Generation of antigen-specific cytotoxic T lymphocytes and regulation of cytokine production takes place in the absence of CD3{zeta}

Jian She1, Melanie C. Ruzek2, Palanivel Velupillai3, Isabel de Aos1,4, Baoping Wang1, Donald A. Harn3, Jaime Sancho4, Christine A. Biron2 and Cox Terhorst1

1 Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
2 Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02912, USA
3 Department of Tropical Public Health, Harvard School of Public Health, Boston, MA 02215, USA
4 Department of Cellular Biology and Immunology, Instituto de Parasitologia y Biomedicina, Consejo Superior de Investigaciones Cientificas (CSIC), Granada, Spain

Correspondence to: C. Terhorst


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The TCR-associated protein CD3{zeta} plays a major role in regulating the state of responsiveness to peptide–MHC complexes on the surface of antigen-presenting cells. In this paper the requirement of CD3{zeta} in the generation of cytotoxic T cells was compared with its requirement in cytokine gene activation in two mutant mice: ZKO mice with a disrupted CD3{zeta} gene and ZTG mice in which a truncated CD3{zeta} segment was expressed as a transgene on the ZKO background. Upon infection of ZTG mice with lymphocytic choriomeningitis virus (LCMV), antigen-specific cytotoxic T lymphocyte (CTL) responses were detected, identical to responses in wild-type mice. In addition, antigen-specific CTL responses to allogeneic class I and class II MHC in ZTG animals were indistinguishable from those in wild-type animals. However, CTL responses to the same major antigens were not detectable in ZKO mice. We conclude that the signal transduction pathways leading to CTL development and cytokine production can be triggered through TCR in the absence of functional CD3{zeta}, provided the remainder of the TCR–CD3 complex is expressed at high levels on the cell surface. Surprisingly, IFN-{gamma} production in response to LCMV followed the same kinetics in ZKO, ZTG and wild-type mice. However, in vitro studies showed that cytokine production in general was abnormally regulated in T lymphocytes from ZKO mice, in contrast to ZTG T cells. Taken together, these studies support the hypothesis that development of CTL can take place in the absence of functional CD3{zeta}. However, CTL development requires stronger TCR-initiated signal transduction events than induction of cytokine genes.

Keywords: cytokines, cytotoxicity, infectious immunity virus, TCR, transgenic/knockout


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Signals transduced by the TCR regulate the early development and selection of thymocytes. The signal-transducing potential of the TCR complex is conferred by multiple subunits (the CD3{gamma}, {delta}, {varepsilon} and {zeta} or Fc{varepsilon}RI{gamma} chain) (13) that share a conserved structural element, the immunoreceptor tyrosine-based activation motif (ITAM) (4). Whereas CD3{gamma}, {delta} and {varepsilon} each contain a single ITAM, CD3{zeta} contains three ITAM within its cytoplasmic tail. ITAM sequences are conserved but non-identical, and the induced individual motifs perform in part unique and in part equivalent functions (58). It has been suggested that ITAM, through their ability to facilitate activation of cytoplasmic protein tyrosine kinases (PTK), mediate all known TCR effector functions (9,10). After TCR engagement, phosphorylation of ITAM leads to the recruitment of SH2 domain-containing proteins to the TCR complex and initiation of the T cell activation cascade (1114).

The first level PTK, c-fyn (15) or c-lck (12,16), cause phosphorylation of CD3{gamma}, {delta}, {varepsilon} (17) and/or {zeta} (18). The tandem PTK, ZAP70 (11,19) or Syk (2025), bind to the phosphorylated ITAM and phosphorylate a key membrane-associated protein LAT (26). A number of downstream signaling molecules such as Grb2 (27), which connect with Vav (28,29), SLP-76 (30,31), Cbl (32) and SOS (33), or phospholipase C (PLC)-{gamma} (34) and/or the p85 subunit (35) of phosphoinositide 3-kinase bind to phosphorylated LAT. Recruitment of these and other signaling molecules lead to T cell proliferation, or activation-induced cell death or induction of cytokine genes. Although it has been suggested that the CD3{zeta} chain may function as the predominant TCR signaling structure and that its triple ITAM may serve as a control switch for TCR signal amplification (2,3642), CD3{gamma}, {delta} or {varepsilon} can in principle initiate the same signal transduction pathways (43).

Disruption of the CD3{zeta} gene in CD3{zeta}/{eta}null (ZKO) mice (4447) results in only a partial block in T cell development, suggesting that at least part of the functions of CD3{zeta} can be taken over by the other CD3 proteins. Love et al. (7) recently showed that efficient positive selection is dependent upon the presence of all these CD3{zeta} ITAM. T cells from CD3{zeta}/{eta}null:tg{zeta}{Delta}67-150 (ZTG) mice, which are ZKO mice in which a truncated form of CD3{zeta} is introduced as a transgene (48), lack all three {zeta} chain ITAM but express high levels of TCR on the surface. In these animals positive selection of thymocytes has been proven to be inefficient as judged by crossing the ZTG with transgenic mice of a specific TCR {alpha}ß pair (7). In fact efficient positive selection takes place only if all three {zeta} ITAM are present. Nonetheless ZTG and ZKO mice have large numbers of peripheral CD4+ and CD8+ T lymphocytes in their spleens and lymph nodes, probably due to expansion in the periphery (4951).

In the current paper these ZTG and ZKO peripheral T lymphocytes were used to examine the functional role of {gamma}, {delta} and {varepsilon} in the absence of the CD3{zeta} ITAM. ZTG and ZKO mice were used to study the generation of lymphocytic choriomeningitis virus (LCMV) and alloantigen-specific cytotoxic T cells, and for their ability to regulate induction of cytokine genes. Surprisingly, in ZTG mice signal transduction pathways leading to cytotoxic T lymphocyte (CTL) development and cytokine production can be triggered through TCR in the absence of functional CD3{zeta}. Although CTL were not generated in ZKO mice, their peripheral T cells still produced IFN-{gamma} in response to LCMV following the same kinetics as wild-type mice. In vitro studies showed that cytokine production in ZTG T lymphocytes was regulated in a normal fashion, but was abnormal in ZKO mice. These studies therefore support the hypothesis that development of CTL and regulation of induction of cytokine genes can take place in the absence of functional CD3{zeta}. These results lend further support to the notion that the CD3 are of critical importance during thymocyte development but are redundant in the peripheral T cell.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
CD3{zeta}/{eta}null (ZKO) mice, which were generated and maintained at the Beth Israel Deaconess Medical Center Animal Facility on a C57BL/6x129/Sv background (45), were used in all our experiments. A breeding pair of CD3{zeta}/{eta}null and CD3{zeta}/{eta}null:tg{zeta}{Delta}67-150 (ZTG) mice was kindly provided by Dr Paul E. Love (National Institutes of Health, Bethesda MD) (48). ZTG mice were bred on either an H-2b/b or H-2b/d background. Conventional wild-type DBA/2NTacfBR, C57BL/6NTacfBR and 129/SvEvTacfBR mice were purchased from Taconic Laboratory (Germantown, NY). Where indicated, mice were either uninfected or infected i.p. with LCMV, Armstrong strain, clone E350, on day 0 for 6, 8 or 12 days.

mAb
The following antibodies directed at murine cell surface markers were used in this study: anti-CD16/32 (2.4G2, ATCC), anti-TCR{alpha}ß (H57-597), anti-CD3{varepsilon} (145-2C11, 500A2), anti-B220 (RA3-6B2), anti-CD4 (RM4-5), anti-CD8{alpha} (53-6.7), anti-CD11b (Mac-1, M1/70), anti-CD25 (7D4), anti-CD28 (37.51), anti-CD69 (H1.2F3), anti-CTLA-4 (UC10-4F10-11), anti-Ly-6G (Gr-1, RB6-8C5), anti-IFN-{gamma} (XMG1.2) (52), rat anti-mouse IL-4R (Genzyme, Cambridge, MA) and Red-670-conjugated streptavidin (Gibco/BRL, Grand Island, NY), affinity-purified goat anti-hamster IgG (Cappel, Durham, NC), agarose-conjugated anti-phosphotyrosine, 4G10 (Upstate Biotechnology, Lake Placid, NY), affinity-purified rabbit anti-human CD3{varepsilon} antibody (Dako, Carpinteria, CA), purified rabbit anti-ZAP-70 and agarose-conjugated rabbit anti-ZAP-70 (Santa Cruz, Santa Cruz, CA), and horseradish peroxidase-conjugated anti-phosphotyrosine (PY-Plus) (Zymed, San Francisco, CA). Antibodies were purchased from PharMingen (San Diego, CA) unless otherwise indicated.

Cell preparations and purification
Single-cell suspensions of splenic and lymph node cells were isolated. Cells were either used from individual mice or pooled together from 10 mice of each group, and red blood cells were lysed. Cell yields and viability were determined using Trypan blue exclusion. For T lymphocyte purification, B cells, monocytes and granulocytes were depleted from single-cell suspensions by incubating with biotinylated anti-CD11b and anti-Ly-6G mAb at 10 µg/1x107 cells in 100 µl DMEM (Gibco/BRL) medium for 15 min on ice, washing once in PBS, and incubating with a mixed solution of super-paramagnetic microbeads conjugated with anti-B220 mAb and streptavidin (Miltenyi Biotec, Sunnyvale, CA) each at 10 µl/1x107 cells in 100 µl DMEM medium for 15 min on ice. After a final washing, the T lymphocytes were purified by passage through a high gradient MACS separator (53). Samples of purified cells were stained with anti-CD4–FITC and anti-CD8{alpha}–phycoerythrin (PE) mAb, and purity determined by FACS analysis. In our experience, this protocol leads to cell suspensions containing >96% T lymphocytes in wild-type, ZTG mice and >70% T lymphocytes in ZKO mice.

Flow cytometric analysis
For cell surface phenotypic analysis, freshly isolated, purified T lymphocytes and cultured T lymphocytes were stained with mAb as specified. Three color immunofluorescence analyses were performed with PE, FITC and Red-670 fluorochrome-conjugated mAb. A total of 0.5–1x106 cells was incubated with the biotinylated antibody at 5 µg/ml in PBS plus 1% BSA for 20 min on ice, washed once in PBS/2% FBS, and then incubated with both the PE- and FITC-conjugated antibodies as well as Red-670-conjugated streptavidin at 5 µg/ml in PBS plus 1% BSA (Sigma, St Louis, MO) for 20 min on ice. After two washes in PBS plus 2% FBS, cells were resuspended in 100 µl PBS on ice. Flow cytometry was immediately performed with a FACScan (Becton Dickinson, San Jose, CA). Appropriate fluorochrome-conjugated isotype-matched Ig were used as negative controls. For each color, a single-positive control was used in all experiments. To block Fc receptor-mediated binding of the mAb, cell culture supernatants from the 2.4G2 hybridoma were added to cells and incubated for 20 min before antibody staining. For each sample 20,000 events were collected. Data were analyzed on a computer with WinList (Verity Software House, Topsham, ME) or CellQuest (Becton Dickinson) programs.

Intracellular staining for IFN-{gamma}
Methods used for intracellular staining of IFN-{gamma} were described previously (54,55). In experiments with freshly isolated populations, cells were resuspended at 1x106 cells/ml in media containing 10% FBS and stimulated with immobilized anti-CD3{varepsilon} for a total of 6 h in LCMV-treated experiments or 48 h in non-LCMV-treated experiments, with Brefeldin A (Sigma) added to 5 µg/ml during the last 2 h of incubation. Cells were collected and washed in cold PBS containing 0.5% BSA and 0.006% NaN3. Single-cell suspensions were surface stained as indicated above with FITC-conjugated and biotinylated antibodies, plus streptavidin–Red-670 or streptavidin–Per-CP (Becton Dickinson). After cell surface staining, cells were washed and fixed with 1–2% paraformaldehyde-PBS for 30 min After additional washes, cells were permeabilized with 0.25–1% saponin (Sigma) in PBS plus 2% FBS or 0.5% BSA. Cells were incubated in permeabilization buffer with PE-conjugated anti-IFN-{gamma} mAb for 20 min To verify specificity for cytokine, staining with PE-conjugated anti-IFN-{gamma} was subjected to blocking with purified IFN-{gamma} prior to and throughout incubation with cells. Isotype-matched Ig were used as negative controls. After final washes, flow cytometry analyses were immediately performed with a FACScan (Becton Dickinson).

T cells activation, immunoprecipitation and Western blotting
Analyses were performed as described with modifications (45). Briefly, purified T lymphocytes were stimulated at 1x107 cells/100 µl by treatment with anti-CD3{varepsilon} mAb at 10 µg for various periods of time at 37°C with shaking (Eppendorf, Thermomixer 5436) or by incubation with anti-CD3{varepsilon} mAb at 5 µg (1x107 cells/100 µl) for 2 min and antibody cross-linking with goat anti-hamster IgG at 10 µg (1x107 cells/100 µl) for 3 min at 37°C with shaking. At termination of antibody stimulation, cells were washed with PBS with 1 mM PMSF or not washed respectively, and lysed in 1 ml of 1% NP-40 lysis buffer (150 mM NaCl, 10 mM triethanolamine, 1 mM EGTA, 10.8 mM iodoacetamide, 1 mM PMSF, 1 µg of leupeptin, pepstatin and antipain, 50 mM sodium fluoride, and 1 mM sodium orthovanadate; Sigma). Lysates were clarified by centrifugation at 14,000 r.p.m. in a microcentrifuge (Eppendorf, Model 5415C) for 20 min at 4°C. CD3{varepsilon} and ZAP-70 proteins were then isolated by immunoprecipitation as previously described (18). Proteins were separated by electrophoresis on 12.5% SDS–polyacrylamide gels, followed by transfer to PVDF membranes (Immobilon-P; Millipore, Bedford, MA) in 48 mM Tris, 39 mM glycine, 0.0375% (w/v) SDS and 20% methanol. Filters were blocked by incubation for 1 h in blocking buffer (5% BSA) dissolved in TNA (10 mM, Tris, pH 7.2, 0.9% NaCl and 0.05% NaN3). Tyrosine-phosphorylated proteins were detected by reaction with horseradish peroxidase-conjugated anti-phosphotyrosine (PY-Plus; Zymed) (dilution 1:40,000). For CD3{varepsilon} and ZAP-70 Western blot, affinity-purified rabbit anti-human CD3{varepsilon} antibody (dilution 1:1000; Dako) and purified rabbit anti-ZAP-70 antibody (dilution 1:1000; Santa Cruz) were used respectively. Detection of labeled proteins was carried out with an ECL protocol (Amersham, Little Chalfont, UK).

Determination of the cytokine production by lymphocytes
T lymphocytes (2x106 cells/ml, normalized for equivalent numbers of CD4+ and CD8+ cells) from ZKO, ZTG and/or wild-type mice were cultured at 37°C in 96-well U-type plates in the presence of 10 µg IgG/ml (30 µl/well) of the indicated mAb or 1 µg/ml superantigen. The mAb of anti-CD3{varepsilon} and anti-TCRß were coated on plates (4°C overnight), and of anti-CD28 and anti-CTLA-4 were added to the culture medium. To block consumption of IL-2 and IL-4, 60 µg/ml anti-IL-2R (PharMingen) or 10 µg/ml anti-IL-4R (Genzyme) mAb was added in the culture medium respectively. To block cytokine production, cyclosporine A (CsA; 10 µM; Sandoz, East Hanover, NJ) was added to culture medium. Supernatant fluids were harvested 48 h after the initiation of cultures and diluted (1:4) in PBS/10% FBS. The cells were used for intracellular staining for cytokines. For generation of media conditioned with freshly isolated splenic leukocytes from infection experiments, 107 cells/ml were incubated in 12-well cluster plates at 37°C without exogenous stimulation and supernatant fluids were harvested after 24 h.

A two-site capture ELISA for IL-10 was performed as described (56). In brief, polystyrene microtiter plates (Costar, Cambridge, MA) were coated with IL-10-specific mAb (JES5-2A5; PharMingen) at 2 µg/ml (50 µl/well) overnight at 4°C, blocked and washed. Culture supernatants were then added to wells for capture of IL-10, followed by addition of biotinylated anti-IL-10 mAb (SXC-1; PharMingen). Bound secondary antibody was detected by addition of avidin–peroxidase conjugate (Sigma) followed by tetramethyl-benzidine substrate (Kirkegaard & Perry, Bethesda, MD). The reaction was terminated by addition of phosphoric acid (0.4 M) to each well after which absorbance was measured at 450 nm in a UVMax automated plate reader (Molecular Devices, Menlo Park, CA). Serial dilutions of recombinant IL-10 (PharMingen) were included to construct a standard curve for relative IL-10 concentration.

IL-2, IL-4 and IFN-{gamma} also were detected by a standard sandwich ELISA. ELISA plates were coated with anti-IL-2 (S4B6) or anti-IL-4 (11B11) (PharMingen), at 2 µg/ml (50 µl/well) overnight at 4°C. Wells were washed and blocked with PBS/10% FBS, then incubated overnight with culture supernatant fluids and serial dilutions of recombinant IL-2 or IL-4 (PharMingen). Plates were washed with PBS/0.05% Tween 20, followed by addition of biotinylated anti-IL-2 (JES6-5H4) or anti-IL-4 (BVD6-24G2) (PharMingen). Finally, the plates were incubated with avidin–peroxidase and developed with substrate as described for IL-10. IFN-{gamma} was detected in two different ELISA protocols. The first used capture (R4-6A2) and biotinylated detection (XMG1.2); anti-IFN-{gamma} mAb were from PharMingen and recombinant standard IFN-{gamma} (R & D Systems, Minneapolis, MI) as described above. The second was carried out as previously described (57). It used the XMG1.2 antibody (provided as the hybridoma line by Dr Robert Coffman, DNAX, Palo Alto, CA) for cytokine capture and a polyclonal rabbit anti-IFN-{gamma} (a gift from Dr Phillip Scott, University of Pennsylvania, Philadelphia, PA) for detection. Peroxidase-conjugated donkey anti-rabbit antibodies were purchased from Jackson ImmunoResearch (West Grove, PA) for use as the tertiary reagent. Recombinant IFN-{gamma}, purchased from PharMingen, was the standard for this assay, and the limit of detection for this assay was 40 pg/ml.

Mixed lymphocyte cultures (MLC) and mixed lymphocyte reactions (MLR)
Splenic leukocytes were collected from H-2b/b and H-2d/d mice. Cells (4x106 cells/ml) from the H-2b/b mice (wild-type, ZTG and ZKO) were used as effector populations and mixed with equivalent numbers of stimulator cells from H-2d/d mice. The MLC were incubated in complete RPMI 1640 medium for 5 days before assays. The supernatant fluids were collected and analyzed for IFN-{gamma} by ELISA, and cells were collected and used for cytotoxicity assays.

Cytotoxicity assays.
Target cells labeled with 51Cr (DuPont-New England Nuclear, Boston, MA) were incubated with lymphocyte effector cells for 4.5–5 h at 37°C in 96-well microtiter plates as described (57,58). Spontaneous release of 51Cr was determined by incubating target cells with medium, whereas maximum release was determined by adding 1% Nonidet P-40 to target cells. In all experiments spontaneous lysis was <15% of maximum release. Percentage lysis was calculated as: 100x(c.p.m. test sample supernatant – c.p.m. spontaneous release)/(c.p.m. maximum release – c.p.m. spontaneous release). For MLR-specific lytic activity, cytotoxicity was determined as the difference between the lysis of P815 and EL-4 target cells in a 4 h 51Cr-release assay. The percentage of specific lysis was normalized to represent equal amounts of T cells per target ratios. E:T ratios were 35:1, 12:1, 4:1 and 1.3:1. LCMV-specific lytic activity was determined with cells freshly isolated from infected mice as the difference between the lysis of infected and uninfected histocompatible target cells. Target cells, NIH 3T3 (H-2d), MC57G (H-2b) and L929 (H-2k), were either uninfected or infected with LCMV at a 0.01 m.o.i. 2 days prior to use. E:T of 200:1, 100:1, 33:1, 11:1 and 3:1 were examined. All assays were performed in quadruplicate and replicate samples had SD of <10% of the mean. Samples were quantitated in either a Wallac Microbeta1 1450 Counter (Wallac, Gaithersburg, MD) following the manufacturer's suggested protocol or a Isoflex Automatic Gamma Counter (ICN Micromedic Systems, Huntsville, AL). Calculations and statistical analysis of the data was done used the Excel program (Microsoft, Redmond, WA).

Determination of the Ca2+ response
Thymocytes, splenic leukocytes and lymph node cells were incubated in complete DMEM medium containing 1% FBS (5x106 cells/ml) at 37°C for 45 min in the presence of 5 µg/ml Indo-1 penta-acetoxymethyl ester and 0.3% pluronic F-127 (Molecular Probes, Eugene, OR), with gentle mixing of the cells a couple of times during the incubation period. Dye-loaded cells were washed and resuspended (2x107 cell/ml), and splenic leukocytes and lymph node cells were stained with dialysis of anti-B220–PE antibody (20 µg/ml) and thymocytes were stained with dialysis of anti-CD8{alpha}–PE antibody (20 µg/ml). The cells were washed and resuspended in ice-cold medium of choice at 1.5x106 cells/ml for analysis after gating with B220 cells of splenic leukocytes and lymph node cells or with CD8{alpha}+ cells of thymocytes. The dye-loaded cells fluorescence intensity was measured by a FACStar machine. As the base lines were stabilized, cells were added to with an anti-CD3{varepsilon} mAb (145-2C11, 20 µg/ml; PharMingen) plus goat anti-hamster IgG (60 µg/ml; Cappel). Maximum fluorescence was determined by adding 5 µg/ml ionomycin plus PMA.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of CTL in mice lacking the signal transduction elements of CD3{zeta}
To characterize the function of CD3{zeta} in antigen-specific responses, the abilities of ZTG and ZKO mice to mount CTL responses were evaluated during infections with LCMV. Animals were infected i.p. with 2x104 p.f.u. LCMV, Armstrong strain, clone 350 and splenic CTL responses were examined on day 8 after infection, a time associated with high level CTL induction in wild-type mice. As shown in Fig. 1Go(A), ZTG mice, reconstituted with the truncated CD3{zeta} chain, developed strong virus-specific CTL responses and these were comparable to those seen in the wild-type mice. Because CTL could only be detected against LCMV-infected targets of the same MHC haplotype as ZTG animals (H-2dxH-2b), i.e. 3T3 or MC57G, but not H-2k, i.e. L929, the responses were MHC restricted. In contrast to cells isolated from infected ZTG mice, cells from LCMV-infected ZKO mice displayed no detectable specific cytotoxic activity against virus-infected target cells expressing either matched, i.e. the H-2b positive MC57G target cells, or un-matched, i.e. H-2d positive 3T3 or H-2k positive L929 target cells, MHC molecules. To ascertain if CTL responses were present with altered kinetics of induction in ZKO mice, virus-specific CTL activity was evaluated in ZKO and wild-type C57BL/6 mice on days 6, 8 and 12 after infection. In contrast to the wild-type mice, with a positive cytotoxic response beginning at day 6, peaking at day 8 and subsiding by day 12 after infection, virus-specific cytotoxic activity was not observed at any time point after LCMV infection of ZKO mice (Fig. 1BGo).




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Fig. 1. Induction of LCMV-specific CTL in ZTG and wild-type mice, but not in ZKO mice. (A) Splenic leukocytes were isolated from uninfected mice or from mice that were infected with LCMV for 8 days. Because the ZTG mice (circle) were H-2b/d and ZKO mice (triangle) were H-2b/b, therefore, 129/Sv (square, H-2b/b) and DBA (diamond, H-2d/d) were used as wild-type controls. CTL activity against uninfected (broken lines) or LCMV-infected (solid lines) MC57G (H-2b), NIH-3T3 (H-2d) and L929 (H-2k) target cells was determined in a 5 h 51Cr-release assay, as described in Methods. Specific lysis of virus-infected as compared to uninfected cells is presented. The data represent the means ± SD of two to three mice per group. The results are representative of three independent experiments. (B) Splenic cells were isolated from uninfected mice or from mice infected with LCMV for 0 (square), 6 (diamond), 8 (circle) or 12 (triangle) days. CTL activity was assayed as described in (A) using uninfected or LCMV-infected MC57G target cells. Data presented are specific lysis of virus-infected target cells and represented as means ± SD of three mice per group.

 
The above results beg the question as to whether, in the absence of measurable CTL responses, other parameters of T cell activation occurred in response to the LCMV virus. To examine this, the potential of T cells to produce IFN-{gamma}, a cytokine classically associated with LCMV responses in mice, was tested. Measurements of IFN-{gamma} secretion using an intracellular staining technique were carried out on purified CD4+ and CD8+ T cells harvested from ZKO, ZTG or wild-type mice on days 0, 6, 8 and 12 post-infection with LCMV (59). As shown in Fig. 2Go, significant levels of IFN-{gamma} production from both subsets were detected. Measured cytokine levels from ZKO T cells were slightly lower than those detected in wild-type 129/Sv T cells and significantly lower than IFN-{gamma} production generated by C57BL/6 wild-type T cells. However, these data reveal that ZKO T cells were capable of responding to infection and therefore to LCMV antigens by induction of IFN-{gamma} production.



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Fig. 2. Intracellular staining for IFN-{gamma} production in T lymphocytes from LCMV-infected mice. C57BL/6, 129/Sv and ZKO mice were infected with LCMV for 0, 6, 8 or 12 days, activated splenic cells were collected and intracellular staining with anti-IFN-{gamma} mAb was conducted as described in Methods. The mean value and standard error of percentage of IFN-{gamma} expression in CD4 or CD8 T lymphocytes were calculated after three-color analysis in a cytofluometer (FACScan).

 
Although the data suggested that high expression of the TCR–CD3 complex was required for CTL induction, other factors, such as ineffective positive selection or small frequencies of LCMV-specific precursor T cells in the ZKO mice, could have contributed to the lack of responses. As an independent measurement of CTL induction with a large pool of responsive precursor T lymphocytes, allogeneic CTL responses were examined. Cells from ZKO and ZTG mice were set up in MLR against MHC-allogeneic stimulator cells. As shown in Fig. 3Go, ZTG CTL responses were comparable to those of wild-type mice. Again, none of the cultures with cells from ZKO animals developed significant allo-specific responses to H-2d-bearing targets after in vitro stimulation with BALB/c (H-2d) splenic leukocytes. Furthermore, CTL responses from ZKO mice could not be increased by in vivo priming animals with BALB/c splenic leukocytes prior to in vitro stimulation. Collectively, these results clearly show that antigen-specific CTL functions can develop in the absence of the signal transduction elements or ITAM of CD3{zeta}. However, in order to generate significant CTL responses, T lymphocytes require high levels of cell surface expression of the TCR–CD3 complex lacking functional CD3{zeta}.



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Fig. 3. Allogeneic CTL can be generated by ZTG, but not by ZKO T lymphocytes. Splenocytes (2x106/ml) from C57BL/6, ZTG (H-2b/b) and ZKO mice were cultured with irradiated splenocytes (2x106/ml) of BALB/c (H-2d/d) mice for 5 days. For the CTL response, cytotoxicity was measured in a 4 h 51Cr-release assay using P815 target cells. The percentage of specific lysis has been normalized to represent equal amounts of T cell per target ratios. (A) Allogeneic MLC were set up with splenocytes from two C57BL/6 (solid line), five ZTG (broken line) or 14 ZKO (dotted line) mice and irradiated BALB/c splenocytes. (B) Before the MLC, three ZKO mice were first injected i.p. with irradiated splenocytes (1x107 per mouse) from BALB/c mice. This was repeated once more after 10 days.

 
Cytokine genes are induced in T cells lacking CD3{zeta}
Since the CD3{zeta} ITAM appear to be critical regulators of T cell responsiveness, the ability to induce the IFN-{gamma} gene in the absence of the CD3{zeta} gene prompted us to examine regulation of these cytokine genes in greater detail. The CD3{varepsilon} expression of ZTG and ZKO mice differs dramatically as shown in Fig. 4Go(A) and Table 1Go. Therefore, all cells used in the following experiments had been enriched by negative selection with super-paramagnetic microbeads (see Methods). Intracellular staining for IFN-{gamma} showed that a small but significant percentage of CD4 and CD8 cells from ZTG and wild-type mice [on average 3.5 and 2.7% respectively (n = 12)] could be induced in vitro by anti-CD3 antibodies to express this cytokine (Fig. 4BGo). In contrast, as shown in a representative experiment in Fig. 4, Goa much higher percentage of IFN-{gamma}-producing cells was detected in cultures derived from ZKO mice than in cultures derived from ZTG or wild-type mice. On average 12.3% of CD4 and 15.1% of CD8 cells from ZKO mice (n = 12) expressed IFN-{gamma}. Since similar results were obtained upon stimulation with anti-TCR, or the superantigens staphylococcal enterotoxin (SE) A or B (data not shown), we conclude that IFN-{gamma} production in ZTG mice was regulated normally, but that in ZKO T lymphocytes this cytokine gene was activated in an abnormal fashion upon engagement of the TCR–CD3 complex.




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Fig. 4. The expression of CD3{varepsilon} and IFN-{gamma} production by CD4 and CD8 T lymphocytes. (A) Splenocytes from C57BL/6, ZTG and ZKO mice were stained for CD3{varepsilon} mAb. Results are represented as histograms of log10 fluorescence against relative cell number. (B) Splenocytes from C57BL/6, ZTG and ZKO mice were cultured for 48 h with 10 µg/ml immobilized anti-CD3{varepsilon} mAb (145-2C11) in a 24-well plate. Activated cells were incubated with blocking anti-Fc{gamma}R mAb supernatant and stained with anti-CD4 and anti-CD8{alpha} mAb, and were then stained for intracellular expression of IFN-{gamma} as described in Methods. Isotype control mAb for IFN-{gamma} were used for negative control. CD4 and CD8 cells were gated by three-color analysis and the percentages of IFN-{gamma}+ cells are indicated in each histogram.

 

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Table 1. The percentage of CD4+ and CD8+ in splenic T lymphocytes of wild-type, ZTG and ZKO mice
 
The in vitro observation that IFN-{gamma} production was extremely high on a per cell basis was compatible with the results of in vivo stimulation of adult ZKO mice with anti-CD3. Ten ZKO mice treated with 10 µg/g body wt of highly purified anti-CD3{varepsilon} (500-A2) (60,61) died within 30 h of injection probably because of high levels of tumor necrosis factor (TNF)-{alpha}/IL-1 (data not shown). ZTG or control animals (C57BL/6x129/Sv) treated in the same fashion with 500-A2 or ZKO mice treated with an isotype-matched IgG did not show this dysregulated response. Based on our in vitro studies, it is likely that the absence of CD3{zeta} resulted in an anti-CD3{varepsilon}-induced, IFN-{gamma}-initiated cascade of events.

As shown in Table 2Go, purified T cells from wild-type, ZTG and ZKO mice produced similar levels of IL-2 in response to in vitro stimulation via anti-CD3{varepsilon}, anti-TCRß or superantigens. All responses were measured in the presence of anti-IL-2R antibody, which blocked the autocrine pathway. Thus, all measurements were independent of the level of surface expression of the IL-2R (see below). In contrast to wild-type or ZTG T cells, T lymphocytes derived from ZKO mice showed only moderate enhancement of IL-2 production by activation of the CD28 co-stimulatory pathway. Incubation of ZKO T cells with anti-CD3{varepsilon} in the presence of anti-CTLA-4 resulted in a similar level of IL-2 production as found with anti-CD3{varepsilon} or anti-TCRß. Again incubation of ZTG or wild-type T cells showed a decrease in IL-2 production by co-engagement of the CTLA-4 and CD3 molecules. These data demonstrated that T cells from ZKO mice produce quantities of IL-2 comparable to those produced by ZTG or wild-type cells. The co-stimulatory molecules CD28 and CTLA-4 barely affected IL-2 production. Although the IL-4 secretion results were similar in T lymphocytes from wild-type, ZTG and ZKO mice (Table 3Go), IL-4 production by ZKO T lymphocytes was somewhat variable between experiments (n = 8). IL-10 secretion was remarkably low in ZKO T cells (10% of wild-type levels) (Table 4Go). Once again, in ZTG mice (data not shown) IL-4 and IL-10 levels were identical to those of wild-type T cells. Cytokine production was always inhibited by CsA suggesting that the calcium–calcineurin pathways were utilized in both ZTG and ZKO T cells.


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Table 2. IL-2 is produced at wild-type levels by activated ZTG and ZKO T lymphocytes
 

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Table 3. IL-4 is produced by activated ZKO T lymphocytes
 

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Table 4. IL-10 is produced by activated ZKO T lymphocytes
 
Taken together, the in vitro data show that in ZTG T cells which lack functional CD3{zeta}, but which express high levels of TCR–CD3, cytokine production was controlled in a normal fashion. The data suggested that regulation of cytokine responses in the ZKO T cells which expressed low levels of TCR were in part abnormal.

Cell surface activation markers are induced in absence of CD3{zeta} ITAM
To further examine the extent of activation of T lymphocytes that lack functional CD3{zeta}, expression of CD25 or CD69 was analyzed in purified T cells cultured for different time periods with anti-TCR–CD3 mAb or superantigens. In all cases ZTG T lymphocytes responded as wild-type T cells as shown for instance in Fig. 5Go(A). After 48 h of incubation with anti-CD3{varepsilon} or TCRß mAb, 36 or 44% (respectively) of T lymphocytes from ZKO mice expressed CD69 and CD25 on their surface (Fig. 5AGo). Similarly, upon stimulation with the superantigen SEA or SEB, 24 or 30% (respectively) of cells from ZKO mice were CD25+CD69+ as compared with 48% (SEA) or 13% (SEB) in wild-type mice. These results demonstrate that T lymphocytes can be activated via the antigen receptor to express activation markers in the absence of functional CD3{zeta} (ZTG T lymphocytes). Moreover, this process did not require high TCR–CD3 surface expression (ZKO T lymphocytes).




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Fig. 5. CD25, CD28, CTLA-4 and CD69 are expressed on the surface of activated T lymphocytes. Splenocytes from C57BL/6, ZTG and ZKO mice were cultured for 48 h with anti-CD3{varepsilon} and anti-TCRß mAb, SEA and SEB superantigens. (A) Electronically gated CD4+ and CD8{alpha}+ (both PE-conjugated mAb) T lymphocytes were analyzed for CD25 and CD69 expression. The percentages of CD25+ and CD69+ cells are indicated in each histogram. (B). CD4 and CD8 cells were gated by three-color analysis, and the percentages of CD28+ and CTLA-4+ cells are indicated.

 
It is interesting to note that in ZTG and wild-type T lymphocytes significantly larger numbers of cells constitutively expressed CD28 and CTLA-4 as compared with those in ZKO T lymphocytes: in ZKO T lymphocytes only 2.8% of CD4+ cells and 1.9% of CD8+ cells were CD28+. Likewise, in these cells only 4.2% of CD4+ cells and 6.4% of CD8+ cells were CTLA-4+ (Fig. 5BGo). These data could explain why anti-CD28 co-stimulation further increased anti-TCR or -CD3 induced IL-2 production in ZTG T lymphocytes and not in ZKO T cells (Table 2Go). Similarly, the absence of CTLA-4 from the surface of ZKO T cells and its presence on the surface of ZTG T cells could explain the differential responses to anti-CTLA-4. Collectively, these data demonstrate that in T lymphocytes lacking functional CD3{zeta} engagement results in induction of activation markers. Absence of cell surface expression of CTLA-4 and CD28 may be the primary causes for aberrant regulation of cytokine production in ZKO T lymphocytes. Perhaps more importantly, in ZTG T lymphocytes cytokine production is regulated in a normal fashion in part because of the correct induction of the CTLA-4/CD28 regulatory module.

CD3{varepsilon} initiates signal transduction in ZKO T lymphocytes
Occupancy of the TCR–CD3 complex causes the initial activation of Fyn and/or Lck, leading to tyrosine phosphorylation of a number of protein substrates, including the invariant subunits of the TCR–CD3 complex (mainly CD3{zeta} and CD3{varepsilon}). This is followed by recruitment of ZAP-70 to the tyrosine phosphorylated CD3 ITAM, and subsequent ZAP-70 tyrosine phosphorylation and activation (1). To examine whether in the absence of CD3{zeta} this cascade could be initiated by CD3{varepsilon}, purified T lymphocytes of wild-type and ZKO mice were stimulated with the anti-CD3{varepsilon} mAb 2C11 followed by cross-linking with a goat anti-hamster IgG. Anti-CD3{varepsilon} immunoprecipitates were then analyzed by immunoblotting with an anti-phosphotyrosine mAb. As shown in Fig. 6Go(A), in T lymphocytes from both ZKO and wild-type mice CD3{varepsilon} resulted in increased tyrosine phosphorylated upon CD3 ligation (Fig. 6AGo, upper panel, lanes 6 and 3 respectively).



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Fig. 6. Tyrosine phosphorylation of CD3{varepsilon} and ZAP-70. Splenic T lymphocytes from wild-type and ZKO mice were purified by negative selection as described in Methods. Purified CD4+ and CD8+ cells from wild-type and ZKO mice were cross-linked at 1x107 cells/ml with CD3 (145–2C11) or/and goat anti-hamster IgG under the indicated time course. The cells were lysed, and immunoprecipitation and Western blots were conducted as described in Methods. (A) Purified CD4+ and CD8+ cells from wild-type and ZKO mice were incubated at 1x107 cells/ml with anti-CD3{varepsilon} mAb (145-2C11) 5 µg/ml, 2 min, and then goat anti-hamster IgG added at 10 µg/ 100 µl PBS, 2 min at 37°C with shaking. The cells were lysed in buffer containing 1% NP-40. Lysates were conducted Immunoprecipitation with anti-CD3{varepsilon} mAb and Western blot with anti-PT (PY-Plus) mAb (upper part). The whole lysates were subject to Western blot with affinity-purified rabbit anti-human CD3{varepsilon} antibody (lower part). (B) Purified CD4+ and CD8+ cells from wild-type and ZKO mice were stimulated at 1x107 cells/ml with anti-CD3{varepsilon} mAb 10 µg/100 µl for a indicated period of time at 37°C. After final washing, the cells were lysed in buffer containing 1% NP-40. Lysates were subjected to immunoprecipitation with anti-CD3{varepsilon} mAb and Western blot with purified rabbit anti-ZAP-70 antibody (upper part), and immunoprecipitation with agarose-conjugated rabbit anti-ZAP-70 antibody and Western blot with anti-PT (PY-Plus) mAb (middle part). The whole lysates were subjected to Western blotting with purified rabbit anti-ZAP-70 antibody (lower part).

 
The results on CD3-induced CD3{varepsilon} phosphorylation prompted us to examine the kinetics of ZAP-70 tyrosine phosphorylation upon CD3 ligation in T lymphocytes from ZKO mice. To this end, ZAP-70 immunoprecipitates from lysates of T lymphocytes from ZKO or wild-type mice were analyzed by Western blotting with an anti-phosphotyrosine mAb. As shown in Fig. 6Go(B), in T lymphocytes from ZKO mice CD3 cross-linking induced a slow but significant increase in ZAP-70 tyrosine phosphorylation (Fig. 6BGo, middle panel, lanes 5–8). In contrast, the same stimulus in T lymphocytes from wild-type mice induced a more potent increase in ZAP-70 tyrosine phosphorylation followed by a faster ZAP-70 dephosphorylation process (Fig. 6BGo, middle panel, lanes 1–4). In both T lymphocytes from ZKO and wild-type mice (Fig. 6BGo, upper panel) increased amounts of ZAP-70 were co-immunoprecipitated in anti-CD3 immunoprecipitates from 2C11-stimulated cells. In both ZKO and T lymphocytes, the kinetics of ZAP-70 tyrosine phosphorylation (Fig. 6BGo, middle panel) and of ZAP-70 association with CD3{varepsilon} (not shown) correlated with the kinetics of CD3{varepsilon} tyrosine phosphorylation. These results agree with previous observations that in CD3-stimulated T cells ZAP-70 binding to CD3 chains coincides with increased tyrosine phosphorylation and kinase activity (62). Therefore, the above results (Fig. 6A and BGo) strongly suggest that in ZKO T lymphocytes CD3 engagement by agonist antibodies resulted in activation of the CD3{varepsilon}/ZAP-70 signaling pathway despite their very low TCR surface expression.

One immediate consequence of triggering the TCR–CD3 complex involves the induction of PLC-{gamma}1-mediated hydrolysis of inositol phospholipids to generate diacylglycerol and inositol polyphosphates, which induce protein kinase C activation and elevate intracellular calcium levels respectively. To further understand which down-stream signals are generated upon CD3 ligation in ZKO T cells, calcium flux experiments were conducted using a number of agonist antibodies. The data show that CD3 ligation in T lymphocytes from lymph nodes and thymocytes of ZTG and wild-type mice induced increased levels of intracellular calcium, while the same stimulus did not induce a calcium response in T lymphocytes from ZKO mice (Fig. 7Go). This could be explained either because the relative insensitivity of the method used to measure the calcium flux signals or because the signals delivered in CD3-stimulated ZKO T cells are calcium–calcineurin-independent. To test this hypothesis the calcineurin-selective inhibitor CsA was used in TCR–CD3-induced cytokine production assays. The results showed that in the presence of 1 µM CsA cytokine production was totally blocked in all cultured cells, including the T lymphocytes from ZKO mice (Tables 2–4GoGoGo). These results strongly suggest that the activation signal induced by TCR–CD3 ligation in T lymphocytes in ZKO mice relies on the calcium–calcineurin pathway. Collectively, these data indicated that the earliest signaling events after TCR–CD3 stimulation could take place in the absence of CD3{zeta}.



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Fig. 7. Ca2+ response in thymocytes and lymph node cells from wild-type and ZTG, but not from ZKO mice. Thymocytes and lymph node cells from wild-type, ZTG and ZKO mice were cultured in single-cell suspension (5x106 cells/ml) at 37°C and loaded with Indo-1. Ca2+ mobilization in response to CD3 cross-linking [145- 2C11 (first arrow) plus goat anti-hamster IgG (second arrow)] was determined as described in Methods.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Because large proportions of CD8+ and CD4+ T cells are specific for LCMV during an acute infection (59,63,64), this virus was used in ZTG and ZKO mice to measure antigen-specific responses. Analyses of the in vivo responses to LCMV in mice that lack the three ITAM motifs (ZTG) of CD3{zeta} clearly demonstrate that a wild-type-like cytotoxic T cell response to the virus can occur. Moreover, allogeneic CTL can be generated from ZTG precursor T lymphocytes. In addition to CTL responses, T cell expansion and IFN-{gamma} production in response to LCMV take place with the same kinetics as in wild-type mice. However, as shown in the ZKO mouse, high levels of TCR expression are required for CTL development. Both these observations and the in vitro allogeneic MLC/CTL studies showed that generation of CTL needed a stronger TCR–CD3-mediated signal than induction of cytokine genes. We therefore conclude that CD3{zeta} ITAM-mediated signals are dispensable for the generation of CTL and cytokine production, although the CD3{zeta} transmembrane region is important for CTL generation by promoting surface expression of the TCR. Antigen-driven responses in ZTG mice therefore appeared to be controlled in a normal fashion. This suggested that in a mature T lymphocyte the regulatory role of CD3{zeta} could be taken over by CD3{gamma}, {delta} and {varepsilon}. Since their ITAM can be phosphorylated and de-phosphorylated by the same sets of enzymes, it is likely that CD3{gamma}, {delta} and {varepsilon} have the ability to control signal transduction pathways in mature T lymphocytes. Since the CD3{zeta} ITAM are, however, obligatory for precise regulation of positive selection in the thymus (7,3641), it is plausible that the full complement of CD3 is necessary during that phase of thymic development only.

LCMV infection of the ZTG mice led to extensive T cell expansion at day 8 after infection. However, in ZKO mice the same infection resulted in a detectable induction of T cell IFN-{gamma} production without extensive T cell expansion. The reduced expansion is consistent with the known effects of the {zeta} chain deficiency on T cell proliferation in vitro (4447) and with the observation that virus-induced expansion is a prerequisite for CTL development (5759,63,64). However, given the low frequency of antigen-specific cells in naive mice, it is difficult to reconcile the appearance of T cells producing IFN-{gamma} with a lack of cell expansion, if all of the cytokine-expressing cells are antigen-specific. Virus-specific T cells were shown to be contributing by evaluation of IFN-{gamma} production following short-term stimulation with virus-infected, histocompatible cells (data not shown). Although the response was not as dramatic as that observed with cells from infected normal mice, it was enhanced by the presence of the class I compatible antigen-presenting cells. Thus, although there are several mechanisms which might contribute to induction of T cell IFN-{gamma} production in the ZKO mice, the most likely is a severely impaired but low level T cell expansion during infection.

Cytokine production in mice lacking the CD3{zeta} ITAM (ZTG) appears to be controlled by the CD28/CTLA-4 control mechanism as in wild-type mice. In contrast, cytokine production in the ZKO mice cannot be regulated by CD28/CTLA-4 as these markers are expressed at extremely low levels in ZKO T cells and are not induced. Therefore, neither the positive signal mediated through CD28 nor the negative signal mediated by CTLA-4 through the SHP-2 tyrosine phosphatase can be delivered (65). This would explain why ZKO T lymphocytes do not respond to in vitro co-stimulatory signals upon CD28 or CTLA-4 stimulation with specific mAb, whilst ZTG T lymphocytes do. The in vitro observations that IFN-{gamma} production is extremely high on a per cell basis are compatible with the results of in vivo stimulation of adult ZKO mice with anti-CD3. Ten ZKO mice treated with 10 µg/g body wt of highly purified anti-CD3{varepsilon} (500-A2) (60,61) died within 30 h of injection probably because of high levels of TNF/IL-1 (data not shown). ZTG or control animals (C57BL/6x129/Sv) treated in the same fashion with 500-A2 or ZKO mice treated with an isotype-matched IgG did not show this dysregulated response. Based on our in vitro studies it is likely that the absence of CD3{zeta} resulted in an anti-CD3{varepsilon}-induced, IFN-{gamma}-initiated cascade of events.

The observation that generation of CTL and cytokine gene induction occurred in a normal fashion in ZTG and ZKO T lymphocytes led us to analyze some of the early signaling events triggered by the TCR–CD3 complex. Ligation of the TCR–CD3 complex in purified T lymphocytes from ZKO mice induced CD3{varepsilon} and ZAP-70 tyrosine phosphorylation with delayed kinetics as compared with T lymphocytes from wild-type mice. These results strongly suggest that in ZKO T lymphocytes CD3 engagement by agonist antibodies results in activation of the ZAP-70/LAT signaling induction events despite the absence of CD3{zeta}. However, in ZKO T lymphocytes we could not detect anti-CD3-mediated calcium fluxes despite the fact that productive cytokine production induced by TCR–CD3 cross-linking seemed to require a calcium–calcineurin-dependent signaling pathway. These results suggest that in the absence of CD3{zeta} stimulation of ZKO cells with anti-CD3{varepsilon} mAb induces CD3{varepsilon}/ZAP-70 tyrosine phosphorylation and low levels of intracellular calcium and additional signal sufficient to induce cytokine production. Alternatively, in these cells engagement of the remaining CD3 ITAM may provide some additional signaling function necessary for cytokine production but separate from calcium mobilization.

Studies concerned with the fine specificity of T cell responses to peptide and antigen-presenting cells have revealed an important role for phosphorylation of CD3{zeta} in modulating anergic responses (3641). The fact that T cell responses are grosso modo intact in mature ZTG and ZKO T cells suggests that perhaps the balance of (de)-phosphorylation of CD3{varepsilon}, {gamma} or {delta} could take over CD3{zeta}'s regulatory functions. Moreover, studies in T–T hybridomas developed the notion that the TCR–CD3 complex consists of two autonomous signal transduction modules made up of the {gamma}{delta}{varepsilon} and {zeta} subunits respectively. Interestingly, T–T hybridoma studies did not reveal that generation of CTL could exist in the absence of CD3{zeta}. In the T–T hybridoma model the TCR–CD3 surface requires the presence of CD3{zeta}. Likewise, CD3{zeta}-deficient cells are defective in TCR-mediated T cell signaling and cytokine production (8,66). Our studies support the hypothesis that development of CTL and expression of the regulatory elements CD28/CTLA-4 require higher levels of TCR expression than induction of cytokine genes, indicating that the former functions may need a stronger signal than the latter. These data agree with the notion that TCR density and therefore ITAM number may determine the outcome of transduced signals through association with different effectors. Moreover, the observations about the functional capabilities of T cells derived from ZTG and ZKO mice add to our understanding of the distinct and overlapping roles of the ITAM of the four CD3 molecules.


    Acknowledgments
 
This work was funded in part by the National Institutes of Health grants AI-15066 and AI-17651 (to C. T.), CA-41268 (to C. A. B.), AI-27448 (to D. A. H.), and by a grant (SAF96-0117) from the Interministerial Commission of Science and Technology, Spain, to J. S. and grant CRG 960778 from the North Atlantic Treaty Organization (NATO), Brussels to J. S. and C. T. We thank Dr Jan de Vries (DNAX) for help with the initial experiments, and Dr Stephen Simpson and other members of the Terhorst lab for helpful discussions and for a critical reading of the manuscript.


    Abbreviations
 
CsAcyclosporine A
CTLcytotoxic T lymphocytes
ITAMimmunoreceptor tyrosine-based activation motif
LCMVlymphocytic choriomeningitis virus
MLCmixed lymphocyte culture
MLRmixed lymphocyte reaction
PEphycoerythrin
PLCphospholipase C
PTKprotein tyrosine kinase
SEstaphylococcal enterotoxin
TNFtumor necrosis factor
ZKOCD3{zeta}/{eta}null mice
ZTGCD3{zeta}/{eta}null:tg{zeta}{Delta}67-150 mice

    Notes
 
Transmitting editor: J. Kearney

Received 1 October 1998, accepted 4 February 1999.


    References
 Top
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 Introduction
 Methods
 Results
 Discussion
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