol. 155:4052.[Abstract]
  6. Kuchroo, V. K., Das, M. P., Brown, J. A., Ranger, A. M., Zamvil, S. S., Sobel, R. A., Weiner, H. L., Nabavi, N. and Glimcher, L. H. 1995. B7-1 and B7-2 costimulatory molecules activate differentially the Th1/Th2 developmental pathways: application to autoimmune disease therapy. Cell 80:707.[ISI][Medline]
  7. Tan, K. N., Min, H. K., Pan, L., Ferrara, J. L. and Myrick, K. V. 1996. Biological characteristics of an immunoregulatory activity secreted by an autoreactive CD4+ T cell clone that suppresses autoimmune diabetes in non-obese diabetic mice. Int. Immunol. 8:689.[Abstract]
  8. Chernajovsky, Y., Adams, G., Podhajcer, O. L., Mueller, G. M., Robbins, P. D. and Feldmann, M. 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.[ISI][Medline]
  9. Chernajovsky, Y., Adams, G., Triantaphyllopoulos, K., Ledda, M. F. and Podhajcer, O. L. 1997. Pathogenic lymphoid cells engineered to express TGF ß1 ameliorate disease in a collagen-induced arthritis model. Gene Ther. 4:553.[CrossRef][ISI][Medline]
  10. Mageed, R. A., Adams, G., Woodrow, D., Podhajcer, O. L. and Chernajovsky, Y. 1998. Prevention of collagen-induced arthritis by gene delivery of soluble p75 tumour necrosis factor receptor. Gene Ther. 5:1584.[CrossRef][ISI][Medline]
  11. Nakajima, A., Seroogy, C. M., Sandora, M. R., Tarner, I. H., Costa, G. L., Taylor-Edwards, C., Bachmann, M. H., Contag, C. H. and Fathman, C. G. 2001. Antigen-specific T cell-mediated gene therapy in collagen-induced arthritis. J. Clin. Invest. 107:1293.[Abstract/Free Full Text]
  12. Dreja, H., Annenkov, A. and Chernajovsky, Y. 2000. Soluble complement receptor 1 (CD35) delivered by retrovirally infected syngeneic cells or by naked DNA injection prevents the progression of collagen-induced arthritis. Arthritis Rheum. 43:1698.[CrossRef][ISI][Medline]
  13. Moritani, M., Yoshimoto, K., Li, S., Kondo, M., Iwahana, H., Yamaoka, T., Sano, T., Nakano, N., Kikutani, H. and Itakura, M. 1996. Prevention of adoptively transferred diabetes in nonobese diabetic mice with IL-10-transduced islet-specific Th1 lymphocytes. J. Clin. Invest. 98:1851.[Abstract/Free Full Text]
  14. Shaw, M. K., Lorens, J. B., Dhawan, A., DalCanto, R., Tse, H. Y., Tran, A. B., Bonpane, C., Eswaran, S. L., Brocke, S., Sarvetnick, N., Steinman, L., Nolan, G. P. and Fathman, C. G. 1997. Local delivery of interleukin 4 by retrovirus-transduced T lymphocytes ameliorates experimental autoimmune encephalomyelitis. J. Exp. Med. 185:1711.[Abstract/Free Full Text]
  15. Mathisen, P. M., Yu, M., Johnson, J. M., Drazba, J. A. and Tuohy, V. K. 1997. Treatment of experimental autoimmune encephalomyelitis with genetically modified memory T cells. J. Exp. Med. 186:159.[Abstract/Free Full Text]
  16. Kramer, R., Zhang, Y., Gehrmann, J., Gold, R., Thoenen, H. and Wekerle, H. 1995. Gene transfer through the blood-nerve barrier: NGF-engineered neuritogenic T lymphocytes attenuate experimental autoimmune neuritis. Nat. Med. 1:1162.[ISI][Medline]
  17. Reth, M. 1989. Antigen receptor tail clue. Nature 338:383.[ISI][Medline]
  18. Annenkov, A. E., Moyes, S. P., Eshhar, Z., Mageed, R. A. and Chernajovsky, Y. 1998. 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} subunit. J. Immunol. 161:6604.[Abstract/Free Full Text]
  19. Annenkov, A. and Chernajovsky, Y. 2000. Engineering mouse T lymphocytes specific to type II collagen by transduction with a chimeric receptor consisting of a single chain Fv and TCR {zeta}. Gene Ther. 7:714.[CrossRef][ISI][Medline]
  20. Annenkov, A. E., Daly, G. M. and Chernajovsky, Y. 2002. Highly efficient gene transfer into antigen-specific primary mouse lymphocytes with replication-deficient retrovirus expressing the 10A1 envelope protein. J. Gene Med. 4:133.[CrossRef][ISI][Medline]
  21. Holmdahl, R., Rubin, K., Klareskog, L., Larsson, E. and Wigzell, H. 1986. Characterization of the antibody response in mice with type II collagen-induced arthritis, using monoclonal ant-type II collagen antibodies. Arthritis Rheum. 29:400.[ISI][Medline]
  22. Ledbetter, J. A. and Herzenberg, L. A. 1979. Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol. Rev. 47:63.[ISI][Medline]
  23. Miller, E. J. 1972. Structural studies on cartilage collagen employing limited cleavage and solubilization with pepsin. Biochemistry 11:4903.[ISI][Medline]
  24. Ho, I. C., Hodge, M. R., Rooney, J. W. and Glimcher, L. H. 1996. The proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4. Cell 85:973.[ISI][Medline]
  25. Costa, G. L., Benson, J. M., Seroogy, C. M., Achacoso, P., Fathman, C. G. and Nolan, G. P. 2000. Targeting rare populations of murine antigen-specific T lymphocytes by retroviral transduction for potential application in gene therapy for autoimmune disease. J. Immunol. 164:3581.[Abstract/Free Full Text]
  26. Cai, Z., Kishimoto, H., Brunmark, A., Jackson, M. R., Peterson, P. A. and Sprent, J. 1997. Requirements for peptide-induced T cell receptor downregulation on naive CD8+ T cells. J. Exp. Med. 185:641.[Abstract/Free Full Text]
  27. Itoh, Y., Hemmer, B., Martin, R. and Germain, R. N. 1999. Serial TCR engagement and down-modulation by peptide:MHC molecule ligands: relationship to the quality of individual TCR signaling events. J. Immunol. 162:2073.[Abstract/Free Full Text]
  28. Hemmer, B., Stefanova, I., Vergelli, M., Germain, R. N. and Martin, R. 1998. Relationships among TCR ligand potency, thresholds for effector function elicitation, and the quality of early signaling events in human T cells. J. Immunol. 160:5807.[Abstract/Free Full Text]
  29. Valitutti, S., Muller, S., Cella, M., Padovan, E. and Lanzavecchia, A. 1995. Serial triggering of many T-cell receptors by a few peptide–MHC complexes. Nature 375:148.[CrossRef][ISI][Medline]
  30. Letourneur, F. and Klausner, R. D. 1991. T-cell and basophil activation through the cytoplasmic tail of the T-cell-receptor {zeta} family protein. Proc. Natl Acad. Sci. USA 88:8905.[Abstract]
  31. Irving, B. A. and Weiss, A. 1991. The cytoplasmic domain of the Fc receptor {zeta} chain is sufficient to couple to receptor-associated signal transduction pathway. Cell 64:891.[ISI][Medline]
  32. Vivier, E., Rochet, N., Ackerly, M., Petrini, J., Levine, H., Daley, J. and Anderson, P. 1992. Signalling functions of reconstituted CD16:{zeta}:{gamma} receptor complex isoforms. Int. Immunol. 4:1313.[Abstract]
  33. Eshhar, Z., Waks, T., Gross, G. and Schindler, D. G. 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]
  34. Brocker, T. and Karjalainen, K. 1995. Signals through T cell receptor-{zeta} chain alone are insufficient to prime resting T lymphocytes. J. Exp. Med. 181:1653.[Abstract]
  35. Romeo, C., Amiot, M. and Seed, B. 1992. Sequence requirements for induction of cytolysis by the T cell antigen/Fc receptor {zeta} chain. Cell 68:889.[ISI][Medline]
  36. Brocker, T., Peter, A., Traunecker, A. and Karjalainen, K. 1993. New simplified molecular design for functional T cell receptor. Eur. J. Immunol. 23:1435.[ISI][Medline]
  37. Pages, F., Ragueneau, M., Rottapel, R., Truneh, A., Nunes, J., Imbert, J. and Olive, D. 1994. Binding of phosphatidylinositol-3-OH kinase to CD28 is required for T-cell signalling. Nature 369:327.[CrossRef][ISI][Medline]
  38. King, P. D., Sadra, A., Teng, J. M., Xiao-Rong, L., Han, A., Selvakumar, A., August, A. and Dupont, B. 1997. Analysis of CD28 cytoplasmic tail tyrosine residues as regulators and substrates for the protein tyrosine kinases, EMT and LCK. J. Immunol. 158:580.[Abstract]
  39. Schafer, P. H., Wadsworth, S. A., Wang, L. and Siekierka, J. J. 1999. p38{alpha} mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells. J. Immunol. 162:7110.[Abstract/Free Full Text]
  40. Gonzalo, J.-A., Delaney, T., Corcoran, J., Goodearl, A., Gutierrez-Ramos, J. C. and Coyle, A. J. 2001. Cutting edge: the related molecules CD28 and inducible costimulator deliver both unique and complementary signals required for optimal T cell activation. J. Immunol. 166:1.[Abstract/Free Full Text]
  41. Riley, J. L., Blair, P. J., Musser, J. T., Abe, R., Tezuka, K., Tsuji, T. and June, C. H. 2001. ICOS costimulation requires IL-2 and can be prevented by CTLA-4 engagement. J. Immunol. 166:4943.[Abstract/Free Full Text]
  42. Tamada, K., Shimozaki, K., Chapoval, A. I., Zhai, Y., Su, J., Chen, S.-F., Hsieh, S.-L., Nagata, S., Ni, J. and Chen, L. 2000. LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response. J. Immunol. 164:4105.[Abstract/Free Full Text]
  43. Tamura, H., Dong, H., Zhu, G., Sica, G. L., Flies, D. B., Tamada, K. and Chen, L. 2001. B7-H1 costimulation preferentially enhances CD28-independent T-helper cell function. Blood 97:1809.[Abstract/Free Full Text]
  44. Tseng, S.-Y., Otsuji, M., Gorski, K., Huang, X., Slansky, J. E., Pai, S. I., Shalabi, A., Shin, T., Pardoll, D. M. and Tsuchiya, H. 2001. B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells. J. Exp. Med. 193:839.[Abstract/Free Full Text]
  45. Wang, S., Zhu, G., Chapoval, A. I., Dong, H., Tamada, K., Ni, J. and Chen, L. 2000. Costimulation of T cells by B7-H2, a B7-like molecule that binds ICOS. Blood 96:2808.[Abstract/Free Full Text]
  46. Yamashita, M., Hashimoto, K., Kimura, M., Kubo, M., Tada, T. and Nakayama, T. 1998. Requirement for p56lck tyrosine kinase activation in Th subset differentiation. Int. Immunol. 10:577.[Abstract]
  47. Hehner, S. P., Li-Weber, M., Giaisi, M., Droge, W., Krammer, P. H. and Schmitz, M. L. 2000. Vav synergizes with protein kinase C{theta} to mediate IL-4 gene expression in response to CD28 costimulation in T cells. J. Immunol. 164:3829.[Abstract/Free Full Text]
  48. Fowell, D. J., Shinkai, K., Liao, X. C., Beebe, A. M., Coffman, R. L., Littman, D. R. and Locksley, R. M. 1999. Impaired NFATc translocation and failure of Th2 development in Itk-deficient CD4+ T cells. Immunity 11:399.[ISI][Medline]
  49. Fanger, C. M., Neben, A. L. and Cahalan, M. D. 2000. Differential Ca2+ influx, KCa channel activity, and Ca2+ clearance distinguish Th1 and Th2 lymphocytes. J. Immunol. 164:1153.[Abstract/Free Full Text]
  50. Sloan-Lancaster, J., Steinberg, T. and Allen, P. 1997. Selective loss of the calcium ion signaling pathway in T cells maturing toward a T helper 2 phenotype. J. Immunol. 159:1160.[Abstract]
  51. Crabtree, G. R. and Clipstone, N. A. 1994. Signal transmission between the plasma membrane and nucleus of T lymphocytes. Annu. Rev. Biochem. 63:1045.[CrossRef][ISI][Medline]
  52. Geisler, C. 1992. Failure to synthesize the CD3-{gamma} chain. Consequences for T cell antigen receptor assembly, processing, and expression. J. Immunol. 148:2437.[Abstract/Free Full Text]
  53. Geiger, T. L., Nguyen, P., Leitenberg, D. and Flavell, R. A. 2001. Integrated src kinase and costimulatory activity enhances signal transduction through single-chain chimeric receptors in T lymphocytes. Blood 98:2364.[Abstract/Free Full Text]




This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Request Permissions
Google Scholar
Articles by Annenkov, A. E.
Articles by Chernajovsky, Y.
PubMed
PubMed Citation
Articles by Annenkov, A. E.
Articles by Chernajovsky, Y.