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
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Abstract |
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Keywords: protein kinase, signal transduction, T lymphocytes, TCR
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Introduction |
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The protein tyrosine kinases (PTK) such as Fyn and Lck are known to play important roles in the transduction of the TCRCD3 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 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 Fc
R
heterodimer in the TCR complex with concomitant decrease in T cells with
homodimer.
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Methods |
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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 (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 SDSPAGE 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)-
1 (anti-PLC-
1, mouse IgGs) and polyclonal rabbit IgG antibody against the
chain of the high-affinity IgE receptor (antiFc
R
) were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-
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
and FITCanti-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-FcRII/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 -methylmannoside. These Th cell clones used in the present experiments were confirmed to produce IL-2 and IFN-
, 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-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 TrisHCl (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 TrisHCl (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 GSepharose and incubated with anti-Fyn, anti-ZAP-70, anti-PLC-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 ProteinG Sepharose respectively.
Immunoblotting
The immune complexes precipitated with Protein A or Protein GSepharose were washed with TNE or Brij buffer and resolved by SDSPAGE 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 TrisHCl (pH 7.5) and 150 mM NaCl] and incubated with anti-PY, anti-Fyn, anti-ZAP-70, anti-PLC-1, anti-
, antiFc
R
or anti-TCR
. 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 streptavidinalkaline 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 GSepharose were washed with TNE buffer and kinase buffer [50 mM HEPESNaOH (pH 7.4), 20 mM MnCl2 and 10 mM MgCl2 for Fyn, and 50 mM HEPESNaOH (pH 7.4) and 10 mM MnCl2 for ZAP-70]. The immunoprecipitates were suspended in 20 µl of kinase buffer containing 10 µCi of [-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 TrisHCl (pH 6.8), 9% SDS, 15% 2-mercaptoethanol and 30% glycerol]. Then, the mixture was boiled for 5 min and subjected to SDSPAGE under reducing conditions, followed by autoradiography.
Flow cytometric analyses of TCR complex and Fc
R
chains
Intracellular staining of and Fc
R
chains in the TCRCD3 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-
or antiFc
R
chain and then with FITCgoat 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 phycoerythrinstreptavidin after incubation with 300 µg/ml mouse IgG, permeabilized with 0.5% Triton X-100, and then stained with anti-
or antiFc
R
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).
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Results |
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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. 1A). 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. 1B
). These results confirmed the impairment in IL-2 production of old T cells reported previously (27,28).
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We next assayed these young and old T cells stimulated with anti-CD3 for tyrosine phosphorylation of PLC-1 in terms of Western blotting with anti-PY on the materials immunoprecipitated with anti-PLC-
1. In young T cells, tyrosine phosphorylation of PLC-
1 was increased at 5 min, peaked at ~15 min and then decreased. However, the phosphorylation of PLC-
1 in old T cells was not detected for 30 min (Fig. 3A
), although the amount of PLC-
1 immunoprecipitated from old T cells was comparable to that from young T cells (Fig. 3B
).
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Taken together, the above results indicate an impaired efficiency of tyrosine phosphorylation of PTK in the TCRCD3 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. 4). 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-
1 phosphorylation.
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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. 5A.
Fc
R
heterodimer in the TCRCD3 complex of old T cells
The chain in the TCRCD3 complex is known to associate with Fyn to play a critical role in TCR activation signal transduction. We therefore analyzed
chain expression in young and old T cells by flow cytometry after intracellular staining with anti-
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
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. 6A
. We analyzed young and old mice individually, six mice each, and results were essentially the same to those in Fig. 6A
. Proportions of the T cells with low
chain expression in young and old T cells from six mice each are summarized (Fig. 6A
). We analyzed the surface expression of the TCRCD3 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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We next examined whether the FcR
is associated with
in the TCRCD3 complex. The TCRCD3 complexes were immunoprecipitated with anti-CD3, resolved by SDSPAGE under non-reducing conditions and immunoblotted with antiFc
R
. Fc
R
was detected in the precipitates from both young and old T cells. The amount of Fc
R
precipitated from old T cells was about 2-fold more than that from young T cells, although TCR
in the precipitates was comparable to each other in amount (Fig. 7
). When the membrane was re-blotted with anti-
, the
chain was detected at exactly the same position to that of the Fc
R
protein (Fig. 7
), indicating that Fc
R
was associated with
in the TCRCD3 complexes. These experiments were repeated 4 times with essentially the same results.
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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. 8).
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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. 10), indicating that the efficiency in Ca2+ signal generation in old Th1 clones is lower than that in young Th1 clones.
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Taken together, these results support the above notion on primary CD4+ T cells that CD4+ T cells in which the chain is partly replaced by the Fc
R
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
R
chain expression in these old Th1 clones as described above.
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Discussion |
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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-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-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
chain expression in the TCRCD3 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 TCRCD3 complex expression. The chain in the TCRCD3 complex was reported to play an important role in TCR signal transduction (34) and also in transportation of the TCRCD3 complex to the cell surface (3537). The cells with the
Fc
R
heterodimer have been reported to be present in both CD4+ and CD8+ T cell populations from tumor-bearing mice, and the Fc
R
chain was also suggested to transport the TCRCD3 complex to the cell surface (30). Human CD4+ T cells infiltrating into carcinoma were also reported to lose the
chain in the TCRCD3 complex (38). Our present results indicate that small numbers of young T cells bear the
Fc
R
heterodimer in the TCRCD3 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
chain in the TCRCD3 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
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
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 Fc
R
heterodimer (41). CD16 on NK cells also associates with the
Fc
R
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-
and antiFc
R
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
R
chain more than young T cells, especially in the cells with naive phenotypes, at the expense of
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
Fc
R
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 FcR
chain did not associate with Fyn (45). Moreover, the Fc
R
chain contains only one immunoreceptor tyrosine-based activation motif YXXL, while the
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
Fc
R
heterodimer in the TCRCD3 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
homodimer with the
Fc
R
heterodimer in the TCRCD3 complex, which may result from oxidative stress or other mechanisms in milieu.
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Acknowledgments |
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Abbreviations |
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anti-CD3 anti-CD3![]() |
[Ca2+]i intracellular Ca2+ concentration |
ConA concanavalin A |
Fc![]() ![]() ![]() |
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 |
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Notes |
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6 Present address: Department of Biology, Sunchon National University, Sunchon 540-742, Republic of Korea
Received 19 February 2000, accepted 8 May 2000.
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References |
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