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The Journal of Immunology, 2007, 178: 1443-1449.
Copyright © 2007 by The American Association of Immunologists, Inc.

Age-Associated Decline in Effective Immune Synapse Formation of CD4+ T Cells Is Reversed by Vitamin E Supplementation1

Melissa G. Marko2,*, Tanvir Ahmed2,3,*, Stephen C. Bunnell{dagger}, Dayong Wu*, Heekyung Chung*, Brigitte T. Huber{dagger} and Simin Nikbin Meydani4,*,{dagger}

* Nutritional Immunology Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111; and {dagger} Department of Pathology, Sackler Graduate School of Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Aging is associated with reduced IL-2 production and T cell proliferation. Vitamin E supplementation, in aged animals and humans, increases cell division and IL-2 production by naive T cells. The immune synapse forms at the site of contact between a T cell and an APC and participates in T cell activation. We evaluated whether vitamin E affects the redistribution of signaling proteins to the immune synapse. Purified CD4+ T cells, from the spleens of young and old mice, were treated with vitamin E before stimulation with a surrogate APC expressing anti-CD3. Using confocal fluorescent microscopy, we observed that CD4+ T cells from old mice were significantly less likely to recruit signaling proteins to the immune synapse than cells from young mice. Vitamin E increased the percentage of old CD4+ T cells capable of forming an effective immune synapse. Similar results were found following in vivo supplementation with vitamin E. When compared with memory cells, naive T cells from aged mice were more defective in immune synapse formation and were more responsive to vitamin E supplementation. These data show, for the first time, that vitamin E significantly improves age-related early T cell signaling events in naive CD4+ T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Age-associated deterioration of the T cell-mediated immune response contributes to a higher incidence of morbidity and mortality from infectious diseases and various forms of cancer. Declines in T cell function have been proposed to be the central defect in age-associated immune senescence (1, 2, 3). We along with others have reported that the age-related impairment of T cell function is primarily manifested by decreases in IL-2 production and cell proliferation in both humans and experimental rodents (4, 5, 6). IL-2 is a cytokine that promotes clonal expansion of activated T cells, which represents an essential step in mounting an adaptive immune response. Age-related changes in the T cells themselves include an increase in the proportion of Ag-experienced memory cells and a decrease in the number of naive cells (7, 8). Furthermore, recent evidence has indicated that naive T cells from aged animals possess a reduced ability to produce IL-2, to proliferate and to proceed through the cell cycle (6).

IL-2 expression is regulated by a series of signaling events initiated by the TCR (9, 10). These signals are initiated in the immune synapse, an adhesive junction between a T cell and an APC (11). Within seconds of Ag engagement, the TCR initiates a tyrosine phosphorylation cascade that triggers the redistribution of various signaling molecules to the TCR. Over several minutes, the tyrosine kinases Lck, Fyn, and Zap70, and the adaptor proteins linker for activation of T cells (LAT)5 and Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) all enter the immune synapse. These molecules contribute to the recruitment and activation of various effector proteins, including Vav, phospholipase C{gamma} (PLC{gamma}), and the serine/threonine kinase protein kinase C{theta} (PKC{theta}). These enzymes trigger calcium influxes and activation of the MAPK pathways, driving the synthesis or nuclear translocation of the transcription factors including NF-AT, AP-1, and NF-{kappa}B. These events collaborate to drive IL-2 expression and T cell proliferation. Recently, several age-related defects have been reported in early signal transduction events. These changes affect TCR proximal events, including tyrosine phosphorylation of the CD3{zeta} chain (12), the phosphorylation, activation, and localization of Lck (13, 14, 15, 16, 17), the activation of Fyn (18), and the phosphorylation and activation of Zap70 (15, 19). Effects on downstream signaling events have also been reported. Age-related changes have been observed in the initiation of calcium influxes (20, 21), the activation of MAPK pathways (including the ERK and JNK pathways) (22, 23, 24, 25), and the synthesis, translocation, and activation of the transcription factors NF-AT (26), AP-1 (25, 27), and NF-{kappa}B (28, 29). Most recently, reports have indicated that immune synapse formation is impaired in aged T cells (30, 31).

Reactive oxygen species (ROS) contribute to the age-related decline in T cell function. ROS damage specific cell compartments, including the lipid moieties of membranes and enzymatic and structural proteins. Vitamin E is the most biologically active fat-soluble antioxidant, capable of neutralizing free radical damage to unsaturated fatty acids and therefore contributes to membrane stability and proper function (32). We have previously demonstrated a beneficial effect of vitamin E on T cell-mediated immune function in the aged, in both human clinical trials and in animal studies. The effects include improvement in delayed type hypersensitivity response, in vitro T cell proliferation, and IL-2 production (33, 34, 35) and improved resistance to respiratory infections (36, 37). We further demonstrated that naive, but not memory, T cells from old mice displayed the greatest age-related defects, and were uniquely responsive to vitamin E supplementation, resulting in increased cell divisions and IL-2 production in response to TCR-mediated stimulation in these cells (6). In the present study, we examine the mechanisms by which vitamin E enhances T cell function in old mice by testing the hypothesis that vitamin E induces its effect through improving effective immune synapse formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Young (4–6 mo) and old (22–26 mo) pathogen-free C57BL/6 mice obtained from the National Institute on Aging colonies at Harlan Sprague Dawley were fed autoclaved Harlan Teklad 7012 mouse chow or semisynthetic diets (see below) and water, ad libitum. Mice were housed in filtered cages and maintained at a constant temperature (23°C) with a 12-h light-dark cycle. Mice were euthanized via CO2 asphyxiation and spleens were aseptically removed and placed in sterile, endotoxin-free RPMI 1640 (BioWhittaker) medium supplemented with 25 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete RPMI; all from Invitrogen Life Technologies). All handling and animal conditions were approved by the Animal Care and Use Committee of the Jean Mayer U.S. Department of Agriculture-Human Nutrition Research Center on Aging at Tufts University. Mice exhibiting tumors, splenomegaly, grossly visible skin lesions, or significant pathology were excluded from the study.

Purification of CD4+ spleen cells by negative selection

Single-cell suspensions were acquired by gently disrupting spleens between two sterile frosted glass slides. RBC were lysed using a hemolytic ammonium chloride-based Gey’s reagent. Splenocytes depleted of RBCs were washed with RPMI complete medium and resuspended in degassed buffer composed of 1 x PBS, 2 mM EDTA, and 0.5% BSA (Sigma-Aldrich) (6). The cell suspension was incubated with a mixture of biotin-conjugated mAbs against CD8a (Ly-2), CD11b (Mac-1), CD45R (B220), DX5, and Ter119 to indirectly label CTLs, B cells, NK cells, dendritic cells, macrophages, granulocytes, and erythroid cells. The biotin-labeled, non-CD4+ T cells were then directly bound with an anti-biotin secondary Ab conjugated to superparamagnetic microbeads (all Abs from Miltenyi Biotec). After a 15-min incubation at 4°C, cells were washed and CD4+ T cells negatively selected using a MACS separation column (Miltenyi Biotec) equipped with a flow restrictor. To assess purity, negative selected cell aliquots were stained for PE-conjugated anti-CD4 along with other lymphocyte markers, such as anti-CD8 and anti-CD19 (from BD Pharmingen), and analyzed on a FACSCalibur flow cytometer (BD Biosciences). Cytofluorgraphic analysis demonstrated that ≥93% of the enriched cell population expressed CD4, a coreceptor specific for Th cells and the greatest T cell subset derived from mice spleens to demonstrate age-related changes (21). Cells purified from both young and old mice were not pooled in any experiment, thus each mouse represents an individual sample or n = 1.

Sorting of naive and memory T cells

Purified CD4+ T cells from both young and old mice were stained with 1 µg/ml PE-conjugated anti-CD44 mAb (BD Pharmingen) diluted in staining buffer (PBS/1% FBS) for 30 min, washed twice in staining buffer, and resuspended in 300 µl of staining buffer for cell sorting on a MoFlo (DakoCytomation) (Tufts Laser Cytometry Research Core Facility). Cells were sorted based on the expression patterns of the CD44 Ag on the cell surface with naive cells expressing low levels of CD44 and memory cells expressing high levels of CD44. Cells were collected in 15-ml conical tubes containing 1 ml of RPMI 1640/10% FBS on ice. Purity of cells was confirmed using anti-CD62L (a naive T cell marker) in addition to anti-CD44; it was found that sorting yielded 86–90% purity of naive CD4+ T cells and ~85% purity of memory CD4+ T cells, which is similar to other reports (38).

Vitamin E supplementation

In vitro supplementation. A 37 mg/ml stock solution of natural vitamin E was made by dissolving RRR-{alpha}-tocopherol (Henkel) in ethanol. To enhance cellular uptake, the vitamin E stock solution was mixed in FBS at a final concentration of 1 mg/ml and incubated for 1 h in a 37°C water bath, protected from the light, and periodically vortexed. Purified CD4+ T cells from young and old mice, adjusted to a final concentration of 2 x 106 cells/ml, were incubated with vitamin E at a final concentration of 20 µg/ml (46 µM) or 0.06% ethanol (vehicle control) in RPMI 1640 complete medium plus 10% FBS in a 37°C and 5% CO2 incubator for 4 h as described before (6). This level of vitamin E is equivalent to the average plasma {alpha}-tocopherol levels measured in humans taking a daily vitamin E supplement of 200 IU, which we have shown to be both safe and optimal for improving the immune response of the elderly (35, 39).

In vivo supplementation. For in vivo vitamin E supplementation, old mice (22–26 mo) were fed semisynthetic nutritionally adequate diets containing 30 (adequate level) or 500 (supplemental level) parts per million (ppm) dl-{alpha}-tocopheryl acetate for 8 wk as previously described (33). CD4+ T cells were purified from spleens as described above.

APC culture

The hybridoma cell lines expressing cell surface hamster mAbs specific for either murine CD3{epsilon} (clone 145-2C11) and ICAM-1 and B7 or the negative control mAb IgG that reacts with DNP hapten, anti-DNP (clone UC8), were both obtained from American Type Culture Collection and maintained in IMDM with 10% FBS at 37°C and 5% CO2. These hybridomas act as surrogate APCs, providing a primary signal through the TCR-CD3 complex specifically in the case of the 145-2C11 clone, thereby forming the immune synapse, which represents the initial step in T cell activation, leading to IL-2 production and cell proliferation as described by Tamir et al. (30). We, along with others (30), have found by flow cytometry that the 145-2C11 clones possess surface IgG, allowing for sufficient cell-to-cell contact with primary T cells. Tamir et al. (30) reported age-associated defects in early T cell signaling using this system, which was comparable to those observed using a pigeon cytochrome c (PCC) TCR-specific transgenic murine model activated by PCC-stimulated APCs, further endorsing the hybridoma model.

Slide preparation and microscopy

Slides were prepared and analyzed by confocal microscopy following a protocol and criteria for analysis adapted from Tamir et al. (30). CD4+ T cells, at a concentration of 4 x 106 cells/ml, were cocultured for 30 min at 37°C with either 145-2C11 anti-CD3{epsilon} hybridoma cells or UC-8 anti-DNP hybridoma cells (negative control) at a concentration of 2 x 106 cells/ml to achieve a 2:1 ratio of T cells:APCs. Fifty microliters of the cell mixture suspension was gently spread onto prewarmed poly (L-lysine)-coated slides and incubated for 30 min at room temperature to promote adherence. Slides were then fixed with 3.7% formaldehyde in PBS for 20 min at room temperature followed by washing three times with PBS. Cells were permeabilized with 0.1% Triton X-100/PBS for 10 min, washed three times with PBS, and were then blocked overnight in 1% BSA/PBS at 4°C.

Slides were stained with rabbit polyclonal Abs against Vav, LAT, Zap70, and rabbit IgG (Santa Cruz Biotechnology) at 2 µg/ml in blocking solution for 1 h at room temperature in a dark humidified chamber. Slides were washed three times with PBS and counterstained for 1 h at room temperature with 20 µg/ml FITC-conjugated goat anti-rabbit (Sigma-Aldrich) secondary Ab diluted in blocking solution, followed by another three washes. Coverslips were mounted to the slides using Slowfade light antifade kit (Molecular Probes), dried for at least 1 h at room temperature, edges were sealed with nail polish, and dried overnight protected from light at room temperature. After drying overnight, slides were coded for blind analysis and stored at 4°C protected from light until microscopic analysis.

Randomly selected conjugates were analyzed by both confocal and fluorescence microscopy. Single-color confocal analysis and imaging was performed using a Noran Odessey XL laser scanning confocal microscope system (Tufts University Confocal Microscope Core Facility). Further quantification of cell conjugates were analyzed under fluorescent microscopy using an Olympus BH2 fluorescence microscope following criteria previously established (30) including tight cell-to-cell contact between only one T cell and one APC found in the same plane. At least 50 conjugates were counted in each slide and rated as either positive or negative for signaling protein redistribution. The percentage of effective immune synapse is reported as the number of positive conjugates divided by the total number of conjugates found per slide.

Statistical analysis

Data were analyzed for the significant effects of age and vitamin E by Kruskal-Wallis one-way ANOVA using Systat 10 statistical software. Results are presented as means ± SEM. Significance was set at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD4+ T cells from old mice are defective in immune synapse formation

Fig. 1 shows representative confocal images of T cells stimulated by hybridomas (clone 145-2C11) that express Abs specific for the CD3{epsilon} subunit of the TCR/CD3 complex. A control staining performed with nonimmune serum is shown in Fig. 1A. Stains for Zap70 (Fig. 1B), LAT (Fig. 1C), Vav (Fig. 1D), and PLC{gamma} (Fig. 1E) reveal that these proteins are recruited to the immunological synapse. The degree of redistribution can be interpreted as a change in the intensity of fluorescence or green staining at the contact site of the T cell (small cell) and APC (larger cell). Conjugate formation did occur when a control hybridoma was used in place of the 2C11 hybridoma. This control APC (clone UC8) does not express an Ab specific for the TCR/CD3 complex. As expected no redistribution of signaling molecules was observed in these conjugates (Fig. 1, F–J).


Figure 1
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  		FIGURE 1. Single-cell analysis of Zap70, LAT, Vav, and PLC{gamma} redistribution in APC-stimulated CD4+ T cells. CD4+ T cells were cocultured for 30 min at 37°C with 145-2C11 anti-CD3 hybridoma cells and control UC8 anti-DNP hybridoma cells. Cells were gently spread onto slides and incubated for 30 min, followed by 3.7% formaldehyde fixing. The cells were then stained with Abs to Zap70, LAT, Vav, and PLC{gamma} followed by anti-rabbit FITC-conjugated secondary Ab. The immunofluorescent images were recorded. In each image, the T cells are the smaller cells. Each confocal image is taken in the same plane of the conjugated T cell. A, A representative of CD4+ T cell-145-2C11 hybridoma conjugate without Zap70, LAT, Vav, or PLC{gamma} redistribution. B–E, A representative of CD4+ T cell-145-2C11 hybridoma conjugates in which Zap70, LAT, Vav, and PLC{gamma} redistribution has occurred. F–J, A representative of CD4+ T cell-UC8 hybridoma conjugates without Zap70, LAT, Vav, or PLC{gamma} redistribution. Arrows show the area of greatest concentration.

 
CD4+ T cells from old mice were less likely to form effective immune synapses than CD4+ T cells from young mice. This effect was observed with four key signaling molecules, Zap70 (14 ± 2.0% vs 28 ± 2.8%, p = 0.018), LAT (12 ± 0.9% vs 27 ± 2.6%, p = 0.015), Vav (18 ± 1.8% vs 36 ± 4.4%, p = 0.018), and PLC{gamma} (19 ± 0.9% vs 26 ± 2.1%, p = 0.024) (Fig. 2). On average, the percentage of CD4+ T cell conjugates displaying effective immune synapse formation is 46% lower for aged T cells than for young T cells. These findings were not due to differences in the number of cell conjugates formed. Both age groups formed the same number of total conjugates. We also investigated the redistribution of the tyrosine kinases Fyn and Lck, the adaptor protein SLP-76 and the serine/threonine kinase PKC{theta}, but we found no significant changes with age in these molecules (data not shown).


Figure 2
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  		FIGURE 2. In vitro vitamin E supplementation restores redistribution of signaling molecules to the immune synapse in CD4+ T cells from old mice. CD4+ T cells were incubated with vitamin E (RRR-{alpha}-tocopherol) or vehicle control (0.06% ethyl alcohol) for 4 h in a 37°C and 5% CO2 incubator, then stimulated with 145-2C11 anti-CD3 hybridoma cells for 30 min, fixed, and stained with Abs against Zap70, LAT, Vav, and PLC{gamma}, followed by anti-rabbit FITC-conjugated secondary Ab, and analyzed by confocal microscopy. The broken line represents the fraction (average 8.3%) of conjugates that scored false positives using rabbit IgG in place of the primary Ab. Bars show mean ± SEM of percentage of CD4+ T cells forming immune synapse, which were positive for the indicated signaling molecule. (n = 5 for Zap70 and Vav; n = 4 for LAT; n = 3 for PLC{gamma} in each age group). *, A significant age difference in the redistribution of a signaling molecule to the immune synapse at p < 0.05; #, a significant vitamin E effect on redistribution of a signaling molecule to the immune synapse at p < 0.05 by Kruskal-Wallis one-way ANOVA.

 
Vitamin E supplementation restores defective redistribution of signaling proteins to the immune synapse in CD4+ T cells from old mice

When CD4+ T cells were supplemented in vitro with vitamin E (RRR-{alpha}-tocopherol)-enriched medium followed by stimulation by the murine CD3{epsilon} hybridoma, the percentage of conjugates displaying effective immune synapses increased, on average, by 54% in cells from old animals. The increased redistribution of Zap70, LAT, Vav, and PLC{gamma} into the immune synapse was significantly increased by vitamin E supplementation (Zap70: 14 ± 2.0% vs 26 ± 2.8%, p = 0.021; LAT: 12 ± 2.6% vs 40 ± 5.8%, p = 0.046; Vav: 18 ± 1.8% vs 38 ± 5.5%, p = 0.013; PLC{gamma}: 19 ± 0.8% vs 33 ± 2.4%, p = 0.004, in control and vitamin E supplemented cells, respectively) (Fig. 2). Young cells supplemented in vitro with vitamin E-enriched medium did not exhibit an increase in the redistribution of Zap70, LAT, Vav, or PLC{gamma} into the T cell/APC contact area (Zap70: 28 ± 2.8% vs 26 ± 2.4%, p = 0.851; LAT: 27 ± 2.6% vs 28 ± 2.3%, p = 0.581; Vav: 36 ± 4.4% vs 40 ± 7.5%, p = 0.575; PLC{gamma}: 26 ± 2.1% vs 29 ± 5.0%, p = 0.269, in control and vitamin E supplemented cells, respectively) (Fig. 2).

To ensure the validity of these in vitro data, we fed two groups of old mice (22–26 mo) a semisynthetic nutritionally adequate diet, containing 30 (adequate level) or 500 ppm vitamin E (dl-{alpha}-tocopheryl acetate), for 8 wk. Thereafter, CD4+ T cells were isolated from the spleens of these mice and stimulated with the murine CD3{epsilon} hybridoma. As can be seen in Fig. 3, cells from old mice fed 500 ppm vitamin E had significantly higher redistribution of LAT and Vav into the T cell/APC contact area compared with old mice fed 30 ppm vitamin E (LAT: 18 ± 1.6% vs 32 ± 3.4%, p = 0.009; Vav: 19 ± 1.6% vs 38 ± 2.5%, p = 0.005, in control and vitamin E-supplemented cells, respectively). In addition, cells from old mice fed 500 ppm vitamin E showed a trend of a higher redistribution of Zap70 into the T cell/APC contact area compared with old mice fed 30 ppm vitamin E (16 ± 1.3% vs 27 ± 4.7%, p = 0.095, in control and vitamin E-supplemented cells, respectively) (Fig. 3).


Figure 3
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  		FIGURE 3. In vivo vitamin E supplementation of old mice increases redistribution of signaling molecules to the immune synapse. Old mice were fed semisynthetic nutritionally adequate diets containing 30 ppm (adequate) or 500 ppm (supplemented) vitamin E (dl-{alpha}-tocopheryl acetate) for 8 wk. CD4+ T cells were isolated from these mice and stimulated with 145-2C11 anti-CD3 hybridoma cells for 30 min, fixed, and stained with Abs to Zap70, LAT, and Vav, followed by anti-rabbit FITC-conjugated secondary Ab, and analyzed by confocal microscopy. The broken line represents the fraction (average 8%) of conjugates that scored false positives using rabbit IgG in place of the primary Ab. Bars show mean ± SEM of percentage of CD4+ T cells forming immune synapse, which were positive for the indicated signaling molecule. (n = 5 for LAT and Vav; n = 4 for Zap70 in each age group). *, A significant vitamin E effect on redistribution of a signaling molecule to the immune synapse at p < 0.05; #, p < 0.1 for vitamin E-enhancing redistribution of a signaling molecule to the immune synapse by Kruskal-Wallis one-way ANOVA.

 
Naive, but not memory, CD4+ T cells from old mice exhibit significant defective redistribution of signaling proteins to the immune synapse

A major shift occurs in the T cell compartment of the immune system with age, including a gradual decline in the number of naive T cells and an increase in the number of Ag-primed memory T cells (7, 8). Previously, we demonstrated that naive T cells exhibit the greatest age-related defect, which is specifically reversed by vitamin E supplementation, enhancing IL-2 production and cell proliferation in these cells from old, but not young, mice (6). Vitamin E, however, did not have an effect on percent naive or memory T cells (6). This led to the hypothesis that vitamin E primarily enhances immune synapse formation by affecting the naive CD4+ T cells of old mice.

CD4+ T cells purified from young and old mice were labeled with PE-conjugated anti-CD44 mAb, because this transmembrane glycoprotein is expressed at a low level in naive cells (CD44low), but at high levels in memory cells (CD44high). Based on these phenotypic characteristics, the cells were sorted by a MoFlo into naive and memory cell subsets and supplemented with vitamin E for 4 h. The cells were then stimulated by the murine CD3{epsilon} hybridoma for 30 min, stained for Zap70, LAT and Vav, and analyzed by confocal microscopy, as previously discussed. Aged naive CD4+ T cells (CD44low) were on average 35% less likely to form effective immune synapses compared with young naive CD4+ T cells (Fig. 4). The redistribution of the signaling proteins Zap70 and Vav was significantly lower in the naive cells from old mice compared with naive cells from the young mice (Zap70: 18 ± 2.0% vs 25 ± 1.8%, p = 0.026; Vav: 20 ± 1.7% vs 38 ± 3.5%, p = 0.003, in old and young mice, respectively). A smaller redistribution of the adaptor protein LAT was also seen in naive T cells from old mice compared with young mice (LAT: 20 ± 4.1% vs 28 ± 4.5%, in old and young mice, respectively), but this difference did not reach statistical significance. Unlike aged naive cells, redistribution of the signaling proteins Zap70, LAT, and Vav was not significantly different between young and old memory CD4+ T cells (CD44high) (Zap70: 28 ± 4.8% vs 29 ± 4.3%, p = 0.900; LAT: 19 ± 3.6% vs 24 ± 4.3%, p = 0.434; Vav: 27 ± 4.6% vs 33 ± 4.8%, p = 0.394, in old and young mice, respectively) (Fig. 5).


Figure 4
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  		FIGURE 4. In vitro vitamin E supplementation increases redistribution of signaling molecules to the immune synapse in naive CD4+ T cells from old mice. Sorted CD44low naive CD4+ T cells were incubated with vitamin E (RRR-{alpha}-tocopherol) or vehicle control (0.06% ethyl alcohol) for 4 h in a 37°C and 5% CO2 incubator and then stimulated with 145-2C11 anti-CD3 hybridoma cells for 30 min, fixed, and stained with Abs against Zap70, LAT, and Vav, followed by anti-rabbit FITC-conjugated secondary Abs, and analyzed by confocal microscopy. The broken line represents the fraction (average 9.8%) of conjugates that scored false positives using rabbit IgG in place of the primary Ab. Bars show mean ± SEM of percentage of naive CD4+ T cells forming immune synapse, which were positive for the indicated signaling molecule. (n = 7 for LAT and Vav; n = 6 for Zap70 in each age group). *, A significant age difference in the redistribution of a signaling molecule to the immune synapse at p < 0.05; #, a significant vitamin E effect on redistribution of a signaling molecule to the immune synapse at p < 0.05 by Kruskal-Wallis one-way ANOVA.

 

Figure 5
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  		FIGURE 5. In vitro vitamin E supplementation increases LAT, but not Vav or Zap70, redistribution to the immune synapse in old memory CD4+ T cells from old mice. Sorted CD44high CD4+ memory T cells were incubated with vitamin E ({alpha}-tocopherol) or vehicle control (0.06% ethyl alcohol) for 4 h in a 37°C and 5% CO2 incubator, followed by stimulation with 145-2C11 anti-CD3 hybridoma cells for 30 min. The cells were then fixed, and stained with Abs against Zap70, LAT, and Vav, followed by anti-rabbit FITC-conjugated secondary Abs, and analyzed by confocal microscopy. The broken line represents the fraction (average 8.5%) of conjugates that scored false positives using rabbit IgG in place of the primary Ab. Bars show mean ± SEM of percentage of memory CD4+ T cells forming immune synapse, which were positive for the indicated signaling molecule. (n = 7 for Vav, and LAT; n = 6 for Zap70 in each age group). #, A significant vitamin E effect on redistribution of a signaling molecule to the immune synapse at p < 0.05 by Kruskal-Wallis one-way ANOVA.

 
Vitamin E supplementation improved immune synapse formation mainly in old naive CD4+ T cells

Naive CD4+ T cells from old mice, supplemented with vitamin E, showed increased immune synapse formation compared with those treated with vehicle control (on average 39% higher). Redistribution of the signaling proteins Zap70, LAT, and Vav were all significantly increased in old naive T cells, supplemented with vitamin E, compared with old naive T cells, treated with a vehicle control (Zap70: 18 ± 2.0% vs 28 ± 1.0%, p = 0.005; LAT: 20 ± 4.1% vs 33 ± 3.0%, p = 0.038; Vav: 20 ± 1.7% vs 35 ± 4.3%, p = 0.017, in vehicle and vitamin E-supplemented old naive T cells, respectively) (Fig. 4). Naive T cells from young mice supplemented with vitamin E did show a significant increase in the redistribution of Zap70 to the immune synapse. It is unsure, however, if this enhancement results in a functional change downstream because we do not see an increase in IL-2 production or proliferation in young T cells supplemented with vitamin E (6). When memory T cells from old mice were supplemented with vitamin E, redistribution of the adaptor protein LAT (19 ± 3.6% vs 30 ± 5.1%, p = 0.001, in vehicle and vitamin E-supplemented old memory T cells, respectively), but not the guanine nucleotide exchange factor Vav or the protein kinase Zap70, was significantly increased (Zap70: 28 ± 4.8% vs 31 ± 4.6%, p = 0.531; Vav: 27 ± 4.6% vs 35 ± 5.0%, p = 0.114, in vehicle and vitamin E-supplemented old memory T cells, respectively) (Fig. 5). Thus, it is the naive T cells that exhibit the greatest age-related defect in immune synapse formation. We demonstrated here, for the first time, that supplemental vitamin E has a direct immunoenhancing effect; reversing the functional defect of aged naive T cells through increased redistribution of signaling proteins to the immune synapse.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previously, we demonstrated that naive T cells from aged mice possess a reduced ability to produce IL-2 and to proliferate and that supplementation with vitamin E, a potent lipid soluble antioxidant, was able to reverse these age-associated functional defects (6). In the current study, we investigated the mechanism by which vitamin E reverses the age-associated functional defects in naive T cells. Specifically, we evaluated the recruitment of signaling molecules to the immune synapse, an important structure implicated in the regulation of T cell activation and proliferation.

Impairment of T cell function as a result of aging has been well-documented. Studies have shown multiple defects in the early signaling cascade initiated by TCR stimulation in old mice, resulting in various functional defects (12, 21, 22, 23). These defects in signaling have been attributed to a decrease in the redistribution of key signaling molecules, such as LAT and Vav, to the immune synapse formed at the site of the T cell-APC interaction (30). To investigate the effect of vitamin E on immune synapse formation, we supplemented purified CD4+ T cells from young and old mice, in vitro, with vitamin E. In agreement with previous studies (30), we demonstrate that CD4+ T cells from old mice have on average a 46% lower chance of forming an effective immune synapse compared with cells from young mice, as measured by the ineffective redistribution of the tyrosine kinase Zap70 and signaling proteins LAT, Vav, and PLC{gamma} (Fig. 2). In this study, however, we did not see a significant age effect on redistribution of Fyn, Lck, SLP-76, and PKC{theta}. Previous studies by Miller and colleagues (30, 38), using the same system, reported declines in PKC{theta} clustering to the immune synapse in CD4+ T cells from old mice. However, the authors also demonstrated that a percentage of cells relocalized PKC{theta} without association with LAT, possibly indicating that the defect in LAT may not be associated with a defect in PKC{theta} and that the key age-associated defect is specific for LAT (30). Our unpublished data confirms this finding that LAT activation, specifically its phosphorylation, is defective with age. In addition, vitamin E supplementation increased the redistribution of the four key signaling molecules, Zap70, LAT, Vav, and PLC{gamma} to the immune synapse on average by 54%, but had no effect on CD4+ T cells from young mice (Fig. 2). Similar results were found when mice were supplemented in vivo, i.e., CD4+ T cells from old mice fed 500 ppm vitamin E, had significantly higher effective immune synapse formation (on average 45% higher) compared with those fed 30 ppm (basal or adequate levels) of vitamin E (Fig. 3).

The intrinsic shift in the proportion of T cells that have not encountered Ag (naive) to those that have (memory) is a major change that influences T cell function in the aged (7, 8). Age-related functional changes in both the memory (40, 41) and naive (31, 42) T cell subpopulations have been reported. In particular, using TCR-transgenic mice specific for PCC, Garcia and Miller (31) reported that naive T cells isolated from old mice were unable to translocate signaling proteins to the immune synapse and that those that did form a synapse had less cytoplasmic migration of NFAT, a transcription factor essential for IL-2 transcription.

Previously, we showed that vitamin E increases IL-2 production by old naive, but not memory, T cells. Here, we hypothesized that vitamin E enhances the redistribution of early signaling proteins to the immune synapse of naive T cells, thereby resulting in enhanced IL-2 transcription and ultimately proliferation. Significant age-related declines in immune synapse formation, as indicated by redistribution of Zap70 and Vav, were only observed within the naive (CD44low) T cell subpopulation; namely, old naive CD4+ T cells were on average 35% less likely to form an effective immune synapse (Figs. 4 and 5). However, both naive and memory T cells showed a nonsignificant decrease in LAT redistribution (20.4 vs 28.3% in old vs young naive cells and 19.4 vs 24.0% old vs young memory cells). These data suggest that the age-related defects in Zap70 and Vav redistribution observed in mixed CD4+ T cells are mainly due to changes in naive T cells. In contrast, both naive and memory cells may contribute to the defective redistribution of LAT in mixed CD4+ T cells. In vitro vitamin E supplementation of old naive CD4+ T cells reversed these age-related declines in protein redistribution, enhancing immune synapse formation on average by 39% (Fig. 4). Although vitamin E supplementation did not have an effect on redistribution of Zap70 or Vav in memory cells, it did have a significant effect on that of LAT.

Increased generation of oxygen free radicals with age was first postulated in 1956 (43). Subsequent studies implicated oxygen free radicals in age-associated damage to cellular proteins and lipid moieties, supporting the hypothesis that these free radicals promote declines in the health of the aged (44, 45). Reports have established that exposure of T cells to oxidative stress inhibits IL-2 production, which is a hallmark of age-associated defects in T cell activation (46, 47). Dietary (33, 34, 35, 39, 48) and in vitro (6, 49, 50, 51, 52) antioxidants have been shown to partially reverse the age-associated decline in the T cell response. Here, we demonstrate, for the first time, that the effect of the antioxidant, vitamin E, is mediated through improvements in the recruitment of signaling molecules to the immune synapses formed by naive T cells. Naive T cells are more susceptible to ROS than memory T cells. Along these lines naive T cells where shown to have lower levels of the endogenous antioxidant glutathione (53, 54).

The mechanism of vitamin E enhancement of immune synapse formation has not been determined. The antioxidant effect of vitamin E may influence the fine structure of the plasma membrane. The plasma membranes of T cells are not homogenous. In fact, microdomains composed primarily of cholesterol and sphingolipids, commonly known as lipid rafts, are found throughout the plasma membrane and have been shown to be essential for effective immune synapse formation (55). Oxidative stress can prevent lipid raft-associated proteins from entering the microdomains (56), and can prevent the structural modifications required for the entry of these proteins into lipid microdomains (57). Thus, vitamin E could improve immune synapse formation by affecting the association of signaling proteins within these microdomains. Alternatively, vitamin E could maintain the activation or phosphorylation of specific signaling molecules. The observation that the effect of vitamin E is specific to some, but not all signaling proteins, supports this theory.

In summary, our results show that the age-associated defect in the redistribution of signaling molecules to the immunological synapse is reversed by vitamin E. This effect is strongest in naive T cells, which exhibit the age-related defects in protein recruitment and T cell activation. This is the first demonstration of a reversal of a key early signaling defect in aged T cells by a nutrient. These findings have important implications for the development of preventive and therapeutic strategies to reduce age-associated defects in T cells. Further studies are needed to elucidate the mechanism by which vitamin E specifically enhances the recruitment of signaling molecules into the immune synapses formed by the naive T cells of old mice.


   Acknowledgments
 
We are grateful for the assistance of Allen Parmelee with the sorting of naive and memory T cells; Drs. Rob Willson and Lai Ding for assistance in confocal imaging; and Dr. K. Eric Paulson for helpful discussion and suggestions regarding the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the National Institute on Aging Grant R01-AG009140-10A1, Office of Dietary Supplement, the U.S. Department of Agriculture, Agriculture Research Service under Contract Number 58-1950-9-001, a Unilever Health Institute fellowship, and an Ellison Medical Foundation-International Nutrition Foundation fellowship. Back

2 M.G.M. and T.A. had equal contribution to the work presented. Back

3 Current address: International Centre for Diarrheal Disease Research Immunology Laboratory, Laboratory Sciences Division, G.P.O. Box 128, Dhaka 1000 Bangladesh. Back

4 Address correspondence and reprint requests to Dr. Simin Nikbin Meydani, Nutritional Immunology Laboratory, Jean Mayer Human Nutrition Research Center on Aging, Tufts University, 711 Washington Street, Boston, MA 02111. E-mail address: simin.meydani{at}tufts.edu Back

5 Abbreviations used in this paper: LAT, linker for activation of T cells; SLP-76, Src homology 2 domain-containing leukocyte protein of 76 kDa; PKC, protein kinase C; PLC, phospholipase C; ROS, reactive oxygen species; PCC, pigeon cytochrome c; ppm, parts per million. Back

Received for publication October 5, 2006. Accepted for publication November 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Miller, R. A.. 1991. Aging and immune function. Int. Rev. Cytol. 124: 187-215. [Medline]
  2. Ben-Yehuda, A., M. E. Weksler. 1992. Host resistance and the immune system. Clin. Geriatr. Med. 8: 701-711. [Medline]
  3. Makinodan, T., M. M. Kay. 1980. Age influence on the immune system. Adv. Immunol. 29: 287-330. [Medline]
  4. Thoman, M. L., W. O. Weigle. 1981. Lymphokines and aging: interleukin-2 production and activity in aged animals. J. Immunol. 127: 2102-2106. [Abstract]
  5. Nagel, J. E., R. K. Chopra, F. J. Chrest, M. T. McCoy, E. L. Schneider, N. J. Holbrook, W. H. Adler. 1988. Decreased proliferation, interleukin 2 synthesis, and interleukin 2 receptor expression are accompanied by decreased mRNA expression in phytohemagglutinin-stimulated cells from elderly donors. J. Clin. Invest. 81: 1096-1102. [Medline]
  6. Adolfsson, O., B. T. Huber, S. N. Meydani. 2001. Vitamin E-enhanced IL-2 production in old mice: naive but not memory T cells show increased cell division cycling and IL-2-producing capacity. J. Immunol. 167: 3809-3817. [Abstract/Free Full Text]
  7. Ernst, D. N., M. V. Hobbs, B. E. Torbett, A. L. Glasebrook, M. A. Rehse, K. Bottomly, K. Hayakawa, R. R. Hardy, W. O. Weigle. 1990. Differences in the expression profiles of CD45RB, Pgp-1, and 3G11 membrane antigens and in the patterns of lymphokine secretion by splenic CD4+ T cells from young and aged mice. J. Immunol. 145: 1295-1302. [Abstract]
  8. Miller, R. A.. 1996. The aging immune system: primer and prospectus. Science 273: 70-74. [Abstract]
  9. Rothenberg, E. V., S. B. Ward. 1996. A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation. Proc. Natl. Acad. Sci. USA 93: 9358-9365. [Abstract/Free Full Text]
  10. Carlin, L. M., K. Yanagi, A. Verhoef, E. N. Nolte-’t Hoen, J. Yates, L. Gardner, J. Lamb, G. Lombardi, M. J. Dallman, D. M. Davis. 2005. Secretion of IFN-{gamma} and not IL-2 by anergic human T cells correlates with assembly of an immature immune synapse. Blood 106: 3874-3879. [Abstract/Free Full Text]
  11. Dustin, M. L., D. R. Colman. 2002. Neural and immunological synaptic relations. Science 298: 785-789. [Abstract/Free Full Text]
  12. Garcia, G. G., R. A. Miller. 1997. Differential tyrosine phosphorylation of {zeta} chain dimers in mouse CD4 T lymphocytes: effect of age. Cell Immunol. 175: 51-57. [Medline]
  13. Tinkle, C. W., D. Lipschitz, U. Ponnappan. 1998. Decreased association of p56lck with CD4 may account for lowered tyrosine kinase activity in mitogen-activated human T lymphocytes during aging. Cell Immunol. 186: 154-160. [Medline]
  14. Guidi, L., L. Antico, C. Bartoloni, M. Costanzo, A. Errani, A. Tricerri, M. Vangeli, G. Doria, L. Gatta, C. Goso, et al 1998. Changes in the amount and level of phosphorylation of p56lck in PBL from aging humans. Mech. Ageing Dev. 102: 177-186. [Medline]
  15. Fulop, T., Jr, D. Gagne, A. C. Goulet, S. Desgeorges, G. Lacombe, M. Arcand, G. Dupuis. 1999. Age-related impairment of p56lck and ZAP-70 activities in human T lymphocytes activated through the TCR/CD3 complex. Exp. Gerontol. 34: 197-216. [Medline]
  16. Larbi, A., N. Douziech, G. Dupuis, A. Khalil, H. Pelletier, K. P. Guerard, T. Fulop, Jr. 2004. Age-associated alterations in the recruitment of signal-transduction proteins to lipid rafts in human T lymphocytes. J. Leukocyte Biol. 75: 373-381. [Abstract/Free Full Text]
  17. Hosea, H. J., E. S. Rector, C. G. Taylor. 2005. Age-related changes in p56lck protein levels and phenotypic distribution of T lymphocytes in young rats. Clin. Dev. Immunol. 12: 75-84. [Medline]
  18. Whisler, R. L., S. E. Bagenstose, Y. G. Newhouse, K. W. Carle. 1997. Expression and catalytic activities of protein tyrosine kinases(PTKs) Fyn and Lck in peripheral blood T cells from elderly humans stimulated through the T cell receptor (TCR)/CD3 complex. Mech. Ageing Dev. 98: 57-73. [Medline]
  19. Chakravarti, B., D. N. Chakravarti, J. Devecis, B. Seshi, G. N. Abraham. 1998. Effect of age on mitogen induced protein tyrosine phosphorylation in human T cell and its subsets: down-regulation of tyrosine phosphorylation of ZAP-70. Mech. Ageing Dev. 104: 41-58. [Medline]
  20. Miller, R. A., B. Jacobson, G. Weil, E. R. Simons. 1987. Diminished calcium influx in lectin-stimulated T cells from old mice. J. Cell Physiol. 132: 337-342. [Medline]
  21. Grossmann, A., L. Maggio-Price, J. C. Jinneman, P. S. Rabinovitch. 1991. Influence of aging on intracellular free calcium and proliferation of mouse T-cell subsets from various lymphoid organs. Cell Immunol. 135: 118-131. [Medline]
  22. Gorgas, G., E. R. Butch, K. L. Guan, R. A. Miller. 1997. Diminished activation of the MAP kinase pathway in CD3-stimulated T lymphocytes from old mice. Mech. Ageing Dev. 94: 71-83. [Medline]
  23. Kirk, C. J., R. A. Miller. 1998. Analysis of Raf-1 activation in response to TCR activation and costimulation in murine T-lymphocytes: effect of age. Cell Immunol. 190: 33-42. [Medline]
  24. Liu, B., K. W. Carle, R. L. Whisler. 1997. Reductions in the activation of ERK and JNK are associated with decreased IL-2 production in T cells from elderly humans stimulated by the TCR/CD3 complex and costimulatory signals. Cell Immunol. 182: 79-88. [Medline]
  25. Whisler, R. L., M. Chen, L. Beiqing, K. W. Carle. 1997. Impaired induction of c-fos/c-jun genes and of transcriptional regulatory proteins binding distinct c-fos/c-jun promoter elements in activated human T cells during aging. Cell Immunol. 175: 41-50. [Medline]
  26. Pahlavani, M. A., M. D. Harris, A. Richardson. 1995. The age-related decline in the induction of IL-2 transcription is correlated to changes in the transcription factor NFAT. Cell Immunol. 165: 84-91. [Medline]
  27. Whisler, R. L., L. Beiqing, M. Chen. 1996. Age-related decreases in IL-2 production by human T cells are associated with impaired activation of nuclear transcriptional factors AP-1 and NF-AT. Cell Immunol. 169: 185-195. [Medline]
  28. Trebilcock, G. U., U. Ponnappan. 1996. Evidence for lowered induction of nuclear factor {kappa}B in activated human T lymphocytes during aging. Gerontology 42: 137-146. [Medline]
  29. Trebilcock, G. U., U. Ponnappan. 1998. Nuclear factor-{kappa}B induction in CD45RO+ and CD45RA+ T cell subsets during aging. Mech. Ageing Dev. 102: 149-163. [Medline]
  30. Tamir, A., M. D. Eisenbraun, G. G. Garcia, R. A. Miller. 2000. Age-dependent alterations in the assembly of signal transduction complexes at the site of T cell/APC interaction. J. Immunol. 165: 1243-1251. [Abstract/Free Full Text]
  31. Garcia, G. G., R. A. Miller. 2001. Single-cell analyses reveal two defects in peptide-specific activation of naive T cells from aged mice. J. Immunol. 166: 3151-3157. [Abstract/Free Full Text]
  32. Machlin, L. J., A. Bendich. 1987. Free radical tissue damage: protective role of antioxidant nutrients. FASEB J. 1: 441-445. [Abstract]
  33. Meydani, S. N., M. Meydani, C. P. Verdon, A. A. Shapiro, J. B. Blumberg, K. C. Hayes. 1986. Vitamin E supplementation suppresses prostaglandin E12 synthesis and enhances the immune response of aged mice. Mech. Ageing Dev. 34: 191-201. [Medline]
  34. Meydani, S. N., M. P. Barklund, S. Liu, M. Meydani, R. A. Miller, J. G. Cannon, F. D. Morrow, R. Rocklin, J. B. Blumberg. 1990. Vitamin E supplementation enhances cell-mediated immunity in healthy elderly subjects. Am. J. Clin. Nutr. 52: 557-563. [Abstract/Free Full Text]
  35. Meydani, S. N., M. Meydani, J. B. Blumberg, L. S. Leka, G. Siber, R. Loszewski, C. Thompson, M. C. Pedrosa, R. D. Diamond, B. D. Stollar. 1997. Vitamin E supplementation and in vivo immune response in healthy elderly subjects: a randomized controlled trial. J. Am. Med. Assoc. 277: 1380-1386. [Abstract]
  36. Han, S. N., D. Wu, W. K. Ha, A. Beharka, D. E. Smith, B. S. Bender, S. N. Meydani. 2000. Vitamin E supplementation increases T helper 1 cytokine production in old mice infected with influenza virus. Immunology 100: 487-493. [Medline]
  37. Meydani, S. N., L. S. Leka, B. C. Fine, G. E. Dallal, G. T. Keusch, M. F. Singh, D. H. Hamer. 2004. Vitamin E and respiratory tract infections in elderly nursing home residents: a randomized controlled trial. J. Am. Med. Assoc. 292: 828-836. [Abstract/Free Full Text]
  38. Yang, D., R. A. Miller. 1999. Cluster formation by protein kinase C{theta} during murine T cell activation: effect of age. Cell Immunol. 195: 28-36. [Medline]
  39. Meydani, S. N., M. Meydani, J. B. Blumberg, L. S. Leka, M. Pedrosa, R. Diamond, E. J. Schaefer. 1998. Assessment of the safety of supplementation with different amounts of vitamin E in healthy older adults. Am. J. Clin. Nutr. 68: 311-318. [Abstract]
  40. Hobbs, M. V., W. O. Weigle, D. J. Noonan, B. E. Torbett, R. J. McEvilly, R. J. Koch, G. J. Cardenas, D. N. Ernst. 1993. Patterns of cytokine gene expression by CD4+ T cells from young and old mice. J. Immunol. 150: 3602-3614. [Abstract]
  41. Philosophe, B., R. A. Miller. 1990. Diminished calcium signal generation in subsets of T lymphocytes that predominate in old mice. J. Gerontol. 4: B87-B93.
  42. Linton, P. J., L. Haynes, N. R. Klinman, S. L. Swain. 1996. Antigen-independent changes in naive CD4 T cells with aging. J. Exp. Med. 184: 1891-1900. [Abstract/Free Full Text]
  43. Harman, D.. 1956. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11: 298-300. [Medline]
  44. Stadtman, E. R.. 1992. Protein oxidation and aging. Science 257: 1220-1224. [Abstract/Free Full Text]
  45. Ames, B. N., M. K. Shigenaga, T. M. Hagen. 1993. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 90: 7915-7922. [Abstract/Free Full Text]
  46. Flescher, E., J. A. Ledbetter, G. L. Schieven, N. Vela-Roch, D. Fossum, H. Dang, N. Ogawa, N. Talal. 1994. Longitudinal exposure of human T lymphocytes to weak oxidative stress suppresses transmembrane and nuclear signal transduction. J. Immunol. 153: 4880-4889. [Abstract]
  47. Pahlavani, M. A., M. D. Harris. 1998. Effect of in vitro generation of oxygen free radicals on T cell function in young and old rats. Free Radic. Biol. Med. 25: 903-913. [Medline]
  48. Furukawa, T., S. N. Meydani, J. B. Blumberg. 1987. Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice. Mech. Ageing Dev. 38: 107-117. [Medline]
  49. Wu, D., S. N. Meydani, J. Sastre, M. Hayek, M. Meydani. 1994. In vitro glutathione supplementation enhances interleukin-2 production and mitogenic response of peripheral blood mononuclear cells from young and old subjects. J. Nutr. 124: 655-663. [Abstract/Free Full Text]
  50. Beharka, A. A., D. Wu, S. N. Han, S. N. Meydani. 1997. Macrophage prostaglandin production contributes to the age-associated decrease in T cell function which is reversed by the dietary antioxidant vitamin E. Mech. Ageing Dev. 93: 59-77. [Medline]
  51. Wu, D., M. Meydani, A. A. Beharka, M. Serafini, K. R. Martin, S. N. Meydani. 2000. In vitro supplementation with different tocopherol homologues can affect the function of immune cells in old mice. Free Radic. Biol. Med. 28: 643-651. [Medline]
  52. Franklin, R. A., Y. M. Li, S. Arkins, K. W. Kelley. 1990. Glutathione augments in vitro proliferative responses of lymphocytes to concanavalin A to a greater degree in old than in young rats. J. Nutr. 120: 1710-1717. [Abstract/Free Full Text]
  53. Lohmiller, J. J., K. M. Roellich, A. Toledano, P. S. Rabinovitch, N. S. Wolf, A. Grossmann. 1996. Aged murine T-lymphocytes are more resistant to oxidative damage due to the predominance of the cells possessing the memory phenotype. J. Gerontol. A Biol. Sci. Med. Sci. 51: B132-B140. [Abstract]
  54. Kim, H. J., A. E. Nel. 2005. The role of phase II antioxidant enzymes in protecting memory T cells from spontaneous apoptosis in young and old mice. J. Immunol. 175: 2948-2959. [Abstract/Free Full Text]
  55. Bromley, S. K., W. R. Burack, K. G. Johnson, K. Somersalo, T. N. Sims, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin. 2001. The immunological synapse. Annu. Rev. Immunol. 19: 375-396. [Medline]
  56. Gringhuis, S. I., A. Leow, E. A. Papendrecht-Van Der Voort, P. H. Remans, F. C. Breedveld, C. L. Verweij. 2000. Displacement of linker for activation of T cells from the plasma membrane due to redox balance alterations results in hyporesponsiveness of synovial fluid T lymphocytes in rheumatoid arthritis. J. Immunol. 164: 2170-2179. [Abstract/Free Full Text]
  57. Cemerski, S., J. P. van Meerwijk, P. Romagnoli. 2003. Oxidative-stress-induced T lymphocyte hyporesponsiveness is caused by structural modification rather than proteasomal degradation of crucial TCR signaling molecules. Eur. J. Immunol. 33: 2178-2185. [Medline]



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