An Age-Related Decrease in Rescue from T Cell Death Following Costimulation Mediated by CD28

CELLULAR IMMUNOLOGY ARTICLE NO.

170, 141–148 (1996)

0144

An Age-Related Decrease in Rescue from T Cell Death Following Costimulation Mediated by CD28 CHRISTIAN R. ENGWERDA,1 BARRY S. HANDWERGER,*,†

AND

BARBARA S. FOX*,2

Division of Rheumatology and Clinical Immunology, Department of Medicine, and *Department of Microbiology and Immunology, University of Maryland at Baltimore, Baltimore, Maryland 21201; and †Baltimore VA Medical Center, Baltimore, Maryland 21201 Received December 11, 1995; accepted January 26, 1996

We previously reported that T cell proliferation in response to a primary signal through the T cell receptor (TCR) and a costimulatory signal via the CD28 molecule is impaired in healthy, aged mice. Here we extend these studies to examine factors which may be involved in this defect in T cells from aged mice. To determine if age-related changes in cytokine production might be responsible, splenic T cells from young (2–4 months) and aged (20–26 months) mice were stimulated with immobilized anti-CD3e and soluble antiCD28 mAbs in the presence of exogenous IL-2, IL-4, IFN-g, IL-1a, or IL-6. No improvement in the proliferative response of T cells from aged mice was found following the addition of any cytokine. In addition, the decreased proliferative response of T cells from aged mice was not caused by the enhanced production of IFN-g or other inhibitory factors. Interestingly, despite the age-related reduction in proliferation, no significant difference was found in the percentage of live cells entering the S, G2 , or M phase of the cell cycle in stimulated T cells from young and aged mice. Instead, anti-CD28-mediated costimulation was found to rescue T cells from young mice from anti-CD3e-induced cell death, but did not rescue T cells from aged mice. This failure of T cells from aged mice to respond to costimulatory signals appears to contribute to the decreased proliferation observed from cultures containing these cells, and may be involved in many other age-related alterations in immunological responsiveness. q 1996 Academic Press, Inc.

INTRODUCTION The increased susceptibility of elderly individuals to various infectious and neoplastic diseases has been 1 To whom correspondence should be addressed at Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT. Fax: 44-171-636-8739. Email: [email protected]. 2 Present address: ImmuLogic Pharmaceutical Corporation, 610 Lincoln Street, Waltham, MA 02154.

well documented (1, 2). This age-related decline in immune responsiveness has been associated with many factors, including alterations in the functions of T cells. T cells isolated from elderly humans and mice display a reduced capacity to proliferate in response to a variety of stimuli, as well as a decreased ability to provide help for immunoglobulin (Ig) production by B cells, generate delayed type-hypersensitivity (DTH), and activate cytotoxic T lymphocytes (1, 2). In addition, there is a well documented, age-related change in T cell cytokine production following stimulation (3–10). There are at least two possible explanations for alterations in T cell function with increasing age. There could either be age-related changes in the response of all T cells to stimulation, or an accumulation of a subpopulation of T cells which respond differently. One subpopulation of T cells which increases with age is memory T cells (11). The accumulation of memory T cells may contribute to the age-related alterations in T cell responses because they have different activation requirements than do naive T cells (3, 11–13) and produce a different pattern of cytokines (3–10), including less IL-2, and more IL-4 and IFN-g, consistent with age-related changes in T cell cytokine production. However, we and others have shown that both naive and memory T cells from aged animals exhibit reduced responsiveness to various stimuli, compared with corresponding populations from young animals (11, 14, 15). Therefore, the accumulation of memory T cells is not alone the cause of decreased immune function. Several investigators have reported intrinsic changes in the response of T cells from aged animals to stimulation, including reduced protein kinase C (PKC) activity (16), Ca2/ mobilization (16, 3), and generation of inositol phosphates (16). In addition, age-related alterations in mitogen-induced protein phosphorylation patterns in T cells also have been observed (17, 18). Again, the accumulation of memory T cells does not appear to explain all of these changes because both naive and memory T cell subpopulations from aged mice appear to be affected (17). Because these signaling events are critical to the various outcomes of T cell activation, includ-

141

AID

CI 8108

/

6c0e$$$181

04-26-96 14:43:28

cia

0008-8749/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

AP: Cell Immuno

142

ENGWERDA, HANDWERGER, AND FOX

ing progression into cell cycle and programmed cell death (apoptosis) (19–21), these observations suggest that stimulation of T cells from aged animals could result in different responses, compared with T cells from young animals. Our increasing understanding of the specific activation requirements of T cells and the signaling pathways and functional events associated with them now enable us to examine specific age-related alterations in T cell function associated with specific T cell activation events. T cell activation requires a primary signal delivered through the antigen-specific receptor (TCR) and a second, costimulatory signal. CD28-mediated costimulation is required for IL-2 production and T cell proliferation (reviewed in 22, 23), and may also be involved in the initiation of programmed cell death (24–26). We have reported previously that there is an agerelated decrease in T cell proliferation following stimulation with a primary signal through the CD3e chain and a costimulatory signal via CD28 molecules (15). In this report, we have extended these findings to determine factors involved in reduced proliferation of T cells from aged mice. There are at least three possible reasons why changes in response to stimulation could result in reduced proliferation. First, age-related alterations in cytokine production may be responsible. Second, failure of aged T cells to progress into cell cycle could be involved, or, finally, T cells from aged animals may have an increased propensity to die following stimulation. Here we report that no cytokine we tested was able to improve the proliferative response of T cells from aged mice or to reduce the proliferative response of T cells from young mice. There was an age-related reduction in proliferation following activation with immobilized anti-CD3e and soluble anti-CD28 mAbs; however, no significant age-related difference was found in the percentage of live cells entering the cell cycle. Instead, an increased number of T cells isolated from aged mice failed to be rescued from anti-CD3einduced death by CD28-mediated costimulation in culture. This observation may explain many of the agerelated alterations in T cell function. MATERIALS AND METHODS Animals. Male C57BL/6NCr1BR and CBA/CaHNNia mice were purchased from Charles River Laboratories (Wilmington, MA), under contract with the National Institute of Aging. T cells isolated from two to four young adult (2 to 4 months) or old (20 to 26 months) mice were pooled, separately, and used in paired experiments. Mice found to have tumors, grossly visible skin lesions, or significant pathology were not used. Through the use of a sentinel mouse program, mice were serologically monitored on a regular basis and were free of adventitious bacterial, viral, or mycoplasma infections.

AID

CI 8108

/

6c0e$$$182

04-26-96 14:43:28

Antibodies and cytokines. Purified mAbs used for staining and flow cytometry included phycoerythrin (PE)-conjugated anti-IL-2R a chain (3C7), PE-conjugated rat IgG2b,k (isotype control for 3C7), and FITCconjugated anti-Thy-1.2 (30-H12) (all from Pharmingen, San Diego, CA). Recombinant murine IL-2, IL-4, and interferon-g (IFN-g) were purchased from Pharmingen. Recombinant human IL-1a (Biological Response Modifier Program, NIH, Washington, DC) and IL-6 (Genzyme, Cambridge, MA) were also used in studies. Monoclonal Abs used in T cell cultures included anti-CD28 (37.51) (27), anti-CD3e chain (1452C11) (28), and anti-IFN-g (R46A2) (29) mAbs. Purified and ammonium sulfate-precipitated ascites fluid of anti-CD28 mAb was used. Anti-IFN-g and anti-CD3e mAbs were used as ammonium sulfate-precipitated and unpurified ascites fluid, respectively. Cell preparations. Animals were killed by CO2 asphyxiation and spleens were aseptically removed. Single cell suspensions were prepared in tissue culture medium (TCM) consisting of RPMI 1640 (GIBCO Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Paragon Biotech, Baltimore, MD), 2 mM L-glutamine, 50 mM 2-mercaptoethanol, and antibiotics (50 mg/ml gentamicin and 10 U/ml penicillin). Erythrocytes were lysed at 5 1 107 lymphocytes/ml in lysing buffer (140 mM NH4Cl, 17 mM Tris, pH 7.2) for 5 min at room temperature. Lysis was terminated by adding excess TCM and T cells were purified by incubation on nylon wool (Robbins Scientific, Sunnyvale, CA) for 1 hr at 377C (30). T cells were collected in the effluent and contained ú90% Thy-1/ and õ5% major histocompatability complex (MHC) class II/ cells, as assessed by flow cytometry. These cells did not proliferate in the presence of soluble antiCD3e mAb, indicating that the T cell populations were not contaminated by significant Ig-receptor (FcR)/ cells or nonspecifically activated by purification with nylon wool. Purification also did not alter the ratio of CD44hi:CD44lo or CD4/:CD8/ T cells. T cell proliferation. T cells were cultured in 96-well, flat-bottomed, microculture plates (Corning, Corning, NY) and stimulated with 100 mg/ml immobilized (platebound) anti-mouse CD3e mAb (145-2C11) and 10 mg/ ml soluble anti-mouse CD28 mAb (37.51). Anti-CD3e mAb (145-2C11 ascites), diluted in phosphate-buffered saline (PBS), was added to microculture plates (50 ml) and incubated for 16 hr at 47C. Wells were then washed three times in PBS prior to addition of T cells in TCM. T cells were cultured in triplicate wells, incubated at 377C in humidified 5% CO2 and pulsed with 0.5 mCi of [3H]thymidine for 12 hr prior to harvesting onto glass fiber filters and counting. Flow cytometric analysis of cell surface markers. Cells (1 1 106) were incubated for 30 min on ice with

cia

AP: Cell Immuno

AGE-RELATED INCREASE IN T CELL DEATH

either PE- or FITC-labeled mAb diluted in 100 ml FACS buffer (1% heat-inactivated FCS in PBS, 0.02% NaN3) which contained 3% mouse serum. Labeled cells were analyzed on a Becton Dickinson FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Gates were set on lymphocytes, using forward and side scatter, and data were plotted on a log scale. Cell cycle analysis. The progression of T cells into the S, G2 , and M stages of the cell cycle was measured using techniques, as previously described (31). Briefly, cultured T cells were first stained with FITC-labeled anti-Thy1.2 mAb as described above, and then fixed in 2 ml 70% ethanol at 47C for at least 60 min. Cells were then washed in 1 ml PBS and resuspended in 0.5 ml PBS to which 0.5 ml RNase (Type I-A, Sigma, St. Louis, MO, 1 mg/ml in PBS) and 1 ml propidium iodide (PI, Sigma, 100 mg/ml in PBS) were added. Cells were then mixed gently and incubated in the dark at room temperature for 15 min. Cells were kept at 47C in the dark until analyzed. Gates were set on lymphocytes, using forward and side scatter, and then set on Thy1.2/ cells, using the green fluorescence due to FITC staining. An additional gate was set for cells containing diploid DNA content or greater, using the red fluorescence due to PI staining of nuclear DNA. These cells were then analyzed for progression into cell cycle on a Coulter Elite flow cytometer (Coulter Corp., Hialeah, FL) in the facility of Dr. Marcello Sztein, Center for Vaccine Development, UMAB, using Elite software (Coulter Corp.). Identification of dead T cells. Dead T cells were identified by the presence of fragmented DNA using the technique described by Nicoletti et al. (32). Briefly, cultured T cells were processed and gated as described above, except that cells containing hypodiploid DNA content (fragmented DNA) were also included in the analysis and differentiated on the flow cytometer using Elite software (Coulter Corp.). Statistical analysis. Data were log-transformed to minimize variation. The significance of differences among mean values obtained in cell proliferation, fluorescence intensity (MFI), and percentages of cells in various stages of the cell cycle between age groups was tested using the paired t test. This test was performed with Statview software (Cricket Software, Philadelphia, PA). RESULTS T Cell Proliferation in the Presence of Exogenous T Cell Growth Factors We previously reported that T cell proliferation is impaired in healthy, aged mice when stimulated via the CD3e chain and CD28 molecule (15). To determine if this age-related defect was caused by altered cytokine production, we compared the ability of T cells from

AID

CI 8108

/

6c0e$$$182

04-26-96 14:43:28

143

young and aged mice to proliferate in the presence and absence of exogenously added cytokines. Anti-CD3e mAb stimulation resulted in a small increase in proliferation. This response was increased significantly upon the addition of anti-CD28 mAb and a striking increase in proliferation of T cells from young mice was found in comparison with proliferation of T cells from aged mice, which was consistent with our previous report (15). The addition of either 1000 U/ml IL-2 or 100 U/ ml IL-4, major autocrine T cell growth cytokines, resulted in a small increase in proliferation in cultures of T cells from young and aged mice that were not stimulated with mAbs or were stimulated with antiCD28 mAb alone (Fig. 1). The addition of these cytokines to T cells from both young and aged mice that were stimulated with immobilized anti-CD3e mAb alone resulted in a significant increase (n Å 6; P õ 0.05) in proliferation, similar to levels seen when T cells were stimulated with combined immobilized antiCD3e and soluble anti-CD28 mAbs. When these cytokines were added to T cells stimulated with anti-CD3e and anti-CD28 mAbs, no significant increase in proliferation was found for T cells from either young or aged animals. Following the addition of rIL-2 or rIL-4 to cultures, the proliferation of T cells from aged mice in response to anti-CD3e mAb alone, or the combination of anti-CD3e and anti-CD28 mAbs, was always significantly less (n Å 6; P õ 0.05) than that observed in T cells from young mice. Therefore, the addition of IL-2 or IL-4 did not reverse the age-associated proliferative defect, indicating that changes in production of these T cell growth factors is not alone responsible for the age-related reduction in T cell proliferation. To ensure that these results did not reflect the specific culture conditions used, a range of variables were examined. An age-related decrease in T cell proliferation was still seen when cell densities were varied from 5 1 103 to 105 cells/culture well, the concentrations of cytokines added to cultures were increased 10-fold or decreased 100-fold, or the cytokines were added to cultures 24 hr after initial stimulation (data not shown). The addition of either IL-1a (1000 U/ml) or IL-6 (1000 U/ml) to T cell cultures also failed to overcome the agerelated defect. Similarly, the addition of IFN-g (1000 U/ml) or anti-IFN-g mAb (100 mg/ml) to cultures did not affect the proliferation of T cells from young or aged mice (data not shown). T Cell Proliferation in the Presence of Potential T Cell Inhibitory Factors To determine if some factor produced by T cells from aged mice inhibited T cell proliferation, T cells from aged mice were mixed (1:1) with those from young mice. If an inhibitory factor were produced, the proliferation of the mixed cell cultures would be expected to be comparable to that of T cells from aged mice alone. Figure 2 shows

cia

AP: Cell Immuno

144

ENGWERDA, HANDWERGER, AND FOX

FIG. 1. rIL-2 and rIL-4 are unable to increase proliferation of T cells from aged mice, when costimulated via CD28. T cells (105 cells/ well) isolated from young and aged C57BL/6 mice were cultured with soluble anti-CD28 mAb (10 mg/ml), immobilized anti-CD3e mAb (100 mg/ml), and either rIL-2 (1000 U/ml) or rIL-4 (100U/ml), as indicated, for 48 hr. Data represent the mean and SEM of triplicate cultures. Asterisk indicates that proliferation was less than 500 cpm.

a representative experiment of three performed. Proliferation of the mixed cell culture was the approximate average of that observed in T cells from young and aged mice cultured in isolation. It was also approximately equal to the sum of the proliferative response seen when half the number of T cells (0.5 1 T cells) from young and aged

FIG. 2. T cells from aged mice do not produce factors which inhibit T cell proliferation. T cells (a total of 2 1 104 cells/well or 1 1 104 cells/well (0.51)) isolated from young and aged C57BL/6 mice were cultured with soluble anti-CD28 mAb (10 mg/ml) and immobilized anti-CD3e mAb (100 mg/ml) for 72 hr. Data represent the mean and SEM of triplicate cultures.

AID

CI 8108

/

6c0e$$$183

04-26-96 14:43:28

mice were separately cultured. These observations suggest that T cells from aged mice are not producing a factor which inhibits T cell proliferation. Percentage of T Cells Entering the Cell Cycle In order to test if there was an age-related difference in the progression of T cells into cell cycle, we directly measured the number of live T cells which progressed into S, G2 , or M phases of the cell cycle. Figure 3 shows a representative experiment of five performed. T cells from both young and aged mice did not progress into S, G2 , or M phases of the cell cycle when cultured in TCM or with anti-CD28 mAb for up to 48 hr (data not shown). Following activation with anti-CD3e mAb for 24 hr, 12– 37% of T cells from both young and aged mice had progressed into S, G2 , or M phases of the cell cycle (data not shown). After 48 hr, 30–70% had progressed into the S, G2 , or M phase. Although the addition of anti-CD28 mAb to anti-CD3e mAb-stimulated cultures consistently enhanced the percentage of cells progressing into cell cycle, over five experiments, this enhancement was not statistically significant. No significant age-related difference in the percentage of T cells progressing into cell cycle was found. This surprising result indicated that the age-related defect in proliferation was not reflected in entry of cells into the cell cycle. Percentage of Cells That Die Following Stimulation One possible explanation for the discrepancy between the proliferation data (Fig. 1) and the cell cycle

cia

AP: Cell Immuno

AGE-RELATED INCREASE IN T CELL DEATH

145

FIG. 3. The percentage of T cells from young and aged mice that progress into S, G2 , or M phases of cell cycle is not different. PI DNA fluorescence distributions of T cells (105 cells/well) from young and aged C57BL/6 mice were analyzed following 48 hr of stimulation with soluble anti-CD28 mAb (10 mg/ml) and immobilized anti-CD3e mAb (100 mg/ml), as indicated. T cells were harvested, stained, and analyzed as described under cell cycle analysis of Materials and Methods. The DNA distribution of T cells indicated the presence of at least two populations including G0 /G1 cells defined in the A1 region and S/G2 /M cells defined in the A2 region. Numbers above each region indicate the percentage of cells contained within that region.

data (Fig. 3) is that an increasing number of T cells from aged mice may die in culture following stimulation. These dead cells would affect the outcome of proliferation assays by failing to incorporate [3H]thymidine, but would be excluded from cell cycle analysis because they fall outside the live gates which were used for analysis. We confirmed that a greater percentage of T cells from aged mice did indeed die following stimulation when T cells containing hypodiploid DNA (fragmented DNA) were included in the analysis (Fig. 4). In five experiments, the number of T cells which contained hypodiploid DNA was minimal following culture with either TCM or anti-CD28 mAb alone. At both 24 and 48 hr, 2–10% of cells from both young and aged mice contained fragmented DNA. Others (19) have reported up to 21% of unstimulated T cells contained fragmented DNA following similar culture periods. It is possible that differences in experimental conditions or

AID

CI 8108

/

6c0e$$$183

04-26-96 14:43:28

gating used for analysis may have resulted in the lower values reported here. Over five experiments, the percentage of T cells from young mice that contained hypodiploid DNA increased to 10–18 and 19–46% at 24 and 48 hr, respectively, following activation with anti-CD3e mAb. The percentage of T cells from aged mice that contained hypodiploid DNA similarly increased to 14– 26 and 28–51% at both 24 and 48 hr, respectively, following activation with the same stimulus. The differences between young and aged mice were not statistically significant. Interestingly, the addition of anti-CD28 mAb to antiCD3e mAb-stimulated cultures resulted in a significant (n Å 5; P õ 0.05) reduction in the percentage of T cells from young mice containing hypodiploid DNA at both 24 hr (data not shown) and 48 hr (Fig. 4). These data suggest that anti-CD28 mAb rescued a percentage of T cells from young mice from programmed cell death. In

cia

AP: Cell Immuno

146

ENGWERDA, HANDWERGER, AND FOX

FIG. 4. An age-related decrease in rescue from anti-CD3e-induced T cell death by costimulation mediated by CD28. PI DNA fluorescence distributions of T cells (105 cells/well) isolated from young and aged C57BL/6 mice were analyzed following 48 hr of stimulation with soluble anti-CD28 mAb (10 mg/ml) and immobilized anti-CD3e mAb (100 mg/ml), as indicated. DNA distribution of T cells indicated the presence of at least three populations, including cells with a decreased (hypodiploid) DNA content (region B1), viable G0 /G1 cells (region B2), and viable S/G2 /M cells (region B3). Numbers above each region indicate the percentage of cells contained within that region.

contrast, when T cells from aged mice were examined, addition of anti-CD28 mAb did not result in a significant difference in the percentage of T cells containing hypodiploid DNA. The percentage of T cells from aged mice that contained hypodiploid DNA remained at 15– 23 and 29–48% at 24 and 48 hr, respectively, whereas the percentage of T cells from young mice that contained hypodiploid DNA decreased to 7–10 and 12– 24% at 24 and 48 hr, respectively. Although no agerelated increase in T cell death (cells containing hypodiploid DNA) was found following stimulation with anti-CD3e mAb at both 24 and 48 hr, when T cells were stimulated with combined anti-CD3e and anti-CD28 mAbs, a significantly greater percentage (P õ 0.05) of T cells from aged mice died. This result suggests that signaling through the CD28 molecule rescues T cells from young animals from death induced by a primary signal through the TCR, but not T cells from aged mice.

AID

CI 8108

/

6c0e$$$183

04-26-96 14:43:28

This result was confirmed by staining stimulated T cells with trypan blue (data not shown). In three experiments, a significantly greater (P õ 0.05) percentage of T cells from aged mice were unable to exclude this dye, following stimulation with anti-CD3e and anti-CD28 mAbs for 24 or 48 hr compared to T cells from young mice that were stimulated in an identical manner. DISCUSSION Age-related changes in T cell function have been associated with reduced proliferation in response to a variety of stimuli, accompanied by changes in cytokine production (1, 2). Previously, we demonstrated that proliferation in response to a primary signal through the TCR and a costimulatory signal via the CD28 molecule is impaired in T cells from healthy, aged mice (15). This defect in response to costimulation mediated by

cia

AP: Cell Immuno

147

AGE-RELATED INCREASE IN T CELL DEATH

CD28 was found in both CD4/ and CD8/ T cells and in CD44lo (naive) and CD44hi (memory) T cells. In the present study, we found that the addition of either T cell growth factors or potential T cell growth inhibitors did not change the age-related reduction in proliferation. Others have found that neither IL-2 or IL-4 is able to reverse the decreased proliferation of T cells from aged mice (33). These results strongly suggest that the defect in proliferation of T cells from aged mice is not solely caused by the lack of the production of T cell growth factors, such as IL-2, IL-4, and IL-6, or the excessive production of T cell growth inhibitors. The ligation of the TCR in the absence of costimulatory signals results in death of thymocytes (20) and Th1 clones (34). Data presented in this study indicate that splenic T cells also die more frequently following the ligation of the TCR with anti-CD3e mAb in the absence of costimulatory signals. The induction of cell death in Th1 clones by anti-CD3e mAb can be prevented by the addition of accessory cells to culture (34). However, the precise mechanism of stimulation provided by accessory cells was not identified. Our results suggest that ligands of CD28 on accessory cells—e.g., B7-1 and B7-2—are able to rescue splenic T cells from young mice from anti-CD3e-induced cell death. However, T cells from aged mice are defective in their ability to be rescued from anti-CD3e-induced cell death by CD28-mediated costimulation. Several previous reports have suggested that CD28-mediated costimulation can prevent activation-induced death of T cells. Thus, CD28-mediated costimulation has been shown to prevent mitogen-induced apoptosis of CD4/ T cells from HIV-infected patients (24), prevent apoptosis in the murine thymus (25), and enhance the survival of both human and murine T cells by upregulating expression of the cell survival factor Bcl-xL (26). Together, these data and the results presented in this study suggest that CD28 plays an important role in regulating cell survival following T cell activation and that T cells from aged mice have a defect in this regulatory pathway. Our finding of an age-related decrease in the percentage of T cells that were rescued from cell death following costimulation with anti-CD28 mAb is consistent with several earlier observations. It already has been established that there are age-related alterations in signaling cascades following TCR engagement (3, 16– 18), and apoptosis is known to be an active process involving cellular signaling (19–21). In addition, recent studies indicate that the CD28 cytoplasmic domain participates in signal transduction by binding to phosphoinositide 3-kinase and by lipid kinase activity, as well as triggering both Ca2/-dependent and Ca2/-independent pathways (22, 23). Therefore, it appears that following CD28-mediated costimulation, the signal transduction pathways in T cells from aged animals

AID

CI 8108

/

6c0e$$$183

04-26-96 14:43:28

are altered in such a way that they have a reduced capacity to rescue cells from TCR-mediated cell death. An age-related increase in the number of T cells that die following CD28-mediated costimulation could explain many previous findings. Fewer cells would be available to respond to environmental antigens (e.g., infectious agents), resulting in many of the immune alterations characterized by aging, including decreased T cell proliferative responses and reduced T cell help for B cells, DTH responses, and cytotoxic T cell responses. However, the extent to which an age-related increase in T cell death alters T cell function in vivo is at present unclear. Future studies will be aimed at determining the incidence of death among specific T cell subpopulations. In addition, experiments will be conducted to determine if alterations in specific signaling pathways, or components of these pathways, are involved in the age-related increase in T cell death following costimulation mediated by CD28. Results from these studies could enable strategies to be developed which are aimed at modulating specific T cell signaling pathways to improve the immune responsiveness of elderly individuals. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grant AG-10207. We thank M. Sztein and M. Tanner for assistance with the cell cycle analysis, J. Allison for the 37.51 hybridoma, and N. Dordai for technical support. We also thank P. Kaye and T. Mynott for critical review of the manuscript.

REFERENCES 1. Thoman, M. L., and Weigle, W. O., Adv. Immunol. 46, 221, 1989. 2. Miller, R. A., J. Gerontology 44, B4, 1989. 3. Ernst, D. N., Hobbs, M. V., Torbett, B. E., Glasebrook, A. L., Rehse, M. A., Bottomly, K., Hayakawa, K., Hardy, R. R., and Weigle, W. O., J. Immunol. 145, 1295, 1990. 4. Swain, S. L., McKenzie, D. T., Weinberg, A. D., and Hancock, W., J. Immunol. 141, 3445, 1988. 5. Budd, R. C., Cerrotini, J-C., and MacDonald, H. R., J. Immunol. 138, 3583, 1987. 6. Lee, W. Y., Yin, X-M., and Vitetta, E. S., J. Immunol. 144, 3288, 1990. 7. Bottomly, K., Luqman, M., Greenbaum, L., Carding, S., West, J., Pasqualini, T., and Murphy, D. B., Eur. J. Immunol. 19, 617, 1989. 8. Hobbs, M. V., Ernst, D. N., Torbett, B. E., Glasebrook, A. L., Rehse, M. A., McQuitty, D. N., Thomann, M. L., Bottomly, K., Rothermal, A. L., Noonan, D. J., and Weigle, W. O., J. Cell Biochem. 46, 312, 1991. 9. Hobbs, M. V., Weigle, W. O., Noonan, D. J., Torbett, B. E., McEvilly, R. J., Koch, R. J., Cardenas, G. J., and Ernst, D. N., J. Immunol. 150, 3602, 1993. 10. Ernst, D. N., Weigle, W. O., Noonan, D. J., McQuitty, D. N., and Hobbs, M. V., J. Immunol. 151, 575, 1993. 11. Lerner, A., Yamada, T., and Miller, R. A., Eur. J. Immunol. 19, 977, 1989.

cia

AP: Cell Immuno

148

ENGWERDA, HANDWERGER, AND FOX

12. Miller, R., Jacobson, F., Weil, G., and Simons, E., J. Cell Physiol. 132, 337, 1987. 13. Flurkey, K., Stadecker, M., and Miller, R. A., Eur. J. Immunol. 22, 931, 1992. 14. Philosophe, B., and Miller, R. A., J. Gerontol. Biol. Sci. 45, B87, 1990. 15. Engwerda, C. R., Handwerger, B. S., and Fox, B. S., J. Immunol. 152, 3740, 1994. 16. Proust, J. J., Filburn, C. R., Harrison, S. A., Buchholz, M. A., and Nordin, A. A., J. Immunol. 139, 1472, 1987. 17. Patel, H. R., and Miller, R. A., Eur. J. Immunol. 22, 253, 1992. 18. Shi, J., and Miller, R. A., J. Immunol. 151, 730, 1993. 19. Perandones, C. E., Illera, V. A., Peckham, D., Stunz, L. L., and Ashman, R. F., J. Immunol. 151, 3521, 1993. 20. Smith, C. A., Williams, G. T., Kingstone, R., Jenkinson, E. J., and Owen, J. J. T., Nature 337, 181, 1989. 21. Newell, M. K., Haughn, L. J., Maroun, C. R., and Julius, M. H., Nature 347, 286, 1990. 22. June, C. H., Bluestone, J. A., Nadler, L. M., and Thompson C. B., Immunol. Today 15, 321, 1994.

AID

CI 8108

/

6c0e$$$184

04-26-96 14:43:28

23. Linsley, P. S., and Ledbetter, J. A., Annu. Rev. Immunol. 11, 191, 1993. 24. Groux, H., Torpier, G., Monte, D., Mouton, Y., Capron, A., and Amiesen, J. C., J. Exp. Med. 175, 331, 1992. 25. Shi, Y., Radvanyi, L. G., Sharma, A., Shaw, P., Green, D. R., Miller, R. G., and Mills, G. B., J. Immunol. 155, 1829, 1995. 26. Boise, L. H., Minn, A. J., Noel, P. J., June, C. H., Accavitti, M. A., Lindsten, T., and Thompson, C. B., Immunity 3, 87, 1995. 27. Gross, J. A., St. John, T., and Allison, J. P., J. Immunol. 144, 3201, 1990. 28. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E., and Bluestone, J. A., Proc. Natl. Acad. Sci. USA 84, 1374, 1987. 29. Spitalny, G., and Hall, E., J. Exp. Med. 159, 1560, 1984. 30. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. 31. Shapiro, N. M., ‘‘Practical Flow Cytometry,’’ A. R. Liss, New York, 1988. 32. Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F., and Riccardi, C., J. Immunol. Methods 139, 271, 1991. 33. Nagelkerken, L., Hertogh-Huijbregts, A., Dobber, R., and Drager, A., Eur. J. Immunol. 21, 273, 1991. 34. Liu, Y., and Janeway, C. A., J. Exp. Med. 172, 1735, 1990.

cia

AP: Cell Immuno