Journals of Gerontology Series A: Biological Sciences and Medical Sciences Large Type Edition
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ortiz-Suárez, A.
Right arrow Articles by Miller, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ortiz-Suárez, A.
Right arrow Articles by Miller, R. A.
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 58:B1063-B1073 (2003)
© 2003 The Gerontological Society of America

Antigen-Independent Expansion of CD28hi CD8 Cells From Aged Mice: Cytokine Requirements and Signal Transduction Pathways

Anavelys Ortiz-Suárez1 and Richard A. Miller1,2,3

1 Cellular and Molecular Biology Graduate Program
2 Department of Pathology, University of Michigan School of Medicine, Ann Arbor.
3 University of Michigan Geriatrics Center, and VA Medical Center, Ann Arbor, Michigan.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Memory CD8+ T cells from old mice can proliferate in nonirradiated recipients. Transfer of labeled cells from aged donors into young recipients showed that proliferation of aged donor CD8 cells requires host cells that can both respond to interferon-{gamma} and produce interleukin-15. Reisolation of transferred CD8 cells from host mice showed that LAT (linker for activated T cells) translocation to the immunological synapse, and translocation of NF (nuclear factor)-{kappa}B to the nucleus were diminished in recovered CD8 T cells from old donors, whether they had divided in vivo or not. Cells able to proliferate in vivo could be isolated based on their unusually high levels of CD28 expression, but were found not to differ from other aged CD8 cells in their low levels of LAT and protein kinase C-theta (PKC-{theta}) translocation to the immunological synapse. Thus in vivo proliferation of CD28hi CD8 cells from aged mice cannot be attributed to retention of T-cell receptor signaling.

PERIPHERAL T-lymphocyte numbers are closely regulated by homeostatic mechanisms (1–4). The overall size and composition of the mature T-cell pool are likely to be controlled by multiple factors that influence both proliferation and survival. For instance, T cells undergo "homeostatic" proliferation upon adoptive transfer into T-cell-depleted syngeneic hosts (1–4). Recently, it has been shown that homeostasis of naïve T cells requires signals delivered by major histocompatibility complex (MHC) molecules and the cytokine interleukin (IL)-7 (5–10). In contrast to naïve T cells, homeostasis of memory T cells is known to be regulated independently of contact with MHC molecules (1,3). In addition to proliferating in response to T-cell depletion, memory T cells undergo intermittent cell division even under normal, i.e., nonlymphopenic conditions (11,12). Despite these control circuits, the proportion of memory T cells increases with age in both the CD4 and CD8 lineages (13). Furthermore, the CD8+ T-cell repertoire becomes less diverse in old age due to reduced thymic output (13) and accumulation of clonally expanded memory CD8+ T cells (14–26). These clones can represent as much as 80% of the CD8+ T cells in rare individuals (22). Expanded CD8+ T cells clones appear in middle-aged mice, and longitudinal studies have shown that their frequency increases rapidly after the age of 18 months (27). Most of these mouse CD8+ T-cell clones are long lasting but nevertheless transient, and rarely, if ever, undergo malignant transformation. Hence, the T-cell repertoire of aged mice seems highly unstable, rather than terminally altered, although infrequent stable alterations can sometimes be seen (27). However, the mechanisms that select and maintain such clonal expansions are not yet understood. We have recently shown (28) that the proportion of memory CD8+ T cells able to proliferate in a nonirradiated recipient increases with age, and suggested that this age-dependent change is likely to contribute to the accumulation of expanded CD8 cell clones in old mice (14).

Experiments with agents that stimulate the innate immune system, such as poly I:C and lipopolysaccharide (LPS), have shown that cytokines regulate the antigen-independent turnover of CD8 cells. Injecting these nonspecific stimulators into mice causes a brief burst of T-cell receptor (TCR)-independent (bystander) proliferation, largely restricted to a subset of CD8+ cells with a memory (CD44hi) phenotype (29–32). Poly I:C and LPS stimulate T cells via production of interferons (IFNs), including both IFN-I and IFN{gamma} (31). The inability of IFNs to stimulate purified T cells in vitro suggested that T-cell proliferation in vivo requires IFN-induced synthesis of another cytokine that, in turn, acts directly on CD44hi CD8+ cells. IL-15 seemed a likely candidate because IFNs and IFN-inducing agents induce strong IL-15 mRNA synthesis by antigen-presenting cells (APCs) in vitro (33). Furthermore, IL-15 causes proliferation of purified CD8+ CD44hi T cells (but not CD4+ CD44hi T cells) and mimics the capacity of IFNs to stimulate these cells in vivo (34). Most importantly, IL-15 has been implicated in the normal turnover of CD8+ CD44hi T cells in vivo (35–37).

Like memory phenotype CD8+ T cells (38,39), cells of the expanded clones divide slowly in nonirradiated mice by a process that is probably antigen independent, driven by IL-15, and inhibited by IL-2 (39). However, the proliferating progeny of the large CD8+ T cell clones accumulate, in vivo, more rapidly than the progeny of nonclonal CD8+ T cells (39). It has been speculated (39) that increased sensitivity to the stimulatory effects of IL-15 or lowered sensitive to the inhibitory effects of IL-2 may contribute to the accumulation of clonally expanded CD8 T cells in older mice. However, IL-2 does not seem to exert its inhibitory effects directly on memory CD8+ T cells, but through intermediary cells (40). We have recently shown that the memory CD8+ T cells in old mice that are able to divide in nonirradiated recipients express high levels of CD28 both prior to and after injection into host recipients, and that these cells produce high levels of IFN{gamma} (28). The experiments described in this article were designed to determine whether IFN{gamma} plays a role in the proliferation of memory CD8+ T cells in old mice, and, in addition, to determine if those cells able to proliferate in vivo differed from other CD8 cells of aged mice in their relatively poor biochemical responses to TCR-mediated stimulation.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Mice
Specific-pathogen-free male (BALB/cByJ x C57BL/6)F1 (CByB6F1) and C57BL/6 (B6) mice were purchased from the National Institute on Aging's contract colonies at Harlan (Indianapolis, IN). The generation and description of B6.IL-15-/-, B6.IFN{gamma}-/-, and B6.IFN{gamma}R-/- have been described previously (35,41). B6.IL-15-/- mice were gifts from Dr. Jacques Peschon (Immunex Corp.). B6.IFN{gamma}-/- and B6.IFN{gamma}R-/- mice were purchased from The Jackson Laboratory. Unless otherwise stated, "young" mice were used at 4–8 months of age, and "old" mice were 18–25 months old. Mice were housed for at least 2 weeks after shipment in a specific-pathogen-free holding colony at the University of Michigan, and given free access to food and water. Sentinel animals from this colony were examined quarterly for serological evidence of viral infection; all such tests were negative during the experimental period. Mice that were found to have splenomegaly or macroscopically visible tumors at the time of sacrifice were not used for any experiments.

Cell Lines
B-cell hybridomas expressing monoclonal hamster antibodies (Abs) specific for murine CD3{epsilon} (clone 145-2C11 [2C11]) or DNP (clone UC8) (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 with 10% FCS (fetal calf serum) and 2 mM L-glutamine (RP-FC [RPMI-1640 medium with added FCS]) at 37°C and 10% CO2. Flow cytometric analyses confirmed that both types of hybridoma cells not only express membrane-bound immunoglobulin (Ig) but also display significant surface levels of the costimulatory ligands B7 and ICAM-1 (unpublished data).

For each T-cell stimulation experiment, 2C11 and UC8 cells in log-phase growth were harvested, washed two times with prewarmed (37°) HBSS, and then resuspended at 4 x 106 cells/ml in RP-FC.

Antibodies
All phycoerythrin (PE)- and CyChrome (CyC)-conjugated monoclonal antibodies (mAbs) were purchased from PharMingen (San Diego, CA). These included PE-labeled antibodies to mouse CD4, CD8, CD44, CD28, and immunoglobulin G (IgG) and CyC-labeled antibodies to mouse CD8, CD4, and IgG. Rabbit antimouse IgG was purchased from ICN (Costa Mesa, CA). Rat antimouse nuclear factor (NF)-{kappa}B (clone F-6), rat antimouse LAT (linker for activated T cells), rat antimouse protein kinase C-theta (PKC-{theta}), Alexa-594 goat antirat Fc{gamma}, and Alexa-594 donkey antigoat Fc{gamma} were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Purification of T Cells
To prepare T cells, spleens were gently rubbed between frosted glass slides to obtain single cell suspensions in HBSS containing 0.2% bovine albumin (HBSS-BSA). Erythrocytes were removed by centrifugation over Lympholyte-M (Cedarlane Laboratory, Ontario, Canada), and adherent cells and B cells were depleted by panning over rabbit antimouse IgG Ab-coated plates.

T-Cell Transfer, CFSE Staining, and Flow Cytometry Analysis
Purified cells were labeled with 5 µM CFSE (carboxy fluorescein diacetate succinimide ester) at 1 x 107 cells/ml in PBS/0.1% BSA for 10 minutes at 37°C. Cells were then washed once with cold PBS/0.1% BSA and cold HBSS-BSA. CFSE-labeled cells were injected intravenously into the lateral tail vein of nonirradiated, syngeneic young mice in 200 µl of PBS/0.1% BSA; each recipient received an equal number of donor cells in any single experiment.

At day 12, cells from spleens and pooled lymph nodes (brachial, axillary, and inguinal) of recipient mice were isolated and stained with the appropriate cell surface marker antibodies for 30 minutes on ice. Cells were then washed with PBS/1% BSA and fixed with 4% paraformaldehyde for 30 minutes on ice. Cells were resuspended in PBS/1% BSA for flow cytometric analysis.

Purification of T Cells Based on In Vivo Proliferation History
To study the early phases of T-cell activation, T cells from aged donors and young donors (control) were labeled with CFSE as described above, injected into young recipients, recovered from the spleen and lymph nodes at day 12, and stained with anti-CD8 CyC. CD8+ T cells were then purified by cell-sorting (using a FACSVantage instrument from Becton-Dickinson) on the basis of their CFSE signal. This approach yielded 3 populations of CFSE-labeled CD8+ T cells: 1) those from old donors that had not divided even once in the host (Old N-Div), 2) those from the old donors that had divided after injection (Old Div), and 3) those from the young donors that had not divided (Young N-Div). After sorting, cells were resuspended at 4 x 106 cells/ml in RP-FC, and then incubated at 37° for 15 minutes prior to further analysis.

Purification of CD28hi and CD28lo T Cells
For T-cell stimulation experiments involving CD8+ CD28hi T cells, T cells from aged and young mouse spleens and lymph nodes were stained with anti-CD8 CyC and anti-CD28 PE. T cells were then purified by cell-sorting on the basis of their CD28 levels (CD28hi or CD28lo). This method produced 3 populations of CD8+ cells: 1) CD28hi from old mice, 2) CD28lo from old mice, and 3) CD28lo from young mice. After sorting, cells were resuspended at 4 x 106 cells/ml in RP-FC.

Analysis of T-Cell Signaling Molecules by Confocal Microscopy
Cells purified either by differences in CD28 expression or by differences in proliferative history in vivo were coincubated with equal volumes of 2C11 (anti-CD3) or UC8 (control) hybridoma cells at a 1:1 ratio. In some cases, where indicated, PMA (100 ng/ml) and ionomycin (500 ng/ml) were used to activate the cells. Cell mixtures were incubated at 37°C for 30 minutes and then gently resuspended and spread (50–100 µl/slide) onto prewarmed poly-L-lysine-coated slides (Sigma, St. Louis, MO). Slides were incubated for another 30 minutes at 37° to promote cell attachment. The cells were then fixed in freshly prepared 3.7% formaldehyde/PBS for 15 minutes, and then finally washed three times with PBS. Slides were permeabilized with 0.1% Triton X/PBS for 10 minutes before three washes in PBS. Slides were then placed in blocking solution (1% BSA/0.1% NaN3/PBS) and stored at 4° for at least 24 hours. Slides were then stained with the appropriate rat antimouse antibody for 1 hour at 4°C. The slides were then developed by incubation for 1 hour (each Ab) at 4°C with Alexa-594 goat antirat Fc{gamma} and Alexa-594 donkey antigoat Fc{gamma} (NF-{kappa}B staining only); three washes with PBS were performed between Abs incubations. The slides were washed several times with PBS and mounted as described elsewhere (42). We do not have numerical data on the proportion of cells from each group that could form a conjugate, although our qualitative impression is that this did not differ dramatically among the different experimental groups. Our conclusions are all based on the proportion of conjugates that go on to various stages of activation. We counted at least 100 conjugates (per sample) for each determination, using coded slides to ensure that the evaluations were done without knowledge of the specific experimental group.

Statistical Analyses
To estimate the proportion of injected cells that were able to proliferate in vivo, we used a quantitative analysis that has been described before (43). It relies on the fact that the size of each subset of proliferating T cells is related to the size of the corresponding precursor subset by a function of 2n, where n is the number of division cycles achieved in the recipient mice. Therefore, if a specific fluorescence peak corresponding to "n" mitotic divisions contains "E" cells, then the number of precursor T cells ("P") which must have divided n times to generate them can be estimated as E divided by 2n. The total number of original precursor cells needed to account for the observed progeny can be calculated as the sum of the "P" values for each of the peaks, one for each mitotic cycle, in the histogram. The percentage of cells in the recovered population that has given rise to a clone of proliferating progeny in vivo can then be calculated as the ratio of the sum of the "P" values to the total number of CFSE cells recovered (the sum of "P" plus the number of CFSE-positive nonproliferating cells).

Data are presented in the text and figures as mean ± SEM (standard error of mean), and N indicates the number of individual experiments. Comparisons among different varieties of donor cells and recipient mice were made using analysis of variance.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
We have previously shown using CFSE-tagged cells that the proportion of CD8+ T cells able to proliferate in a nonirradiated recipient increases with age in mice (28). This earlier work showed that the proliferating cells in aged mice were members of a CD8 memory subset, absent from young donors, and characterized by unusually high levels of cell surface CD28, and that these CD28hi cells produced large amounts of IFN{gamma} both before and after transfer to recipient mice. Since IFN{gamma} has been implicated in the in vivo proliferation of memory CD8+ T cells in some systems (29–32), we sought to explore the role of IFN{gamma} in the proliferation of CD8+ T cells from old mice in nonirradiated recipients.

Proliferation of CD8 T Cells From Old Donors in IFN{gamma} and IFN{gamma}R Mutant Host Mice
IFN{gamma} has been shown to be involved in the induction of bystander proliferation of memory CD8+ T cells in vivo (29–32). However, it has been reported that IFN{gamma} failed to cause proliferation of purified CD44hi CD8+ cells in vitro; in fact, under these conditions IFN{gamma} had a strong antiproliferative effect on T cells (33). We therefore hypothesized that if IFN{gamma} produced by aged CD8+ CD28hi T cells played a role in their proliferation in vivo, it would not be in an autocrine fashion but rather by inducing the synthesis of a second cytokine that in turn acted directly on the CD44hi CD8+ cells. T cells from young and old B6 mice were labeled with CFSE and injected into nonirradiated young adult IFN{gamma} receptor knockout (IFN{gamma}R-/-) or wild-type B6 mice. Twelve days later, T cells were isolated from the lymph nodes and spleens of the recipient mice and aliquots stained with PE-CD8. Figure 1 shows the results of a representative experiment. Summary statistics from a series of three such experiments are shown in the top panel of Figure 4. In agreement with previous findings, many of the CFSE-labeled cells from old donors recovered from lymph node (Figure 1) or spleen (not shown) of the wild-type recipient mice had CFSE levels that were 50% or 25% of that shown by the brightest CFSE-positive cells, indicating that these cells had divided once or twice in the previous 12-day period. However, CD8+ T cells from old mice recovered from lymph node (Figure 1) or spleen (not shown) of the IFN{gamma}R-/- recipient mice all had equally high levels of CFSE, indicating a failure to proliferate in the IFN{gamma}R-/- mice. On average only 2% of the injected CD8 cells from old donors had been able to divide in IFN{gamma}R-/- recipient mice, in comparison with 30% from CD8 T cells from old donors injected into wild-type recipients (p =.005). The mean proportion of CD8 T cells from young donors that were able to divide in wild-type recipients was 4%. This was significantly different than the percent of CD8 T cells from old donors that were able to divide in wild-type recipients (p =.008). There was no statistical difference in the proliferation obtained from CD8 T cells from young and old donors injected into IFN{gamma}R-/- mice (p =.414). These data suggest that the proliferation of CD8+ CD28hi T cells from aged mice requires interaction with an IFN{gamma}-responsive cell from the host.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 1. CD8 T cells fail to proliferate in IFN{gamma}R-/- mice. T cells were isolated from young and old C57BL/6J mice, labeled with CFSE (carboxy fluorescein diacetate succinimide ester), and then injected into 2-month-old IFN{gamma}R-/- C57BL/6 or wild-type C57BL/6 recipient mice. T cells from recipients' spleen (not shown) and pooled lymph nodes were isolated 12 days later and stained with {alpha}-CD8-CyC. The left and middle panels show dot plots of CD8 versus CFSE on cells from the lymph nodes of IFN{gamma}R-/- recipient mice injected with CFSE labeled T cells from young and old mice, respectively. The right panel shows a dot plot of CD8 versus CFSE on cells from lymph nodes of wild-type recipient mice injected with CFSE-labeled T cells from an old donor. Similar results were obtained in spleen samples. Each experiment was repeated three times. IFN = interferon; KO = knockout

 


View larger version (28K):
[in this window]
[in a new window]
  		Figure 4. Statistical summaries of proliferation experiments using mutant host mice. The panels show the percentage of CD8+ T cells from old donors (black and gray) and CD8+ T cells from young donors (white and cross-hatched) that had divided by day 12, isolated from the lymph nodes and spleens of the different knockout (black and white) or wild-type (gray and cross hatched) recipient mice. Data from the different knockout recipient mice is presented in the following order from top to bottom: IFN{gamma}R-/-, IFN{gamma}-/-, and IL-15-/-. Values are means ± standard errors. N = 3 for each comparison. KO = knockout; WT = wild type; IFN = interferon; IL = interleukin

 
To determine if IFN{gamma} produced by host cells was involved in the expansion of aged CD8 cells in vivo, we injected CFSE-labeled T cells from young and old donors into IFN{gamma} knockout mice (IFN{gamma}-/-). Figure 2 shows the results of a representative experiment. Summary statistics from a series of three such experiments are shown in the middle panel of Figure 4. The data show that CD8+ T cells from old mice were able to proliferate in IFN{gamma}-/- mice. However, we observed a decrease in proliferation when these recipients were compared with wild-type recipients of the same CD8 cells. On average, 12% of the injected CD8 cells from old donors and recovered from recipient spleens had been able to divide in IFN{gamma}-/- recipient mice, in comparison with 30% from CD8 T cells injected into wild-type recipients (p =.012). There was a statistical difference in the proliferation obtained from CD8 T cells from young and old donors injected into IFN{gamma}-/- mice (p =.002). In addition, the percent of proliferation of CD8 T cells from old donor was statistically different between IFN{gamma}-/- and IFN{gamma}R-/- recipient mice (p =.005). Similar results were seen in lymph node cells recovered from recipient mice. These data suggest that IFN production by the aged CD8+ CD28hi T cells contributes to their own proliferation through the induction of an effector cytokine in the host, but also show that production of IFN{gamma} by host cells can amplify the proliferative response.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Partial role of host interferon-gamma (IFN{gamma})-producing cells in the proliferation of CD8 T cells from aged donors. T cells were isolated from young and old C57BL/6 mice, labeled with CFSE (carboxy fluorescein diacetate succinimide ester), and then injected into 2-month-old IFN{gamma}-/- C57BL/6 or wild-type C57BL/6 recipient mice. T cells from recipients' spleen and pooled lymph nodes were isolated 12 days later and stained with {alpha}-CD8-CyC. The left and middle panels show dot plots of CD8 versus CFSE on cells from the lymph nodes of IFN{gamma}-/- recipient mice injected with CFSE-labeled T cells from old and young mice, respectively. The right panel shows a dot plot of CD8 versus CFSE in cells from lymph nodes of wild-type recipient mice injected with CFSE-labeled T cells from an old donor. Similar results were obtained in spleen samples. Each experiment was repeated three times. KO = knockout

 
IL-15 has been previously implicated in the normal turnover of CD8+ CD44hi T cells in vivo (35–37). To study the role of IL-15 in the CD8+ T cell proliferation in vivo, we injected CFSE-labeled T cells from old and young donor mice into IL-15 knockout (IL-15-/-) recipient mice. Twelve days later, T cells were isolated from the lymph nodes and spleens of the recipient mice and aliquots stained with PE-CD8. Figure 3 shows the results of a representative experiment. Summary statistics from a series of three such experiments are shown in the bottom panel of Figure 4. The data show that CD8+ T cells fail to proliferate in IL-15-/- recipient mice, whether studied in the spleen or lymph node of host mice. We found that, on average, only 1.5% of CD8 T cells from old donors were able to divide in IL-15-/- recipient mice, in comparison with 30% of the CD8 T cells recovered from a wild-type recipient (spleen data; p =.012). There was no statistical difference in the proliferation obtained from CD8 T cells from young and old donors injected into IL-15-/- mice (p =.06). In addition, there was no statistical difference in the percent of proliferation obtained from CD8+ T cells injected into IL-15-/- mice when compared with CD8 T cells injected into IFN{gamma}R-/- mice (p =.06). These experiments suggest that host IL-15 producing cells are required for in vivo proliferation of aged CD8 T cells.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 3. CD8 T cells fail to proliferate in interleukin (IL)-15-/- mice. T cells were isolated from young and old C57B6 mice, labeled with CFSE (carboxy fluorescein diacetate succinimide ester), and then injected into 2-month-old IL-15-/- C57BL/6 or wild-type C57BL/6 recipient mice. T cells from recipients' spleen (not shown) and pooled lymph nodes were isolated 12 days later and stained with {alpha}-CD8-CyC. The left and middle panels show dot plots of CD8 versus CFSE on cells from the lymph nodes of IL-15-/- recipient mice injected with CFSE-labeled T cells from young and old mice, respectively. The right panel shows a dot plot of CD8 versus CFSE on cells from lymph nodes of wild-type recipient mice injected with CFSE-labeled T cells from an old donor. Similar results were obtained in spleen samples. Each experiment was repeated three times. KO = knockout

 
Some groups have reported (44) a decrease in the proportion of memory CD8+ T cells upon injection of T cells into IL-15-/- mice. To determine if the lack of proliferation we observed in IL-15-/- mice was due to disappearance of memory CD8+ T cells in the recipient mice, we injected CFSE-labeled T cells from old and young donor mice into IL-15-/- recipients. Twelve days later cells from the lymph nodes and spleens from recipient animals were stained with anti-CD8 and anti-CD44. Figure 5 shows the results of a representative experiment from a series of three experiments. Memory cells, indicated by relatively high levels of CD44 expression, are still present at day 12 in the IL-15 knockout recipients. The percent of memory cells is higher among CFSE cells recovered from old donors than in the mice receiving cells from young donors, as expected from the known increase with age in the proportion of CD8 memory T cells (13). There were no consistent differences between young and old donors in the proportion of injected T cells that were recovered at day 12 from recipient mice (Table 1).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 5. CFSE (carboxy fluorescein diacetate succinimide ester)-labeled memory CD8 T cells are still present after 12 days in IL-15-/- mice. T cells were isolated from young and old C57BL/6 mice, labeled with CFSE, and then injected into 2-month-old IL-15-/- C57BL/6. T cells from recipients' pooled lymph nodes were isolated 12 days later and stained with {alpha}-CD8-CyC and {alpha}-CD44-PE. The left and right panels show dot plots of CD44 versus CFSE on the gated CD8+ cells from the lymph nodes of IL-15-/- recipient mice injected with CFSE-labeled T cells from young and old mice, respectively. Similar results were obtained in spleen samples. N = 3 experiments. IL = interleukin

 

View this table:
[in this window]
[in a new window]
  		Table 1. Percent of CFSE-Labeled Cells Recovered at Day 12.

 
TCR-Mediated Signaling in CD8 T Cells Recovered After In Vivo Proliferation
Engagement of the TCR induces a series of biochemical events leading to T-cell differentiation and proliferation. These events include translocation of T-cell membrane signaling molecules, including Lck, Fyn, and PKC-{theta}, toward the TCR/APC interaction synapse (45,46), followed by translocation of gene-activating molecules, including NF-{kappa}B, to the cell nucleus. Previous work from this laboratory has shown that virtually all CD4 T cells that are able to relocalize NF-AT (nuclear factor of activated T cells) in response to TCR-mediated stimuli are also able to translocate a wide range of synaptic molecules, but that the proportion of CD4 T cells that relocate NF-AT and synaptic molecules declines with age (47). We speculated that the ability of the CD28hi CD8 T cells to proliferate in vivo might reflect characteristic modes of signal transduction absent from nonproliferative T cells from old donors, as well as from young T cells. To clarify this, we used confocal immunofluorescence microscopy to follow redistribution to the nucleus of NF-{kappa}B, previously shown to play a critical role in the initiation of events leading to T-cell proliferation and differentiation (48).

T cells from aged donors and young donors (control) were labeled with CFSE, injected into young recipients, recovered from the spleen and lymph nodes at day 12, and then purified by cell-sorting on the basis of their CFSE signal. This approach yielded three populations of CFSE-labeled cells: 1) cells, derived from old donors, that had not divided in the host, 2) cells derived from old donors that had divided at least once after injection, and 3) cells from the young donors, which do not divide in vivo. Recovered CFSE-labeled cells from young and old donor mice were incubated with live anti-CD3{epsilon} (2C11) and anti-DNP (UC8, negative control) hybridoma cells to allow the formation of conjugates. Each T cell/2C11 or T-cell/UC8 conjugate was scored as positive (activated) or negative (nonactivated) for NF-{kappa}B relocalization to the nucleus, using coded slides to conceal age and subset information. Figure 6A shows representative images of responsive and unresponsive T cells, and Figure 6B shows a summary of the data. We found that approximately 70% of the conjugated CD8 cells derived from young donors were able to translocate NF-{kappa}B to the nucleus upon activation through the TCR, well above the baseline level (15%) seen in the negative control conjugates using UC8 (anti-DNP) hybridomas as mock stimulators. In contrast, NF-{kappa}B translocation was seen in fewer than 35% of the CFSE-tagged cells recovered from older donors, and the proportion did not differ appreciably between CD8 cells that had or had not divided in vivo. This decrease in NF-{kappa}B relocalization on CD8+ T cells from old donors represented a defect specific for TCR-mediated activation processes, in that we did not observe any differences between cells recovered from young or old donors in responses triggered by phorbol myristate acetate (PMA) and ionomycin, which bypass the T-cell receptor complex. These stimuli induced NF-{kappa}B translocation in a large majority of recovered cells (~80%) regardless of CD8 subset or donor age (Figure 6B, right panel).



View larger version (35K):
[in this window]
[in a new window]
  		Figure 6. NF-{kappa}B Translocation to nucleus decreases in CD8 T cells from old mice. T cells from aged donors and young donors (control) were labeled with CFSE (carboxy fluorescein diacetate succinimide ester), injected into young recipients, recovered from the spleen (not shown) and lymph nodes at day 12, and then purified by cell-sorting on the basis of their CFSE signal producing three populations: 1) cells from old donors that had not divided in the host (Old N-Div), 2) those from old donors that divided after injection (Old Div), and 3) those from the young donors (Yng N-Div). Recovered CFSE-labeled cells from young and old donor mice were then incubated with live anti-CD3{epsilon} (2C11) and UC8 hybridoma cells (negative control) to allow the formation of conjugates. A, left panel: Example of conjugate in which NF-{kappa}B translocation to the nucleus was not induced. A, right panel: Example of conjugate in which NF-{kappa}B has translocated to the nucleus after activation by 2C11 cells. B, left panel: Percent of positive NF-{kappa}B translocation of recovered CFSE-labeled cells from young and old donor mice that were incubated with 2C11 and UC8 hybridoma cells. N = 5. B, right panel: Percent of conjugates that show positive NF-{kappa}B translocation among the recovered CFSE-labeled cells from young and old donors, after activation with phorbol myristate acetate (PMA) and ionomycin. N = 3 experiments. NF = nuclear factor

 
Similar experiments evaluated redistribution of the adapter protein LAT to the immunological synapse. LAT plays an essential role in the transmission of signals from the TCR complex to other key signaling proteins (49–51), which then in combination initiate downstream events such as the activation of Ras, influx of Ca2+, and translocation of NF-{kappa}B to the nucleus. Figure 7 (top panels) shows examples of cells that do and do not redistribute LAT to the APC synapse. In agreement with our NF-{kappa}B data, we found a decrease in LAT relocalization to the immunological synapse in cells from both subsets of T cells from old donors when compared with cells from young donors. The bottom panel of Figure 7 shows a summary of the data. About 60% of conjugated CD8 cells from young donors exhibited redistribution of LAT into the immunological synapse, but the proportion of conjugated CD8 T cells with redistributed LAT was around 30% for both proliferating and nonproliferating CD8 subsets from old donors.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 7. LAT (linker for activated T cells) translocation to synapse after anti-CD3 stimulation. T cells from aged donors and young donors (control) were labeled with CFSE (carboxy fluorescein diacetate succinimide ester), injected into young recipients, recovered from the spleen and lymph nodes at day 12, and then purified by cell-sorting as described in
Figure 6. A and B show representative images in which LAT remains evenly distributed in the T cells (A, left) and in which LAT accumulates at the synapse (B, right). (C) shows summary statistics of three experiments. The bars show cells positive for LAT translocation among the recovered CFSE-labeled cells from young and old donor mice that formed conjugates with anti-CD3 (clone 145-2C11) (black) or with the control hybridoma anti-DNP UC8 (white) as a percentage of conjugates (N = 3) (mean ± SEM [standard error of mean])

 
T-Cell Activation Signals in CD28hi Precursors of Proliferating CD8 Cells From Old Mice
We wanted to see if similar activation defects are apparent in the CD28hi subset of CD8 cells seen in aged mice, because this subset contains the precursors of the cells that are able to divide in vivo. To test this, CD8 T cells from young and old mice were sorted based on their CD28 levels and then examined for translocation to the synapse of LAT and the T-cell-specific protein kinase PKC-{theta}. PKC-{theta} is a key component in the TCR signaling cascade that leads to an upregulation of activating protein-1 (AP-1) (51) and has been shown by immunofluorescent methods to translocate to the immune synapse of TCR-transgenic T cells only in response to agonist peptides (53). The sorting procedure produced 3 groups of cells: 1) CD8+ CD28hi T cells from old mice, 2) CD8+ CD28lo T cells from old mice, and 3) CD8+ CD28lo T cells from young mice. Cells of each subset were activated with 145-2C11 hybridoma cells or with UC8 hybridoma cells as a negative control. The top panel of Figure 8 shows the results of a series of two experiments comparing LAT redistribution in sorted CD8 cells from young and old mice after conjugation to 2C11 stimulators. 2C11 cells induce LAT redistribution in 60% of the conjugated CD8+ CD28lo from young mice, in 30% of the conjugated CD8+ CD28lo from old mice, and in 25% of the conjugated CD8+ CD28hi cells from old mice. Similarly, the frequency of activated T cells with redistributed PKC-{theta} dropped significantly from 55% in CD8+ CD28lo cells from young mice to 28% and 24% in CD8+ CD28lo and CD8+ CD28hi cells from old mice, respectively.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 8. LAT (linker for activated T cells) and protein kinase C-{theta} (PKC-{theta}) translocation in CD28 subsets from old mice. CD8+ T cells from young and old mice were sorted based on their CD28 levels. Three groups of cells were obtained: 1) CD8+ CD28lo T cells from young mice, 2) CD8+ CD28lo T cells from old mice, and 3) CD8+ CD28hi T cells from old mice. Cells from the three groups were activated either with 145-2C11 hybridoma cells (anti-CD3; black) or with UC8 hybridomas (anti-DNP as control; white) to allow the formation of conjugates. We used immunofluorescence assays to follow the redistribution of LAT (top) or PKC-{theta} (bottom) to the immunological synapse, using coded slides to conceal age and subset information. Each T cell/2C11 or T cell/UC8 conjugate was scored as positive or negative for LAT or PKC-{theta} translocation to the immunological synapse. Figure 8 shows a summary of the results (N = 2) (mean ± SEM [standard error of mean])

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The accumulation of large clones of CD8+ T cells (14–26) in aging mice is likely to reflect alterations in the ability of some CD8 cells to regulate division or cell death in the face of homeostatic controls that are still poorly understood. We have recently reported that most of the CD8+ T cells from old mice that can divide in vivo produce IFN{gamma} after isolation from their original host or after in vivo expansion in recipients. Since IFN{gamma} has been implicated in the proliferation of memory CD8+ T cells in some systems (29–32), we explored its potential role in the proliferation of CD8+ cells from old donors after transfer into normal recipients. IFN{gamma} fails to cause proliferation of purified CD44hi CD8+ cells in vitro; in fact, under these conditions IFN{gamma} had a strong antiproliferative effect on T cells (33). Hence, we hypothesized that if IFN{gamma} were involved in CD8 cell proliferation in vivo, it would be through the induction of another cytokine, an effector cytokine that would act directly on CD44hi CD8+ cells.

To study this hypothesis further, we have measured the proliferative capacity of T cells from young and old mice after transfer into IFN{gamma}R-/- recipients by flow cytometry using the fluorescent dye CFSE. We found that the proliferation of CD8+ T cells was prevented in IFN{gamma}R-/- recipients. These results suggest that a response from host cells to IFN{gamma} is necessary for the proliferation of CD8+ T cells in vivo. When we injected CFSE-labeled T cells into IFN{gamma}-/- recipients, we found that CD8+ T cells from old donors are still able to divide in vivo but to a lesser extent than in genetically normal hosts. These data indicate that production of IFN{gamma} by the injected CD8+ CD28hi T cells may promote the proliferation of these cells through the induction of an effector cytokine, but that IFN{gamma} produced from the host may be involved in amplifying the response. However, it is also possible that the absence of IFN{gamma} in these mutant mice leads to a diminished sensitivity of the IFN-responsive cell type needed to promote T-cell expansion in this model (54). By injecting CFSE-labeled T cells into IL-15-/- recipients, we demonstrated that IL-15 is involved in the proliferation of CD8+ T cells from old mice. Thus, IL-15 might be the effector cytokine produced by the host in response to IFN{gamma}. It has been reported that many cell types, including macrophages (but not T cells), can synthesize IL-15 (33). IFN{gamma} has been shown to be a strong inducer of IL-15 mRNA in macrophages (33).

IL-15 has been implicated as a key regulator in the survival and turnover of CD8+ CD44hi T cells in vivo (35–37). Under steady-state conditions, continuous expansion of memory T cells may be limited by contact with specific cytokines, including IL-15. If so, a protracted increase in the level of these cytokines would be expected to cause the numbers of memory T cells to increase above normal. The selective expansions of CD44hi CD8+ cells in IL-15 transgenic mice (55) is consistent with this idea. The data presented in this article support a model in which CD8+ T cells from old mice induce their own proliferation through the IFN{gamma}-induced production of IL-15. The expanded CD8 T-cell clones in aged mice are not malignant, suggesting the presence of other growth-limiting mechanisms that prevent their unbridled proliferation and accumulation. It has been reported that IL-2 suppresses the division of memory CD8+ T cells by inducing cell death of the dividing cells, rather than by inhibition of their division (38,39). The production of IL-2 may check uncontrolled responses by bystander CD8+ memory T cells induced by increased levels of IL-15. The basis for selective inhibition of some, but not all, memory CD8 cell subsets by IL-2 is still unclear, but may involve the activities of a suppressive population of IL-2-dependent regulatory CD25+ CD4+ T cells (40).

To clarify whether the CD8 cells whose proliferative abnormalities cause them to accumulate in aged mice were or were not normally responsive to antigenic stimulation, we used immunofluorescence assays to follow the redistribution of proteins involved in the TCR signaling cascade in cells from old and young donors that were or were not able to divide in a normal host. We found that activation-induced migration of LAT and NF-{kappa}B, two key proteins that act in TCR-initiated signaling pathways, occurred less frequently in CD8 T cells from old donors, whether they were able to divide in vivo or not, than in CD8 T cells from young donors. However, translocation of NF-{kappa}B could readily be induced in CD8 T cells from old donors when activated with PMA and ionomycin, suggesting that the defect in NF-{kappa}B movement was secondary to upstream signaling events in these cells. Our data suggest that intracellular signals are not generated or transmitted efficiently from the TCR complex to early acting components, including LAT, in CD8 T cells from old mice. Here, it is notable that, despite the fact that these CD8 cells from old donors were in a young host for 12 days, they still present defects in TCR activation when compared with CD8 T cells from young donors. This suggests that the defects induced by aging are intrinsic within the cells and are not corrected with a change of environment. We note that the data reported here document that CD8 T cells from aged mice exhibit at least some of the same defects in LAT, PKC-{theta}, and NF-{kappa}B activation pathways that have previously been documented in studies of CD4 cells from older mice (42,47).

In a previous article, we reported a population of memory CD8 cells, with higher levels of CD28, present only in aged mice, from which emerged nearly all of the CD8 cells that were able to divide in vivo. We therefore asked whether CD8+ CD28hi T cells show the same defects as CD8 T cells from old donors recovered from recipients in the translocation of molecules to the immunological synapse. We found that CD8+ CD28hi T cells as well as CD8+ CD28lo T cells from old mice had a decline in the proportion of conjugates that redistribute LAT and PKC-{theta} when compared with CD8+ CD28lo T cells from young donors. This argues against the idea that CD8+ CD28hi T cells might represent a subset of hyperresponsive cells that accumulate in old mice. Instead, they share the same age-associated alteration seen in the majority population of CD8 T cells in old mice. It has been reported that in humans, aging leads to a characteristic loss of CD28, predominantly among CD8+ T cells (15,18–20). In humans, it is this CD28- T-cell population that is most typically susceptible to clonal expansion (15,18–20). This CD28- population consists of a subset of memory T cells with little proliferative response to mitogenic stimulation in vitro (56,57). Several reports suggest that they derive from a CD28+ progenitor (57,58). It is not clear whether elderly humans have a specific CD8+ subset, like the one we documented in aged mice, from which clonal expansions are derived, or whether such a precursor population has unusually high or low levels of CD28 expression.

The decline of LAT and PKC-{theta} in CD8 T cells from old mice is not likely to be due to any alteration in T-cell expression of TCR or CD8. The cell surface concentrations of the TCR/CD3 complex (59–61) and of CD8 (61) have been shown not to be affected by aging in mice. Moreover, previous work from our laboratory showed no change with age in the proportion of CD8 cells that form conjugates with 2C11 stimulators (62).

Phosphorylation of LAT allows it to bind to, and presumably help concentrate and orient, a number of SH2-containing proteins, including Grb2 and phospholipase C-{gamma}, that play a role in the propagation of downstream signals in the early phases of T-cell activation. PKC-{theta} contributes to cell proliferation (53), cell cycle progression (63), and IL-2 production (64–66). Immunofluorescence studies have shown that productive interaction between T-cell clones and APCs leads to PKC-{theta} clustering in ~80% of the conjugated T cells. Interestingly, PKC-{theta} clustering was not induced (53,67) either by antibodies to the TCR complex or by APC-bearing antagonist peptides, nor did T-cell/APC interaction lead to the induction of clustering in any other PKC isoform in cloned T-cell lines. These data suggest that induction of PKC-{theta} clustering might serve as a useful index for early activation events critical to the commitment of a T cell to entry into the mitotic cycle. Hence, we concluded that the defects we observed in LAT, PKC-{theta}, and NF-{kappa}B redistribution in CD8 T cells from old mice are likely to contribute to downstream defects in T-cell activation. The molecular basis for the reduced susceptibility of these cells to homeostatic controls is, however, still undefined by the studies reported here.


   Acknowledgments
 
This work was supported by National Institutes of Health (NIH) grant R01-AG19619. Dr. A. Ortiz-Suárez was supported by the Rackham Merit Fellowship and NIH training grants AI07413 and AG00114. We wish to thank Dr. Gonzalo G. Garcia for advice and comments, and Dr. Jacques Peschon for providing us with the IL-15 knockout mice.

Address correspondence and reprint requests to Dr. Richard Miller, 5316 CCGC, 1500 E. Medical Center Dr., Ann Arbor, MI 48109-0940. E-mail: millerr{at}umich.edu


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received June 19, 2003

Accepted August 25, 2003


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Freitas AA, Rocha BB. Lymphocyte lifespan: homeostasis, selection and competition. Immunol Today.. 1993;14:25-29.[Medline]
  2. Bell EB, Sparshott SM. The peripheral T cell pool: regulation by non-antigen induce proliferation? Sem Immunol.. 1997;9:347-353.[Medline]
  3. Goldrath AW, Bevan MJ. Selecting and maintaining a diverse T-cell repertoire. Nature.. 1999;402:255-262.[Medline]
  4. Surh CD, Sprent J. Homeostatic T cell proliferation. How far can T cells be activated to self-ligands? J Exper Med.. 2000;192:F9-F14.
  5. Viret C, Wong FS, Janeway, Jr. CA. Designing and maintaining the mature TCR repertoire: the continuum of self-peptide: self-MHC complex recognition. Immunity.. 1999;10:559-568.[Medline]
  6. Kieper WC, Jameson SC. Homeostatic expansion and phenotypic conversion of naïve T cell in response to self peptide/MHC ligands. Proc Natl Acad Sci U S A.. 1999;96:13306-13311.[Abstract/Free Full Text]
  7. Ernst BD, Lee S, Chang JM, Sprent J, Surh CD. The peptide ligands mediating positive selection in the thymus control T cell survival and homeostatic proliferation in the periphery. Immunity.. 1999;11:173-181.[Medline]
  8. Goldrath AW, Bevan MJ. Antagonist ligands for the TCR drive proliferation of mature CD8+ T cells in lymphopenic host. Immunity.. 1999;11:183-190.[Medline]
  9. Schulns KS, Kieper WC, Jameson SC, Lefrancois L. Interleukin-7 mediates the homeostasis of naïve and memory CD8 T cells in vivo. Nat Immunol.. 2000;1:426-432.[Medline]
  10. Tan JT, Dudl E, LeRoy E, et al. IL-7 is critical for homeostatic proliferation and survival of naïve T cells. Proc Natl Acad Sci U S A.. 2001;98:8732-8737.[Abstract/Free Full Text]
  11. Tough DF, Sprent J. Turnover of naïve- and memory-phenotype T cells. J Exper Med.. 1994;179:1127-1135.[Abstract/Free Full Text]
  12. Bruno LH, von Boehmer, , Kirberg J. Cell division in the compartment of naïve and memory T lymphocytes. Eur J Immunol.. 1996;26:3179-3184.[Medline]
  13. Miller RA. The aging immune system: primer and prospectus. Science.. 1996;273:70-74.[Abstract]
  14. Callahan JE, Kappler JW, Marrack P. Unexpected expansions of CD8-bearing cells in old mice. J Immunol.. 1993;151:6657-6669.[Abstract]
  15. Posnett DN, Sinha R, Kabak S, Russo C. Clonal populations of T cells in normal elderly humans: the T cell equivalent to "benign monoclonal gammapathy.". J Exper Med.. 1994;179:609-618.[Abstract/Free Full Text]
  16. Clarke GR, Humphrey CA, Lancaster FC, Bouylston AW. The human T cell antigen receptor repertoire: skewed use of Vß gene families by CD8+ T cells. Clin Exper Immunol.. 1994;96:364-369.[Medline]
  17. Wedderburn LR, Patel A, Varsani H, Woo P. The developing human immune system: T-cell receptor repertoire of children and young adults shows a wide discrepancy in the frequency of persistent oligoclonal T-cell expansions. Immunol.. 2001;102:301-309.[Medline]
  18. Fitzgerald JE, Ricalton NS, Meyer AC, et al. Analysis of clonal CD8+ T cell expansions in normal individuals and patients with rheumatoid arthritis. J Immunol.. 1995;154:3538-3547.[Abstract]
  19. Morley JK, Batliwalla FM, Hingorani R, Gregersen PK. Oligoclonal CD8+ T cells are preferentially expanded in the CD57+ subset. J Immunol.. 1995;154:6182-6190.[Abstract]
  20. Ricalton NS, Roberton C, Norris JM, Rewers M, Hamman RF, Kotzin BL. Prevalence of CD8+ T-cell expansions in relation to age in healthy individuals. J Gerontol Biol Sci.. 1998;53:B196-B203.
  21. Ku CC, Kotzin B, Kappler J, Marrack P. CD8+ T-cell clones in old mice. Immunol Rev.. 1997;160:139-144.[Medline]
  22. Ku CC, Kappler J, Marrack P. Characteristics and maintenance of CD8+ T-cell clones found in old mice. Mech Ageing Dev.. 1997;94:41-53.[Medline]
  23. Wack A, Cossarizza A, Heltai S, et al. Age-related modifications of the human T cell repertoire due to different clonal expansions in the CD4+ and CD8+ subsets. Int Immunol.. 1998;10:1281-1288.[Abstract/Free Full Text]
  24. Schwab R, Szabo P, Manavalan JS, et al. Expanded CD4+ and CD8+ T cell clones in elderly humans. J Immunol.. 1997;158:4493-4499.[Abstract]
  25. Maini MK, Wedderburn LR, Hall FC, Wack A, Casorati G, Beverley PC. A comparison of two techniques for the molecular tracking of specific T-cell responses; CD4+ human T-cell clones persist in a stable hierarchy but at a lower frequency than clones in the CD8+ population. Immunology.. 1998;94:529-535.[Medline]
  26. Mosley RL, Koker MM, Miller RA. Idiosyncratic alterations of TCR size distributions affecting both CD4 and CD8 T cell subsets in aging mice. Cell Immunol.. 1998;189:10-18.[Medline]
  27. LeMaoult J, Messaoudi I, Manavalan JS, et al. Age-related dysregulation in CD8 T cell homeostasis: kinetics of a diversity loss. J Immunol.. 2000;165:2367-2373.[Abstract/Free Full Text]
  28. Ortiz-Suárez A, Miller RA. A subset of CD8 memory T cells from old mice have high levels of CD28 and produce IFN{gamma}. Clin Immunol.. 2002;104:282-292.[Medline]
  29. Tough DF, Borrow P, Sprent J. Induction of bystander T cell proliferation by viruses and type interferon in vivo. Science.. 1996;272:1947-1950.[Abstract]
  30. Tough DF, Sun S, Sprent J. T cell stimulation in vivo by LPS. J Exper Med.. 1997;185:2089-2094.[Abstract/Free Full Text]
  31. Sprent J, Zhang X, Sun S, Tough D. T cell proliferation in vivo and the role of cytokines. Philos Trans R Soc London B Ser.. 2000;355:317-322.
  32. Tough D, Zhang X, Sprent J. An IFN-gamma dependent pathway controls stimulation of memory phenotype CD8 T cell turnover in vivo by IL-12, IL-18 and IFN-gamma. J Immunol.. 2001;166:6007-6011.[Abstract/Free Full Text]
  33. Zhang X, Sun S, Hwang I, Tough D, Sprent J. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity.. 1998;8:591-599.[Medline]
  34. Kennedy MK, Park LS, Paxton RJ. IL-15. In: Lotze MT, Thomson AW, eds. The Cytokine Handbook. London: Academic Press; 1998:443–464.
  35. Sprent J, Surh CD. T cell memory. Ann Rev Immunol.. 2002;20:551-579.[Medline]
  36. Kennedy MK, Glaccum M, Brown SN, Peschon JJ. Reversible defects in natural killer and memory CD8 T cell lineages in IL-15-deficient mice. J Exper Med.. 2000;191:771-780.[Abstract/Free Full Text]
  37. LoDolce JP, Boone DL, Chai S, Swain RE, Ma A. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity.. 1998;9:669-676.[Medline]
  38. Ku CC, Murakami M, Sakamoto A, Kappler J, Marrack P. Control of homeostasis of CD8+ memory T cells by opposing cytokines. Science.. 2000;288:675-678.[Abstract/Free Full Text]
  39. Ku CC, Kappler J, Marrack P. The growth of the very large CD8+ T cell clones in older mice is controlled by cytokines. J Immunol.. 2001;166:2186-2193.[Abstract/Free Full Text]
  40. Murakami M., Sakamoto A, Bender J, Kappler J, Marrack P. CD25+ CD4+ T cells contribute to the control of memory CD8+ T cells. PNAS.. 2002;99:8832-8837.[Abstract/Free Full Text]
  41. Huang S, Hendriks W, Althage A, et al. Immune response in mice that lack the interferon-gamma receptor. Science.. 1993;259:1742-1745.[Abstract/Free Full Text]
  42. Eisenbraun MD, Tamir A, Miller RA. Altered composition of the immunological synapse in an anergic, age-dependent memory T cell subset. J Immunol.. 2000;164:6105-6112.[Abstract/Free Full Text]
  43. Gudmundsdottir H, Wells AD, Turka LA. Dynamics and requirements of T cell clonal expansion in vivo at the single-cell level: effector function is linked to proliferative capacity. J Immunol.. 1999;162:5212-5223.[Abstract/Free Full Text]
  44. Judge AD, Zhang X, Fujii H, Surh CD, Sprent J. Interleukin 15 controls both proliferation and survival of a subset of memory-phenotype CD8(+) T cells. J Exper Med.. 2002;196:935-946.[Abstract/Free Full Text]
  45. Viola A, Schroeder S, Sakakibara Y, Lanzavecchia A. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science.. 1999;283:680-682.[Abstract/Free Full Text]
  46. Janes PW, Ley SC, Magee AI. Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J Cell Biol.. 1999;147:447-461.[Abstract/Free Full Text]
  47. Garcia GG, Miller RA. Single-cell analyses reveal two defects in peptide-specific activation of naive T cells from aged mice. J Immunol.. 2001;166:3151-3157.[Abstract/Free Full Text]
  48. Cantrell D. T cell antigen receptor signal transduction pathways. Ann Rev Immunol.. 1996;14:259-274.[Medline]
  49. Zhang W, Irvin BJ, Trible RP, Abraham RT, Samelson LE. Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int Immunol.. 1999;11:943-950.[Abstract/Free Full Text]
  50. Zhang W, Sloan-Lancaster J, Kitchen J, Trible RP, Samelson LE. LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell.. 1998;92:83-92.[Medline]
  51. Finco TS, Kadlecek T, Zhang W, Samelson LE, Weiss A. LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway. Immunity.. 1998;9:617-626.[Medline]
  52. Baier G, Baier-Bitterlich G, Meller N. Expression and biochemical characterization of human protein kinase C-theta. Eur J Biochem.. 1994;225:195-203.[Medline]
  53. Monks CR, Kupfer H, Tamir I, Barlow A, Kupfer A. Selective modulation of protein kinase C-theta during T-cell activation. Nature.. 1997;385:83-86.[Medline]
  54. Dalton DK, Pitts-Meek S, Keshav S, Figari IS, Bradley A, Stewart TA. Multiple defects of immune cell function in mice with disrupted interferon-gamma genes. Science.. 1993;259:1739-1742.[Abstract/Free Full Text]
  55. Marks-Konczalic J, Dobouis S, Tagaya Y. IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice. Proc Natl Acad Sci U S A.. 2000;97:11445-11450.[Abstract/Free Full Text]
  56. Ho, HN, Hultin LE, Mitsuyasu RT, et al. Circulating HIV-specific CD8+ cytotoxic T cells express CD38 and HLA-DR antigens. J Immunol.. 1993;150:3070-3079.[Abstract]
  57. Fagnoni FF, Vescovini R, Mazzola M, et al. Expansion of cytotoxic CD8+ CD28- T cells in healthy agein people, including centenarians. Immunology.. 1996;88:501-507.[Medline]
  58. Azuma M., Phillips JH, Lanier LL. CD28- T lymphocytes: antigenic and Functional Properties. J Immunol.. 1993;150:1147-1159.[Abstract]
  59. Sidman, CL, Luther EA, Marshall JD, Nguyen KA, Roopenian DC, Worthen SM. Increased expression of major histocompatibility complex antigens on lymphocytes from aged mice. Proc Natl Acad Sci U S A.. 1997;84:7624-7628.
  60. Hertogh-Huijbregts A, Vissinga C, Rozing J, Nagelkerken L. Impairment of CD3-dependent and CD3-independent activation pathways in CD4+ and in CD8+ T cells from old CBA/RIJ mice. Mech Ageing Dev.. 1990;53:141-155.[Medline]
  61. Utsuyama M, Hirokawa K. Age-related changes of splenic T cells in mice-a flow cytometric analysis. Mech Ageing Dev.. 1987;40:89-102.[Medline]
  62. Yang D, Miller RA. Cluster formation by protein kinase C theta during murine T cell activation: effect of age. Cell Immunol.. 1999;195:28-36.[Medline]
  63. Resnick MS, Kang BS, Luu D, Wickham JT, Sando JJ, Hahn CS. Differential downstream functions of protein kinase Ceta and -theta in EL4 mouse thymoma cells. J Biol Chem.. 1998;273:27654-27661.[Abstract/Free Full Text]
  64. Baier G, Baier-Bitterlich G, Meller N, et al. Expression and biochemical characterization of human protein kinase C-theta. Eur J Biochem.. 1994;225:195-203.
  65. Baier-Bitterlich G, Uberall F, Bauer B, et al. Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes. Mol Cell Biol.. 1996;16:1842-1850.[Abstract]
  66. Werlen G, Jacinto E, Xia Y, Karin M. Calcineurin preferentially synergizes with PKC-theta to activate JNK and IL-2 promoter in T lymphocytes. EMBO J.. 1998;17:3101-3111.[Medline]
  67. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature.. 1998;395:82-86.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ortiz-Suárez, A.
Right arrow Articles by Miller, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ortiz-Suárez, A.
Right arrow Articles by Miller, R. A.