Molecular mechanism of the impairment in activation signal transduction in CD4+ T cells from old mice

Toshiki Tamura, Takeshi Kunimatsu5, Sung-Tae Yee6, Osamu Igarashi, Masanori Utsuyama1,2, Shin Tanaka3, Shun-ichi Miyazaki4, Katsuiku Hirokawa2 and Hideo Nariuchi

Department of Allergology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
1 Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan
2 Department of Pathology and Immunology, Tokyo Medical and Dental University School of Medicine, Tokyo 113-8510, Japan
3 Laboratory Animal Research Facilities, National Institute for Longevity Sciences, Aichi 474-8511, Japan
4 Department of Physiology, Tokyo Women's Medical College, Tokyo 162-8666, Japan

Correspondence to: T. Tamura


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It is well known that IL-2 production of CD4+ T cells from old mice (old T cells) is impaired. In this study, we have examined TCR complex {zeta} chain expression of old T cells and their TCR downstream signal transduction pathways stimulated with anti-CD3. Activation of protein tyrosine kinases, Fyn and ZAP-70, and turnover of inositol phosphates stimulated with anti-CD3 were severely impaired in old T cells, although levels of these proteins were comparable to those in young T cells. Increase in intracellular Ca2+ concentration in old T cells was also impaired. Old T cells starting the Ca2+ oscillation by the anti-CD3 stimulation were severely decreased in number and the oscillation waves were broader in shape. T cells with {zeta}–Fc{varepsilon}R{gamma} heterodimer in the TCR–CD3 complex were increased in proportion in old T cells with a concomitant decrease in the T cells with {zeta}-{zeta} homodimer. The density of the TCR–CD3 complex on old T cells was confirmed to be comparable to that on young T cells. The impairment in TCR downstream signal transduction pathways and the increase in {zeta}–Fc{varepsilon}R{gamma} heterodimer in the TCR–CD3 complex were confirmed to be the situation in Th1 clones established from old mice. These results indicate that old T cells are impaired in response to TCR stimulation, because T cells with the TCR–CD3 complex containing the {zeta}–Fc{varepsilon}R{gamma} heterodimer are increased in proportion in old T cells.

Keywords: protein kinase, signal transduction, T lymphocytes, TCR


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Age-related reduction in IL-2 production of Th cells is well documented. The reduction is not explained by decreased expression of the TCR–CD3 complex (1). Indeed, the density of the TCR–CD3 complex on old mouse CD4+ T cells (old T cells) has been shown not to be different from that on young mouse CD4+ T cells (young T cells) (2). TCR ligation induces a complex chain of events in resting T cells including the activation of a cascade of protein kinases and an increase in intracellular Ca2+ concentration ([Ca2+]i). Alterations in these intracellular signal transduction pathways have been suggested to underlie functional defects in old T cells (3). Old T cells were shown to generate lower average [Ca2+]i levels after TCR stimulation (4). However, the decline with aging in calcium signal generation in T cells is attributable, at least partly, to a shift in the ratio of naive to memory T cell subsets. Several studies have shown a dramatic decrease in the proportion of naive T cells with a concomitant increase in T cells with memory phenotypes (57), and memory T cells from any age of mice generated lower levels of calcium signal in response to lectin, anti-CD3, or calcium ionophore (8,9). However, the decline in calcium signal generation of old T cells could not be explained completely by the shift of T cell subsets (10).

The protein tyrosine kinases (PTK) such as Fyn and Lck are known to play important roles in the transduction of the TCR–CD3 signal to downstream mechanisms for T cell activation. Phosphorylation of Lck has been reported to be reduced in T cells from elderly (11,12), possibly because of a decrease in the association with CD4 (12). T cells from a substantial proportion of elderly humans were also reported to be impaired in the activation of Fyn in response to anti-CD3 (13). However, contradictory results have been published on the activity of ZAP-70. One report showed the reduction in ZAP-70 activity with aging in human T cells stimulated with mitogens (14). Results from another laboratory showed that the amount of ZAP-70 associated with the TCR complex {zeta} chain was increased with age (15). Fyn and Lck may be functionally interdependent in TCR signal transduction, and subjected to a homeostatic up-regulation of their activation states to compensate for a reduction in one another (16). Fyn activation has been shown to induce cytosolic calcium responses (17), and also activate downstream mechanisms through turnover of inositol phosphates (IP) without participation of Lck in mouse Th1 clones (18) and a human T cell line transfected with Fyn cDNA (19).

In the present experiments, we examined the alteration of Fyn and downstream mechanisms in freshly prepared CD4+ T cells and Th1 clones from aged mice by stimulation with anti-CD3 without inclusion of accessory cells in cultures. Our results indicate that activation of Fyn kinase and other signal transduction machineries downstream to Fyn through IP breakdown are impaired in old T cells, possibly because of an increase in the proportion of T cells with the {zeta}–Fc{varepsilon}R{gamma} heterodimer in the TCR complex with concomitant decrease in T cells with {zeta}{zeta} homodimer.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
C57BL/6 mice (5–8 weeks old) were purchased from Japan SLC (Hamamatsu, Japan) and used as young mice. C57BL/6 mice purchased from Japan SLC at 2–3 months old were housed in a laminar airflow room at the Tokyo Metropolitan Institute of Gerontology (Tokyo, Japan, or at the laboratory animal research facility of the National Institute for Longevity Sciences (Aichi, Japan) and used at 24–28 months old as old mice. In the present experiment, mice were subjected a thorough autopsy and those which bore neoplastic disease or splenomegaly were not used. C3H/He mice (5–8 weeks old) were also purchased from Japan SLC.

Culture media
RPMI 1640 (JRH Biosciences, Lenexa, KS) supplemented with 10% FCS (Sanko Pure Chemicals, Tokyo, Japan), 50 µM 2-mercaptoethanol and 100 µg/ml kanamycin was used for cultures throughout the present experiments. For studying [Ca2+]i, MEM (JRH Biosciences) containing 0.05 or 2% BSA was used.

Reagents and antibodies
Fura-2 acetoxy methylester was purchased from Molecular Probes (Eugene, OR). Enolase was purchased from Sigma (St Louis, MO). Herbimycin A was kindly provided by Dr Y. Uehara (National Institute of Infectious Diseases, Tokyo, Japan). Hybridoma 145-2C11 [anti-CD3{varepsilon} (anti-CD3), hamster IgG] (20) was kindly provided by Dr J. A. Bluestone (National Institutes of Health, Bethesda, MD). Hybridoma 4G10 [anti-phosphotyrosine (anti-PY), mouse IgG2b] (21) was kindly provided by Dr D. K. Morrison (National Cancer Institute, Frederick, MD). These mAb were purified from ascites on a Protein A column. Anti-CD3 Fab was prepared by papain digestion as described previously (22), and the preparation was confirmed to form a single band of 50 kDa on SDS–PAGE stained with Coomassie brilliant blue. The divalent anti-CD3 possibly contaminated in the Fab preparation was estimated to be <0.1% in our preliminary experiments. Monoclonal anti-human p59fyn (anti-Fyn) (Fyn 301, mouse IgG1) cross-reactive to mouse Fyn was purchased from Wako Pure Chemical Industries (Osaka, Japan). Rabbit antiserum to human ZAP-70 (anti-ZAP-70) (23) was provided by Dr T. Yamamoto (Institute of Medical Science, University of Tokyo, Tokyo, Japan). The anti-ZAP-70 was confirmed to cross-react to corresponding mouse molecules. Mixture of monoclonal antibodies to bovine phospholipase C (PLC)-{gamma}1 (anti-PLC-{gamma}1, mouse IgGs) and polyclonal rabbit IgG antibody against the {gamma} chain of the high-affinity IgE receptor (anti–Fc{varepsilon}R{gamma}) were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-{zeta} chain of the TCR complex (H146.968A, hamster IgG) was kindly given by Dr T. Saito (Chiba University, Chiba, Japan), with permission from Dr R. Kubo (Cytel, San Diego, CA). Polyclonal goat IgG antibody to TCR{alpha} and FITC–anti-TCRß (H57-597, hamster IgG) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and PharMingen (San Diego, CA), respectively. Anti-CD44 (KM201, rat IgG2a) and anti-CD45RB (23.G2, rat IgG2a) were used in the form of culture supernatant.

Preparation of primary CD4+ T cells
T cells were purified by passing C57BL/6 spleen cells through nylon wool and Sephadex G-10 columns. CD4+ T cells were purified by treatment with a mixture of anti-CD8 (53.6.72, rat IgG2a), anti-heat stable antigen (M1/69, rat IgG2b), anti-MHC class II (M5/114, rat IgG2b), anti-Fc{gamma}RII/III (2.4G2, rat IgG2b) and anti-rat IgG Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by magnetic separation according to the manufacturer's instructions. Recovered T cells contained >90% CD4+ cells by flow cytometric analysis.

Preparation of Th cell clones
Polyclonal T cell lines were obtained from young and old C57BL/6 mouse splenic CD4+ T cells stimulated in vitro with irradiated allogeneic C3H/He spleen cells. One young Th cell clone, YT10, and old Th clones, OT1, OT12 and OT17, were obtained from these polyclonal T cell lines from young and old mice respectively by limiting dilution repeated at least 3 times. Another Th cell clone, 35-9D, was established from lymph node cells from a young C57BL/6 mouse immunized with ovalbumin as described previously (24). These Th cell clones were maintained by repeated antigen stimulations followed by cultivation in medium containing 10% culture supernatant of rat spleen cells stimulated with concanavalin A (Con A) as a source of IL-2, because all these clones were confirmed to proliferate IL-2 dependently. Con A in the rat spleen cells culture supernatant had been neutralized with 20 mg/ml {alpha}-methylmannoside. These Th cell clones used in the present experiments were confirmed to produce IL-2 and IFN-{gamma}, but not IL-4, IL-5 or IL-6, by antigen stimulation in our preliminary experiments; and therefore classified as Th1. These Th cell clones were used for experiments after cultivation for at least 4 weeks after the last antigen stimulation, and confirmed to be resting and not to proliferate by the addition of exogenous IL-2. There were no significant differences between young and old Th1 clones in terms of TCR and CD3 expression as measured by flow cytometry. Mean fluorescence ratios of anti-TCR/control IgG were 11.9 and 11.1 for young and old Th clones, and those of anti-CD3/control IgG were 11.5 and 11.7 for young and old Th clones respectively.

Stimulation of primary CD4+ T cells or Th1 clones
For the assessment of IL-2 production, primary CD4+ T cells, 5 x 104 cells/culture, were stimulated with plate-bound anti-CD3 for 48 h and Th cell clones, 1 x 104 cells/culture, were stimulated with soluble anti-CD3 Fab for 24 h as described (22) in a flat-bottom 96-well plate, and supernatants were assayed for IL-2 activity. For the assessment of activities of PTK, Fyn and ZAP-70, and tyrosine phosphorylation of PLC-{gamma}1, primary CD4+ T cells, 1 x 106 cells/culture, were stimulated with 1 µg/well of plate-bound anti-CD3 in a flat-bottom 24-well plate, and Th cell clones, 1 x 106 cells/ml, were stimulated with 10 µg/ml of soluble anti-CD3 Fab in a tube as described (22).

Assay for IL-2 activity
IL-2 activity was assayed using CTLL-2 cells as described previously (25). For the assessment of CTLL-2 cell proliferation, cultures were pulsed with 0.25 µCi [3H]thymidine for the last 6 h of 24 h incubation and the incorporated thymidine was counted. The amount of IL-2 that induces 50% of maximum [3H]thymidine incorporation of CTLL-2 cells was defined as 1 U.

Assay for [Ca+]i
[Ca2+]i of individual cells was determined using an image processor ARGUS-50 system, Hamamatsu Photonics (Hamamatsu, Japan), using Fura-2 as described previously (18). In brief, primary CD4+ T cells or Th cell clones, 1 x 106 cells/ml of 2% BSA/MEM, were loaded with Fura-2 by an incubation at 37°C for 1 h with 4 µM Fura-2 acetoxy methylester in a tube. After washing three times, the primary CD4+ T cells and Th cell clones were suspended at 1 x 106 and 5 x 105 cells/ml, respectively, in 0.05% BSA/MEM, and 0.1 ml of the suspension was placed into a 5 mm well of a 35 mm plastic dish with a poly-L-lysine-coated glass cover slip underneath and covered with paraffin oil. The dish was mounted on an inverted microscope and warmed at 37°C. For the stimulation of primary CD4+ T cells, the well was coated with 1 µg/well of anti-CD3. To stimulate Th cell clones, 10 µg/ml of anti-CD3 Fab was added into the well at time zero. UV at 340 and 380 nm was applied to the cells, and the emission fluorescence of Fura-2 was led to a SIT camera. Ca2+ images at 340 nm (F340) and 380 nm (F380) were sequentially accumulated at 20 sec intervals and the ratio of F340/F380 calculated at each time point. A calibration curve correlating the F340/F380 ratio to Ca2+ concentration was obtained by the same procedure using Ca2+-EDTA buffer solutions.

Measurement of IP accumulation
Accumulation of IP, water-soluble inositol derivatives including IP, IP2, IP3 and IP4, in primary CD4+ T cells or Th cell clones was measured as described previously (25). Briefly, primary CD4+ T cells or Th cell clones, 107 cells/ml, were incubated for 18 h in inositol free-MEM (Life Technologies, Grand Island, NY) containing 40 µCi/ml myo[2-3H]inositol, washed, and resuspended in RPMI 1640 medium containing 10% FCS and 10 mM LiCl. The myoinositol-loaded cells, 1 x 106 cells/0.2 ml, were stimulated with 1 µg/well of plate-bound anti-CD3 for primary CD4+ T cells or with 10 µg/ml anti-CD3 Fab for Th cell clones in a flat-bottom 24-well plate. The reaction was terminated by the addition of chloroform/methanol and it was separated into chloroform-soluble and water-soluble fractions. The sample in the aqueous phase was loaded on a Dowex 1 formate column, washed, and the 3H content in the fraction eluted with a mixture of 1 M sodium formate and 0.1 M formic acid was determined in a liquid scintillation counter.

Immunoprecipitation
Primary CD4+ T cells or Th clones were solubilized in 1 ml of cold TNE buffer [50 mM Tris–HCl (pH 8.0) containing 150 mM NaCl, 1% (v/v) Nonidet P-40, 20 mM EDTA, 10 µg/ml aprotinin, 0.4 mM sodium vanadate, and 10 mM sodium pyrophosphate] or Brij buffer [20 mM Tris–HCl (pH 8.1) containing 150 mM NaCl, 1% (w/v) Brij 96, 1 mM PMSF and 10 µg/ml aprotinin]. The cell lysates were centrifuged and the supernatants were precleared with Protein A– or Protein G–Sepharose and incubated with anti-Fyn, anti-ZAP-70, anti-PLC-{gamma}1 or anti-CD3 at 4°C for 1 h, and then the immune complexes formed with anti-CD3 and other antibodies were precipitated with Protein A– and Protein–G Sepharose respectively.

Immunoblotting
The immune complexes precipitated with Protein A– or Protein G–Sepharose were washed with TNE or Brij buffer and resolved by SDS–PAGE under reducing or non-reducing conditions, and then transferred to PVDF microporous membrane (Immobilon; Millipore, Bedford, MA). The membrane was blocked in 5% BSA/TBS [20 mM Tris–HCl (pH 7.5) and 150 mM NaCl] and incubated with anti-PY, anti-Fyn, anti-ZAP-70, anti-PLC-{gamma}1, anti-{zeta}, anti–Fc{varepsilon}R{gamma} or anti-TCR{alpha}. Immunoblots were incubated with appropriate biotinylated antibody, anti-mouse Ig (Amersham Pharmacia Biotech, Little Chalfont, UK), anti-rabbit Ig (Amersham Pharmacia Biotech), anti-hamster IgG (Cedarlane, Hornby, Ontario, Canada) or anti-goat IgG (Jackson Immunoresearch, West Grove, PA) and then incubated with streptavidin–alkaline phosphatase (Amersham Pharmacia Biotech). After the incubation, the membrane was washed with TBS containing 0.1% Tween-20, and developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate substrates.

Immune complex kinase assay
The immune complexes precipitated with Protein G–Sepharose were washed with TNE buffer and kinase buffer [50 mM HEPES–NaOH (pH 7.4), 20 mM MnCl2 and 10 mM MgCl2 for Fyn, and 50 mM HEPES–NaOH (pH 7.4) and 10 mM MnCl2 for ZAP-70]. The immunoprecipitates were suspended in 20 µl of kinase buffer containing 10 µCi of [{gamma}-32P]ATP and incubated at 30°C for 30 min under continuous mixing in the presence of 1 µg of denatured enolase as an exogenous substrate to assay Fyn or ZAP-70 kinase activity, or in the absence of the substrate to assay autophosphorylation. The reaction was stopped by adding 15 µl of 3 x sample buffer [195 mM Tris–HCl (pH 6.8), 9% SDS, 15% 2-mercaptoethanol and 30% glycerol]. Then, the mixture was boiled for 5 min and subjected to SDS–PAGE under reducing conditions, followed by autoradiography.

Flow cytometric analyses of TCR complex {zeta} and Fc{varepsilon}R{gamma} chains
Intracellular staining of {zeta} and Fc{varepsilon}R{gamma} chains in the TCR–CD3 complex of freshly prepared CD4+ T cells or Th clones was carried out as described previously with modifications (26). Briefly, CD4+ T cells were fixed with 3% paraformaldehyde, incubated with 300 µg/ml mouse IgG in PBS and then permeabilized with 0.5% Triton X-100. The cells were stained with anti-{zeta} or anti–Fc{varepsilon}R{gamma} chain and then with FITC–goat anti-hamster IgG or anti-rabbit IgG respectively. For the analysis of naive and memory CD4+ T cells, CD4+ T cells were stained with anti-CD44 or anti-CD45RB, biotinylated anti-rat IgG and phycoerythrin–streptavidin after incubation with 300 µg/ml mouse IgG, permeabilized with 0.5% Triton X-100, and then stained with anti-{zeta} or anti–Fc{varepsilon}R{gamma} as above. They were analyzed on a FACScan using Lysis II software (Becton Dickinson, Mountain View, CA). In preliminary experiments, there were no differences between young and old T cells in membrane permeability after Triton X-100 treatment in terms of intracellular staining for actin by using fluorescein phalloidin (Molecular Probes).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Impaired IL-2 production and TCR signal transduction pathways of old T cells
Purified CD4+ T cells from young and old mice, 10 mice each, were pooled respectively, and stimulated with plate-bound anti-CD3. Aliquots of these cells were assayed for IL-2 in culture supernatants, and others were for kinase activities of Fyn and ZAP-70, and for tyrosine phosphorylation of PLC-{gamma}1.

IL-2 produced by old T cells was apparently lower in amount than that produced by young T cells at any dose of anti-CD3 tested (Fig. 1AGo). Old T cells were confirmed not to increase in IL-2 production by stimulation with 3 µg/well or more anti-CD3 (data not shown). In the above experiments, it is possible that apparent impairment in IL-2 production of pooled old T cells is due to particular T cells from one or more of the old mice. Therefore, we also examined the IL-2 production of CD4+ T cells from individual old and young mice after stimulation with plate-bound anti-CD3, and essentially the same results were obtained (Fig. 1BGo). These results confirmed the impairment in IL-2 production of old T cells reported previously (27,28).



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Fig. 1. IL-2 production of young and old T cells stimulated with plate-bound anti-CD3. CD4+ T cells were purified from young ({circ}) and old (•) mouse spleen cells pooled respectively, 10 mice each (A), or from spleen cells obtained from individual young and old mice (B). Young and old T cells, 5 x 104 cells/culture, were stimulated in a 96-well plate with indicated amounts of plate-bound anti-CD3 for 48 h and the culture supernatants were assayed for IL-2 in terms of [3H] thymidine incorporation of the IL-2-dependent T cell clone CTLL-2. Results in (A) are presented as the mean ± SD of triplicate assays. Each symbol in (B) represents the mean of triplicate assays of CD4+ T cells from individual young (Y) and old (O) mice.

 
We then examined Fyn kinase activity in the aliquot of pooled old T cells stimulated with 1 µg/well plate-bound anti-CD3 as mentioned above in comparison to that in the aliquot of young T cells by immune complex kinase assay. Activity of tyrosine phosphorylation of enolase and Fyn itself increased in young T cells at 5 and 15 min, and decreased thereafter. However, Fyn activity in old T cells was not increased by stimulation in terms of either enolase phosphorylation or autophosphorylation (Fig. 2AGo). No apparent difference was observed between young and old T cells in the amount of Fyn immunoprecipitated from 5 x 106 T cells (Fig. 2BGo).



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Fig. 2. Activation of Fyn and ZAP-70 kinases in young and old T cells stimulated with plate-bound anti-CD3. Young and old T cells, 1 x 106 cells/culture, were stimulated in a 24-well plate with 1 µg/well of plate-bound anti-CD3 for 0, 5, 15 and 30 min, and lysed with TNE buffer. The lysates obtained from 5 x 106 T cells were immunoprecipitated with anti-Fyn (A and B) or anti-ZAP-70 (C and D) and subjected to immune complex kinase assay for Fyn (A) or ZAP-70 (C). Fyn kinase activity was assayed by phosphorylation of an exogenous substrate enolase and autophosphorylation, and ZAP-70 was assayed by autophosphorylation. The immunoprecipitates were also subjected to SDS–PAGE on 4–20% polyacrylamide gradient gels, followed by immunoblotting with anti-Fyn (B) or anti-ZAP-70 (D). Positions of Fyn, enolase and ZAP-70 are indicated by arrows. The value of each band for kinase activity is expressed as the ratio to the value at time 0 in densitometry quantification.

 
Young and old T cells were also assayed for ZAP-70 kinase activity by immune complex kinase assay in terms of autophosphorylation. The ZAP-70 autophosphorylation in young T cells was increased by anti-CD3 stimulation, peaked at 5–15 min and then decreased. However, autophosphorylation in old T cells was not increased for 30 min (Fig. 2CGo), although they contained a comparable amount of ZAP-70 to young T cells (Fig. 2DGo). ZAP-70 kinase activity was also examined by using denatured enolase as an exogenous substrate and similar results obtained to those described above (data not shown).

We next assayed these young and old T cells stimulated with anti-CD3 for tyrosine phosphorylation of PLC-{gamma}1 in terms of Western blotting with anti-PY on the materials immunoprecipitated with anti-PLC-{gamma}1. In young T cells, tyrosine phosphorylation of PLC-{gamma}1 was increased at 5 min, peaked at ~15 min and then decreased. However, the phosphorylation of PLC-{gamma}1 in old T cells was not detected for 30 min (Fig. 3AGo), although the amount of PLC-{gamma}1 immunoprecipitated from old T cells was comparable to that from young T cells (Fig. 3BGo).



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Fig. 3. Tyrosine phosphorylation of PLC-{gamma}1 in young and old T cells stimulated with plate-bound anti-CD3. Young and old T cells, 1 x 106 cells/culture, were stimulated in a 24-well plate with 1 µ/well of plate-bound anti-CD3 for 0, 5, 15 and 30 min, and lysed. The lysates obtained from 5 x 106 T cells were immunoprecipitated with anti-PLC-{gamma}1. The immunoprecipitates were subjected to SDS–PAGE on 4–20% polyacrylamide gradient gel and immunoblotted with anti-PY (A) or anti-PLC-{gamma}1 (B). Positions of PLC-{gamma}1 are indicated by arrows. The value of each phosphorylation band is expressed as the ratio to the value at time 0 in densitometry quantification.

 
The experiments described above were all repeated 3 times using young and old T cells pooled from 10 mice each with essentially the same results.

Taken together, the above results indicate an impaired efficiency of tyrosine phosphorylation of PTK in the TCR–CD3 complex downstream in old T cells.

To confirm the impairment in TCR signal transduction pathways in old T cells, CD4+ T cells from young and old mice, pooled from three mice each, were stimulated with anti-CD3, and assayed for total IP accumulation. The accumulation in old T cells was less than half of that in young T cells at any time point. Although the accumulation in old T cells increased with time, it increased more slowly than that in young T cells (Fig. 4Go). We obtained similar results in three repeated experiments. Thus, the total IP accumulation in old T cells is not as efficient as that in young T cells, in accordance with the impairment in PLC-{gamma}1 phosphorylation.



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Fig. 4. Accumulation of IP in young and old T cells stimulated with plate-bound anti-CD3. Young ({circ}) and old (•) T cells, 1 x 107 cells/ml, were loaded with myo[2-3H]inositol and stimulated in a 24-well plate at 1 x 106 cells/culture with 1 µg/well of plate-bound anti-CD3 for 15, 30, 60 and 120 min, and the accumulation of [3H]IP in the cells was determined in a liquid scintillation counter. Results are presented as the mean ± SD of triplicate assay. The total radio activities of the loaded young and old T cells were 31,679 and 50,749 c.p.m. respectively.

 
Poor [Ca+]i elevation in old T cells
The elevation of [Ca2+]i is known to be an important signal for IL-2 production by CD4+ T cells. Therefore, [Ca2+]i of young and old T cells stimulated with anti-CD3 were compared with each other. Most of the young T cells stimulated with anti-CD3 showed an oscillation in [Ca2+]i with sharp spikes that continued for more than 20 min. Old T cells with [Ca2+]i oscillations, however, were very few in number and their oscillation waves were broad in shape. Some small but wide waves of oscillation were also observed in old T cells (Fig. 5AGo).



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Fig. 5. [Ca2+]i oscillation and inhibitory effect of herbimycin A treatment on the elevation of [Ca2+]i of young and old T cells stimulated with plate-bound anti-CD3. Young and old T cells untreated (A) or treated with 20 nM herbimycin A (B) were loaded with 4 µM Fura-2, and stimulated with 1 µg/well of plate-bound anti-CD3 at time 0. [Ca2+]i of these cells was assayed in a single cell fluorometry imaging system, Argus-50. Each line represents [Ca2+]i of a single cell.

 
To examine whether the increase in [Ca2+]i was caused by the activation of PTK, the effects of treatment with the PTK inhibitor herbimycin A on [Ca2+]i were examined in young and old T cells. The [Ca2+]i elevation in both young and old T cells was apparently suppressed by treatment with 20 nM herbimycin A (Fig. 5BGo), suggesting that [Ca2+]i elevation in both young and old T cells depends on the activation of PTK.

Both experiments described above were repeated 4 times. In each experiment, we analyzed >30 cells on both young and old T cells, and obtained similar results to those shown in Fig. 5AGo.

{zeta}–Fc{varepsilon}R{gamma} heterodimer in the TCR–CD3 complex of old T cells
The {zeta} chain in the TCR–CD3 complex is known to associate with Fyn to play a critical role in TCR activation signal transduction. We therefore analyzed {zeta} chain expression in young and old T cells by flow cytometry after intracellular staining with anti-{zeta} chain. In this experiment, we analyzed naive, memory and total CD4+ T cells, because the proportion of CD4+ T cells with naive T cell markers has been shown to reduce in old mouse spleen cells (57). CD44lowCD45RBhigh naive and CD44highCD45RBlow memory T cells in our young T cell preparations were 77.4 and 5.1%, and those in old T cells were 23.4 and 35.2% respectively. CD44highCD45RBhigh cells which were reported to be effectors (29) existed at a level of 10.0% in young T cells and increased to 17.5% in old T cells. The cells with low {zeta} chain expression were increased in proportion in old T cells, especially in naive T cells. Representative fluorescent profiles of naive, memory and total CD4+ T cells from young and old mice are shown in Fig. 6AGo. We analyzed young and old mice individually, six mice each, and results were essentially the same to those in Fig. 6AGo. Proportions of the T cells with low {zeta} chain expression in young and old T cells from six mice each are summarized (Fig. 6AGo). We analyzed the surface expression of the TCR–CD3 complex by staining with anti-TCR and also with anti-CD3; however, we observed no difference between young and old T cells in either density or fluorescent profile of TCR and CD3 expression (data not shown) as reported.



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Fig. 6. Expression of {zeta} and Fc{varepsilon}R{gamma} chains in young and old T cells. Young and old T cells, total and the cells with naive and memory phenotypes were analyzed for {zeta} (A) and Fc{varepsilon}R{gamma} chain expression (B) by flow cytometry after intracellular staining with anti-{zeta} or anti–Fc{varepsilon}R{gamma} chain. The fluorescent profiles of total, naive and memory T cells from a representative mouse are shown in each panel, and proportions of the cells with low {zeta} chain expression and those with Fc{varepsilon}R{gamma} chain are shown in the table in each panel. Results shown in tables are presented as mean ± SD of six mice.

 
The {zeta} chain in the TCR–CD3 complex of T cells was shown to be replaced by Fc{varepsilon}R{gamma} in tumor-bearing mice (30). Therefore, we analyzed Fc{varepsilon}R{gamma} expression in the young and old T cell preparations above by flow cytometry after intracellular staining with anti–Fc{varepsilon}R{gamma}. Representative fluorescent profiles and proportions of T cells with high Fc{varepsilon}R{gamma} expression from young and old mice, six mice each, are shown in Fig. 6BGo. Although a small population of young T cells expressed Fc{varepsilon}R{gamma} in naive, memory and total CD4+ T cells, T cells expressing Fc{varepsilon}R{gamma} were increased in proportion in old T cells, especially in naive T cells. These results indicate a possibility that a proportion of T cells with the {zeta}–Fc{varepsilon}R{gamma} heterodimer in the TCR–CD3 complex are increased in old T cells.

We next examined whether the Fc{varepsilon}R{gamma} is associated with {zeta} in the TCR–CD3 complex. The TCR–CD3 complexes were immunoprecipitated with anti-CD3, resolved by SDS–PAGE under non-reducing conditions and immunoblotted with anti–Fc{varepsilon}R{gamma}. Fc{varepsilon}R{gamma} was detected in the precipitates from both young and old T cells. The amount of Fc{varepsilon}R{gamma} precipitated from old T cells was about 2-fold more than that from young T cells, although TCR{alpha} in the precipitates was comparable to each other in amount (Fig. 7Go). When the membrane was re-blotted with anti-{zeta}, the {zeta} chain was detected at exactly the same position to that of the Fc{varepsilon}R{gamma} protein (Fig. 7Go), indicating that Fc{varepsilon}R{gamma} was associated with {zeta} in the TCR–CD3 complexes. These experiments were repeated 4 times with essentially the same results.



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Fig. 7. Co-precipitation of {zeta} and Fc{varepsilon}R{gamma} chains in immunoprecipitates with anti-CD3. Young (Y) and old (O) T cells were solubilized with Brij buffer. TCR–CD3 complexes were immunoprecipitated with anti-CD3 on Protein A from the cell lysates, and resolved by SDS–PAGE under non-reducing conditions. The materials on the gel were transferred to a membrane and blotted with anti-TCR{alpha}, and re-blotted with anti-{zeta} and also with anti–Fc{varepsilon}R{gamma} chain. Bands on the membrane were quantified densitometrically. Figures at the bottom represents ratios of the density of the Fc{varepsilon}R{gamma} or {zeta} chain to that of TCR{alpha} in a co-precipitated band of {zeta} and Fc{varepsilon}R{gamma} chains.

 
Taken together, these results indicate that T cells with the {zeta}–Fc{varepsilon}R{gamma} heterodimer in the TCR–CD3 complexes were increased in proportion in old T cells at the expense of the cells with the {zeta}-{zeta} homodimer.

Impairment in IL-2 production and TCR signal transduction pathways in old Th1 clones
We examined IL-2 production and TCR signal transduction pathways in Th1 clones from old mice to confirm the results described above in homogeneous populations, because freshly prepared old T cells were more heterogeneous than young ones in terms of their activation stage. Old Th1 clone OT17 produced only a negligible amount of IL-2 after stimulation with soluble anti-CD3 Fab. IL-2 production of other two old Th1 clones was significantly lower than that of young Th1 clones after anti-CD3 stimulation (Fig. 8Go).



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Fig. 8. IL-2 production of young and old Th1 clones stimulated with soluble anti-CD3 Fab. Young Th1 clones, 35-9D ({circ}) and YT10 ({square}), and old Th1 clones, OT1 (•) , OT12 ({blacksquare}) and OT17 ({blacktriangleup}), 1 x 104 cells/culture, were stimulated in a 96-well plate with the indicated concentrations of soluble anti-CD3 Fab for 24 h and culture supernatants were assayed for IL-2 in terms of [3H]thymidine incorporation of IL-2-dependent T cell clone CTLL-2. Results are presented as the mean ± SD of triplicate assays.

 
We next examined Fyn and ZAP-70 activities of these three old Th1 clones by immune complex kinase assay. Neither Fyn nor ZAP-70 activity was increased for 60 min in any of these old Th1 clones stimulated with soluble anti-CD3 Fab (Fig. 9A and BGo). In young Th1 clones, however, Fyn activity was increased at 5 and 15 min, and ZAP-70 autophosphorylation peaked 15 min after stimulation (Fig. 9A and BGo). The amounts of Fyn and ZAP-70 proteins and mRNA accumulated in these old Th1 clones were comparable to those in young Th1 clones (data not shown).



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Fig. 9. Activation of Fyn, ZAP-70 and PLC-{gamma}1 in young and old Th1 clones stimulated with soluble anti-CD3 Fab. Four millions cells of young, 35-9D and YT10, and old, OT1, OT12, OT17, Th1 clones stimulated with 10 µg/ml of anti-CD3 Fab for 0, 5, 15, 30 and 60 min were lysed with TNE buffer, and the lysates were immunoprecipitated with anti-Fyn or anti-ZAP-70, followed by immune complex kinase assay for Fyn in terms of both enolase phosphorylation and autophosphorylation (A), or ZAP-70 in terms of autophosphorylation (B). The lysates immunoprecipitated with anti-PLC-{gamma}1 were subjected to SDS–PAGE on 4–20% polyacrylamide gradient gel followed by immunoblotting with anti-PY (C). Positions of Fyn, enolase, ZAP-70 and PLC-{gamma}1 are indicated by arrows. The value of each band for kinase activity or phosphorylation is expressed as the ratio to the value at time 0 in densitometry quantification.

 
We next examined the tyrosine phosphorylation of PLC-{gamma}1 in old Th1 clones. PLC-{gamma}1 tyrosine phosphorylation was scarcely detected in two old Th1 clones, OT1 and OT17, after the anti-CD3 stimulation. In one old Th1 clone, OT12, phosphorylation was detected at time 0, but it was not increased by the stimulation (Fig. 9CGo). PLC-{gamma}1 in young Th1 clones was phosphorylated 15 min after the anti-CD3 stimulation. These old Th1 clones contained PLC-{gamma}1 protein at a level comparable to young Th1 clones in amount (data not shown).

Total IP accumulation was scarcely increased in three old Th1 clones stimulated with anti-CD3. [3H]IP incorporation into OT1, OT12 and OT17 stimulated with anti-CD3 for 120 min was 285±52, 518±19 and 520±44 c.p.m., and for unstimulated clones was 233±63, 525±42 and 424±55 c.p.m., whereas that of young clones 35-9D and YT-10 stimulated with anti-CD3 for 120 min was 1551±32 and 4523±392 c.p.m., and for those unstimulated was 310±12 and 840±103 c.p.m. respectively.

We next examined [Ca2+]i elevation of old Th1 clones after anti-CD3 stimulation. Maximum [Ca2+]i values of these old Th1 clones were lower than those of young Th1 clones and most of the oscillation waves of these old clones were broader than those of young Th1 clones (Fig. 10Go), indicating that the efficiency in Ca2+ signal generation in old Th1 clones is lower than that in young Th1 clones.



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Fig. 10. Oscillation of [Ca2+]i of young and old Th1 clones stimulated with soluble anti-CD3 Fab. Young, 35-9D and YT10, and old, OT1, OT12 and OT17, Th1 clones, were loaded with 4 µM Fura-2, and stimulated with 10 µg/ml of anti-CD3 Fab at time 0. [Ca2+]i values of these clones were assayed in a single-cell fluorometry imaging system, ARGUS-50. Each line represents [Ca2+]i of a single cell.

 
{zeta}–Fc{varepsilon}R{gamma} heterodimer in old Th1 clones
We next analyzed the expression of {zeta} and Fc{varepsilon}R{gamma} chains in three Th1 clones established from old mice. All of these old Th1 clones showed lower {zeta} chain expression than young Th1 clones and, inversely, Fc{varepsilon}R{gamma} chain expression in these old Th1 clones was increased (Fig. 11Go). The mean fluorescent ratio of anti-{zeta}/control IgG for young Th1 clones 35-9D and YT10 was 7.8 and 5.1, and for old Th1 clones OT1, OT12 and OT17 was 3.1, 3.4 and 2.7 respectively, and that of anti–Fc{varepsilon}R{gamma}/control IgG for 35-9D and YT10 was 1.0 and 1.2, and for old Th1 clones OT1, OT12 and OT17 was 1.4, 1.9 and 2.4 respectively. TCR–CD3 complexes on old Th1 clones were not significantly different in terms of density from those on young Th1 clones (data not shown).



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Fig. 11. Expression of {zeta} and Fc{varepsilon}R{gamma} chains in young and old Th1 clones. Young and old Th1 clones were stained intracellularly with anti-{zeta} or anti–Fc{varepsilon}R{gamma} chain followed by FITC–anti-hamster IgG or –anti-rabbit IgG respectively and analyzed on a FACScan.

 
We repeated a series of the above experiments using an additional five young and three old Th1 clones, and obtained similar results to those described above except for one old clones (data not shown). The one exceptional old clone responded well to anti-CD3 in terms of both IL-2 production and PTK activation .

Taken together, these results support the above notion on primary CD4+ T cells that CD4+ T cells in which the {zeta} chain is partly replaced by the Fc{varepsilon}R{gamma} chain were increased in proportion in old T cells and that doses of IL-2 produced by old Th1 clones appear to be inversely proportional to the density of Fc{varepsilon}R{gamma} chain expression in these old Th1 clones as described above.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In our present experiments, old T cells were shown to be impaired in terms of the activation of Fyn and downstream signal transducers such as ZAP-70 and PLC-{gamma}1. Consistent with our results, the activation of Fyn kinase in human T cells stimulated with anti-CD3 has been reported to be impaired with aging (13). Oscillation of [Ca2+]i induced by anti-CD3 stimulation was also impaired in old T cells. These findings are consistent with a low efficiency of IL-2 production of old T cells, because these events, including the activation of PTK and [Ca2+]i elevation, are known to play important roles in IL-2 production of CD4+ T cells. In our assay system for [Ca2+]i, it takes different times for T cells to interact with anti-CD3, therefore it is not so important when [Ca2+]i oscillation starts. In our results, phosphorylation levels of Fyn and ZAP-70 in old T cells in their resting conditions were similar to or lower than those in young T cells. However, in previous reports, CD3{zeta} immunoprecipitated from old T cells was shown to associate with ZAP-70 more than that from young T cells and baseline phosphorylation of ZAP-70 was high, although the phosphorylation was not increased by stimulation with anti-CD3 and anti-CD4 (15,31). The difference between our results and theirs in terms of the baseline phosphorylation of ZAP-70 in unstimulated old T cells could partly be explained by the difference in the activation state of the T cells. We purified CD4+ T cells by passing them through nylon wool and Sephadex G-10 columns to deplete highly activated large T cells. The small amount of {zeta} chain in old T cells could also partly explain the increase in the relative amount of ZAP-70 associated per {zeta} chain. In another report, the authors claimed that Fyn activity did not change with aging; however, in their data Fyn kinase activity in old T cells appeared to decrease after anti-CD3 stimulation (32). They stimulated T cells by CD3 cross-linking. The CD3 cross-linking was shown to induce a negative signal against T cell activation (22,33).

We used pooled CD4+ T cells from young and old mice, 10 mice each, in order to examine IL-2 production, kinase activity of Fyn and ZAP-70, and tyrosine phosphorylation of PLC-{gamma}1 in the same T cell preparation. Since CD4+ T cells, especially old T cells, contain heterogeneous populations, we established old Th1 clones and analyzed their TCR downstream signal transduction pathways and obtained similar results in six out of seven clones to those obtained using primary old T cells, suggesting that these old Th1 clones could be useful to analyze the impairment of TCR signal transduction pathway with aging.

We previously showed that young Th2 clones were also impaired in terms of activation of Fyn, ZAP-70, and PLC-{gamma}1 similar to old T cells described in our present experiments. The impairment in activities of these kinases in young Th2 clones, however, was indicated to be caused by a small amount of Fyn protein. The amounts of Fyn protein in Th2 clones were about a third to a fifth of those of young Th1 clones (18). Low Fyn kinase activity in old Th1 clones would be attributable to low {zeta} chain expression in the TCR–CD3 complex. The impaired activity of Fyn kinase would result in a reduction of IL-2 production of these old Th1 clones.

We observed no difference between young and old T cells in terms of the density of TCR–CD3 complex expression. The {zeta} chain in the TCR–CD3 complex was reported to play an important role in TCR signal transduction (34) and also in transportation of the TCR–CD3 complex to the cell surface (3537). The cells with the {zeta}–Fc{varepsilon}R{gamma} heterodimer have been reported to be present in both CD4+ and CD8+ T cell populations from tumor-bearing mice, and the Fc{varepsilon}R{gamma} chain was also suggested to transport the TCR–CD3 complex to the cell surface (30). Human CD4+ T cells infiltrating into carcinoma were also reported to lose the {zeta} chain in the TCR–CD3 complex (38). Our present results indicate that small numbers of young T cells bear the {zeta}–Fc{varepsilon}R{gamma} heterodimer in the TCR–CD3 complex and the T cells with the heterodimer increase in proportion in old T cells. Although the mechanism remains to be studied, the expression of the {zeta} chain in the TCR–CD3 complex was shown to be reduced by direct interaction of CD4+ T cells with activated macrophages (39). Macrophages stimulated with lipopolysaccharide or those from tumor-bearing mice were also indicated to reduce {zeta} chain expression in T cells by oxidative stress or other mechanisms (26). It is rational that old mice had been exposed to various infectious agents or antigens, resulting in macrophage activation to reduce {zeta} chain expression in T cells. The high proportion of CD4+ T cells with memory phenotypes in our old mice could be the cumulative outcome of antigen exposure of T cells, because CD4+ T cells expressing a transgenic TCR specific for pigeon cytochrome c did not undergo a shift to memory phenotype cells during aging (40). Indeed, macrophages in spleen cells from old mice increased in number in comparison to those from young mice (data not shown), which is consistent with the results from tumor-bearing mice (39).

NK1.1+ T cells were shown to express the TCR complex with the {zeta}–Fc{varepsilon}R{gamma} heterodimer (41). CD16 on NK cells also associates with the {zeta}–Fc{varepsilon}R{gamma} heterodimer (42). NK1.1+ T cells were reported to increase in proportion in aged mice (43). CD4+ NK1.1+ T cells in our old mouse spleen cells, however, were not increased in proportion, although CD4 NK1.1+ T cells were slightly but significantly increased. In addition, we obtained essentially the same results presented here by repeating intracellular staining with anti-{zeta} and anti–Fc{varepsilon}R{gamma} chains of old T cells depleted of NK1.1+ T cells and containing NK1.1 CD4+ cells at >98%. The old NK1.1 CD4+ T cells expressed Fc{varepsilon}R{gamma} chain more than young T cells, especially in the cells with naive phenotypes, at the expense of {zeta} chain expression. Moreover, NK1.1+ T cells have been shown to have memory phenotypes on their surface (44). It is, therefore, unlikely that our results on {zeta}–Fc{varepsilon}R{gamma} heterodimer expression in old T cells are ascribed to NK1.1+ T cells contaminating our old T cell preparations.

Our results showed that Fyn kinase and downstream signal transducers were impaired in terms of activity in old T cells, although old T cells contained amounts of Fyn and other signal transducers comparable to those in young T cells. Results from another laboratory showed that the Fc{varepsilon}R{gamma} chain did not associate with Fyn (45). Moreover, the Fc{varepsilon}R{gamma} chain contains only one immunoreceptor tyrosine-based activation motif YXXL, while the {zeta} chain contains three of these motifs. These findings suggest that the transduction of TCR-induced activation signal is not efficient in old T cells, because of the {zeta}–Fc{varepsilon}R{gamma} heterodimer in the TCR–CD3 complex.

Taken together, the results of our present study indicate that the impairment in IL-2 production of old T cells is caused by inefficiency in the signal transduction pathway downstream of the TCR. The inefficiency was also indicated to be caused primarily by the replacement of the {zeta}{zeta} homodimer with the {zeta}–Fc{varepsilon}R{gamma} heterodimer in the TCR–CD3 complex, which may result from oxidative stress or other mechanisms in milieu.


    Acknowledgments
 
This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas, by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture, Japan and by a grant from Japan Human Sciences Foundation for the project to promote development of anti-AIDS pharmaceuticals.


    Abbreviations
 
anti-CD3 anti-CD3{varepsilon}
[Ca2+]i intracellular Ca2+ concentration
ConA concanavalin A
Fc{varepsilon}R{gamma} {gamma} chain of high-affinity IgE receptor
IP inositol phosphate
old T cell sold mouse CD4+ T cells
PLC phospholipase C
PTK protein tyrosine kinase.
young T cells young mouse CD4+ T cells

    Notes
 
5 Present address: Environmental Health Science Laboratory, Sumitomo Chemical Co. Ltd, Osaka 554-8558, Japan Back

6 Present address: Department of Biology, Sunchon National University, Sunchon 540-742, Republic of Korea Back

Transmitting editor: G. Doria

Received 19 February 2000, accepted 8 May 2000.


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 Introduction
 Methods
 Results
 Discussion
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