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,
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Department of Pathology, University of Michigan School of Medicine,
University of Michigan Institute of Gerontology, and
Ann Arbor Department of Veteran Affairs Medical Center, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, linker for activation of T cells (LAT), and Zap-70 in a central
area and c-Cbl, p95vav, Grb-2, PLC
, Fyn, and
Lck distributed more uniformly across the interface area. Two-color
staining showed that those cells that were able to relocalize c-Cbl,
LAT, CD3, or PLC typically relocalized all four of these
components of the activation complex. About 75% of conjugates that
rearranged LAT, c-Cbl, or PLC also exhibited cytoplasmic NF-AT
migration to the T cell nucleus. Aging had two effects. First, it led
to a diminution of 
2-fold in the proportion of T cell/APC conjugates
that could relocalize any of the nine tested proteins to the immune
synapse. Second, aging diminished by 2-fold the frequency of
cytoplasmic NF-AT migration among cells that could generate immune
synapses containing LAT, c-Cbl, or PLC. Thus naive CD4 T cells from
old mice exhibit at least two separable defects in the earliest stages
of activation induced by peptide/MHC complexes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and c-Cbl (11, 12) that together trigger later stages of the signal
transduction cascade. It has recently been proposed that
glycolipid-enriched membrane microdomains, known as rafts or
glycolipid-enriched membranes (GEMS), may play an important role in the
translocation of enzymes and adaptor proteins to the area of TCR-APC
contact (13, 14). In particular, it has been proposed that
the raft domains help to concentrate the constitutively palmitoylated
LAT to the c-SMACs, while excluding other molecules with negative
regulatory roles, such as CD45 (15).
Aging leads to a decline in T cell response to new and previously
encountered Ags (16). Several laboratories have shown that
T cells from healthy old humans and mice exhibit a multitude of defects
at early stages of the T cell signaling pathways, including changes in
serine/threonine and tyrosine phosphorylation (17, 18, 19, 20, 21, 22, 23),
development of calcium signals (24), activation of the
Raf-1/mitogen-activated protein/extracellular signal-related kinase
kinase/extracellular signal-related kinase (19) and c-Jun
N-terminal kinase (JNK) pathways (25, 26) and
translocation of cytoplasmic NF-AT (NF-ATc) to the nucleus
(27). Most of these alterations are demonstrable within
the first 15 min of TCR engagement, and it is not yet clear which of
the defects are primary, and which are merely secondary consequences of
earlier abnormalities. One of the earliest detectable age-related
defects is the decline with age in the phosphorylation of LAT by Zap-70
(28). In vitro kinase assays showed no effects of aging on
Zap-70 protein kinase activity in CD4 T cells from young and old mice
stimulated by anti-CD3/anti-CD4 cross-linking
(29), suggesting that defective LAT phosphorylation might
result from altered accessibility, i.e., differential
compartmentalization of Zap-70 and its substrates. Indeed, confocal
microscopic studies using anti-CD3 hybridoma cells as polyclonal
APC analogs showed that the majority of CD4 T cells from old mice could
not efficiently relocate protein kinase C (PKC)
, LAT, and
p95vav (Vav) to the immune synapse (28, 30, 31), suggesting that at least some of the age-associated
defects in the early stages of signal transduction could be the result
of defects in the formation of immune synapses at the site of T
cell/APC interaction. However, one weakness of this initial study was
its reliance on anti-CD3 stimulation. Interactions between the 2C11
anti-CD3 and the TCR are of much higher affinity, with a much
slower off-rate, than the typical interaction between peptide-bearing
MHC molecules and the TCR (32, 33). Furthermore, the
original studies used responder cells that contained both naive and
memory T cells; because aging leads to major increases in memory T cell
numbers at the expense of naive T cells (for review see Ref.
16), it seemed possible that differences in synapse
formation between naive and memory CD4 might have contributed to the
decline with age in the proportion of cells able to relocalize LAT,
Vav, and PKC to the T cell/APC interface.
To help resolve these issues, we have now used fluorescence confocal microcopy to study the responses of T cells from young and old transgenic mice bearing TCR specific for a peptide fragment of pigeon cytochrome c (PCC), as presented by the CH12 B cell line. The results suggest two age-dependent changes in the early activation process, one that interferes with recruiting of a variety of proteins to the immune synapse, and another, postsynaptic defect that prevents NF-AT translocation even in T cells with apparently normal synapse composition.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeding pairs of the AND line of TCR-transgenic mice, whose T cells respond to PCC, were a generous gift from Dr. Susan Swain (Trudeau Institute, Saranac Lake, NY). Transgene-positive mice were aged in a specific pathogen-free colony at the University of Michigan (Ann Arbor, MI) 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 course of these studies. Transgenic mice that were found to have splenomegaly or macroscopically visible tumors at the time of sacrifice were not used for experiments. Mice used were at 6–8 (young) and 18–20 (old) mo of age. The CH12 B cell line was obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 with 10% FCS and 2 mM L-glutamine at 37°C and 10% CO2.
Abs and reagents
Rabbit polyclonals anti-PLC, Vav, c-Cbl, and Grb-2 were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA), the rabbit
anti-Zap-70, Lck, Fyn, and LAT from Upstate Biotechnology (Lake
Placid, NY) and the anti-CD3 from Dako (Carpinteria, CA). The
mAb anti-c-Cbl was purchased from Transduction Laboratories
(Lexington, KY), and the mAb to NF-ATc1 was purchased from Santa Cruz
Biotechnology. Single-color detection of proteins for confocal
microscopy analysis was performed using rabbit polyclonal Abs and goat
anti-rabbit Ig coupled to FITC (Jackson ImmunoResearch, West Grove,
PA). Two-color analysis was performed using first mAb and goat
anti-mouse Fc coupled to FITC (Jackson ImmunoResearch), then the
rabbit polyclonal and a goat anti-rabbit coupled to Alexa-594
(Molecular Probes, Eugene, OR).
Peptides were synthesized in the Protein Core Facilities of the University of Michigan. The agonist peptide sequence represents aa 88–103 of PCC (ANERADLIAYLKQATK). The nonagonist peptide had the same sequence, with the exception of a single amino acid substitution (K to N) at position 99 (marked in bold), (34).
Cell preparation
CD4+ T cells were obtained from transgenic mice using the negative selection methods described in (28). Flow-cytometry analysis of a typical preparation showed it to be 90–95% positive for both CD3 and CD4.
For each experiment, CH12 cells in log-phase were pulsed with 20 µM agonist or nonagonist peptide for 2 h in fresh medium at 37°C.
Slide preparation and confocal microscopy
A total of 6 x 105 TCR-transgenic
CD4+ T cells (resuspended at 4 x
106 cells/ml in RPMI 1640 plus 1% FCS) was
combined with 3 x 105 CH12 cells
(resuspended at 2 x 106 cells/ml) to
achieve a 2:1 ratio, respectively. Cell mixtures were incubated at
37°C for 20 min and then gently resuspended and spread onto prewarmed
poly(L-lysine)-coated slides (Sigma, St. Louis, MO). Slides were
incubated for another 20 min at 37°C, fixed with 3.7% formaldehyde,
1 mM MgCl2 in PBS (pH 8.5) for 10 min and washed
three times for 5 min each with PBS. Slides were permeabilized with
0.2% Triton X-100 in PBS for 10 min at 4°C, washed with PBS and
blocked overnight with 1% BSA/PBS. For single-color experiments, the
slides were stained with the appropriate primary rabbit Abs diluted in
blocking solution at 1 µg/ml for 1 h at 4°C, washed three
times with PBS and then stained with goat anti-rabbit-FITC at 10
µg/ml in blocking solution for 1 h at 4°C. The slides were
washed four times with PBS, mounted using SlowFade Light antifade
reagents (Molecular Probes) and sealed with nail polish. All slides
were coded for blind analysis, and then stored at 4°C protected from
light. For two-color protocols, the slides were initially stained with
the appropriate mAb diluted in blocking solution at 1 µg/ml for
1 h at 4°C, washed, and incubated with goat anti-mouse
Fc-FITC at 10 µg/ml in blocking solution for 1 h at 4°C.
The second stain was then applied using the method given for single
stains, but using goat anti-rabbit reagent coupled to Alexa-594 as
the second Ab.
Single- and two-color analyses were performed at x100 magnification on a Nikon Diaphot microscope (Nikon, Melville, NY) equipped with a Bio-Rad MRC 600 confocal laser imaging system (Bio-Rad, Hercules, CA). Randomly selected T cell-APC conjugates were analyzed only if the following criteria were first met: 1) tightly formed cell-to-cell contact, 2) T cells in contact with only one APC, and 3) both cells in the same z-axis plane. At least 100 accepted conjugates on each slide were analyzed and scored as either positive or negative for translocation of the protein to the APC interface or for accumulation of NF-ATc in the nucleus. All slides were coded and scored blind, i.e., without knowledge of the age of the T cell donor. Two slides were examined for each sample (n > 200 total conjugates), and the mean of the two values was used for further statistical analysis.
Statistical analyses
Data in the text and tables represent the means ± SD of three mice from each age group, tested as one young and old pair in each set of experiments. Statistical significance was assessed using the paired Student’s t test at p = 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies by Monks et al. (1, 2) have shown that T
cells interacting with APCs form highly organized protein clusters at
the site of cell contact. The TCR becomes concentrated in the c-SMAC of
the interface, with other signaling proteins associated to this cluster
or surrounding it. In our own investigations we made use of the CH12 B
cell line, which expresses the MHC-II in the context of
I-Ek and I-Ak, and high
levels of ICAM-1, B7.1, and B7.2 (data not shown). As our source of T
cells we used the AND line of PCC-specific transgenic mice, in which
the majority of CD4+ T cells remain naive
throughout life, and express the V
11 and V
3 specific for PCC
(35). In preliminary studies using proliferation as
endpoint (I. Dozmorov and R.A. Miller, unpublished observations), we
confirmed previously published data showing that the agonist peptide
sequence ANERADLIAYLKQATK, at 2–20 µM, triggers strong
proliferation of the transgenic T cells, and that a peptide in which N
is substituted for K (shown in boldface) provides a nonstimulatory
control.
Fig. 1
A shows a representative
set of digital images of CD3 localization in transgenic T cells
conjugated to peptide-pulsed CH12 cells. The T cells in each image are
smaller than the CH12 cells, and show strong CD3 fluorescence
signals. The image at left shows a conjugate between a T cell and CH12
bearing the nonagonist control peptide; in this as in other negative
controls, tight conjugation was confirmed by Nomarski optics (not
shown; but see Ref. 28 for examples). In the presence of
the control peptide, CD3 remains distributed evenly around the
outside of the T cell, as expected, consistent with the lack of immune
synapse formation. CH12 cells pulsed with agonist peptide generate two
types of tight conjugates. Some conjugates, like the one shown in the
middle of Fig. 1A, show no redistribution of the
CD3-chain whereas others, such as the one shown at the
right of Fig. 1A, exhibit highly concentrated
-chain staining in the center of the area of cell-to-cell contact.
These latter, positive responses closely resemble the images generated
previously by other authors using parallel methods (2, 5)
and represent the c-SMAC area of the immune synapse.
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Fig. 1
B shows the proportion of conjugates, from three
young and three old mice, that showed strong CD3 relocalization in
responses to agonist and nonagonist peptide stimuli. As expected, the
nonagonist peptide triggers CD3 relocalization in only a small
proportion (<5%) of conjugates from young or old donors. The agonist
peptide triggered -chain redistribution in 48% of T cells from
young mice, and in 20% of the cells from old mice; this difference is
significant at p < 0.03. These data suggest that aging
diminishes the proportion of naive T cells that can form effective
SMACs at the site of interaction with peptide-bearing APC.
The redistribution of multiple proteins to the immune synapse declines with age in naive CD4+ T cells
In some systems (1, 2, 28), the percentage of CD3
molecules relocating to the c-SMAC is much smaller than the fraction of
other TCR-dependent signaling proteins, such as PKC and LAT, that
move to the site of the synapse. Thus the defect in CD3-chain
redistribution in T cells from aged transgenic mice does not exclude
the possibility that the small numbers of TCR molecules at the site of
APC interaction might still be able to complex with other signaling
proteins and form functionally efficient immune synapses. To examine
this problem we performed a series of experiments with nonagonist and
agonist peptides, staining the conjugates for proteins known in other
models to be associated with the TCR complex. We selected three groups
of proteins for these analyses. The first group included PLC and
c-Cbl, enzymes thought to play positive and negative roles,
respectively, in the progress of the TCR signal transduction. The
second group included the tyrosine kinases Lck, Fyn, and Zap-70, which
are responsible for phosphorylation of both CD3 molecules and other
proteins in the TCR signal transduction complex. The third group
included LAT, Grb-2, and Vav, adapter molecules that help to recruit
other elements of the transduction chain. Fig. 2 shows representative digital images
from this series of experiments. The upper left panel of
Fig. 2 shows a negative control, using nonagonist peptide stained for
distribution of c-Cbl; similar negative controls using nonagonist
peptide were obtained for all the Abs used (not shown). The other eight
panels show examples of conjugates in which the indicated target
molecules did indeed relocalize to the synapse in response to agonist
peptide-pulsed CH12 cells.
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(see Fig. 1); these proteins seem likely to be bound
tightly to the TCR/CD3 complex within the c-SMAC. In contrast,
distribution of c-Cbl, PLC, Lck, Fyn, Grb2, and Vav was typically
much more uniform along the area of membrane contact. These differences
in distribution were not altered appreciably by age (not shown).
A series of three experiments, each using an old and a young mouse, was
conducted to determine the effects of age on the proportion of
CD4+ naive T cells that could rearrange these
proteins to the immune synapse. Table I
summarizes these results. Nonagonist peptide controls were included for
c-Cbl, Lck, and LAT, and showed that fewer than 10% of conjugates from
young or old mice were able to induce protein redistribution in
response to the control peptide, in good agreement with the CD3 and
proliferation data.
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rearrangement was noted in 62% of T cells from young mice
(p < 0.05 compared with the 24% of T cells
from old donors); the slightly higher results for PLC may represent
a technical artifact, in that the fluorescent signal was very strong
for this reaction. The effect of aging on c-Cbl relocalization is more
difficult to assess, in part because the c-Cbl localization to the APC
interface was noted in 10% of conjugates generated using the
nonagonist control peptide. The effect of age is somewhat smaller than
for the other molecules examined, and is not statistically significant
(p = 0.15). Taken together, the data suggest
that aging can decrease the translocation of many TCR-associated
proteins to the immune synapse, and are consistent with models in which
altered protein relocation contributes to defects in LAT
phosphorylation (28) and in later stages of activation in
T cells from aged donors. All-or-none redistribution of multiple proteins in the immune synapses of naive CD4 T cells
The close agreement between the proportions of T cells showing
rearrangement of each protein (see Table I) suggests that most T cells
either rearrange all the tested proteins, or none of them. However,
single-color experiments cannot formally exclude the hypothesis that
defects in the ability to relocalize specific coupling molecules might
be differentially distributed among the cells of young or old mice. We
tested this possibility by using a double-staining system, in which
conjugates are examined using both anti-c-Cbl Ab and Abs to CD3,
LAT, or PLC. The method uses a mouse anti-c-Cbl monoclonal and
goat anti-mouse Fc-FITC to avoid cross-reaction with the IgM
expressed on the surface of CH12 cells. No significant background
interference was seen using this secondary Ab alone (not shown). The
second molecule (LAT, CD3, or PLC) was then detected using
specific rabbit antisera followed by goat anti-rabbit coupled to
Alexa-594. Fig. 3 shows typical pairs of
digital images showing both fluorescent channels for individual
conjugates. The examples chosen are ones in which both c-Cbl and the
other tested molecule are rearranged in the same cell; these constitute
the majority of cells examined (see below). The distribution of the
proteins within the synapses is similar to that noted in single-color
experiments (see Fig. 2), with CD3 and LAT more centrally located
than c-Cbl or PLC. Table II presents
results of these two-color tests, from three mice of each age, showing
the proportion of cells with each staining pattern as a percentage of
all cells in which at least one protein was rearranged. Double-positive
cells made up at least 83% of all stained cells for each case. Fewer
than 15% of the positive cells stained for c-Cbl alone, and fewer than
5% stained positive for CD3, LAT, or PLC but not for c-Cbl.
There were no statistically significant differences for any of these
values between the young and old samples. These findings are consistent
with our previous data looking at colocalization of LAT and PKC in
individual CD4 cells from nontransgenic young mice stimulated by an
anti-CD3 hybridoma cell line (28). The observations
show that defects in the ability to relocalize effector proteins to the
immune synapse are not randomly distributed among cells, but instead
that the redistribution reaction may be "all-or-nothing" at least
with respect to the specific proteins we have examined.
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Formation of the immune synapse is followed within minutes by
activation of several downstream pathways whose linkage to the TCR
signal is not yet fully elucidated. Many of these early steps are
diminished by aging, including activation of mitogen-activated protein
kinases of the extracellular signal-related kinase (36)
and JNK families (25, 26), and activation of RAF-1
(19), together with impaired activation of transcriptional
factors such as NF-ATc (27, 28). To see whether defects in
the assembly of protein complexes at the site of TCR/APC interaction
might contribute to age-dependent alterations in these downstream
events, we performed a series of experiments examining both NF-ATc
translocation and relocalization of LAT, PLC, or c-Cbl in individual
T cells. Fig. 4 includes a set of digital
images derived from these experiments, showing examples of conjugates
in which relocalization of LAT, PLC, or c-Cbl to the synapse either
is, or is not, accompanied by NF-ATc migration to the nucleus in the
conjugated T cell. Conjugates positive for NF-ATc translocation but
negative for the membrane rearrangement were extremely rare (not
shown).
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summarizes these results. For
naive CD4 cells from young mice, 75% of the cells that scored
positive for the synaptic protein also exhibited NF-ATc translocation
(scored as +/+). Approximately 24% of the responding conjugates showed
a relocalization of the synapse component but not translocation of
NF-ATc (-/+), and fewer than 3% showed translocation of NF-ATc alone
(+/-). The data show that young T cells that are able to form synapses
involving LAT, c-Cbl, and PLC typically proceed to NF-ATc migration.
In contrast, fewer than 32% of those CD4 naive T cells from old mice
that relocalize LAT, c-Cbl, or PLC also exhibit NF-ATc migration to
the nucleus. The majority (70%) of old T cells that show synaptic
responses fail to proceed to NF-ATc translocation, with rearrangement
of NF-ATc alone as rare among aged T cells as in the ones from younger
donors. The proportion of LAT+ cells that also
score as NF-ATc-positive is significantly different
(p = 0.03) between young and old mice, and the
effects of age in the experiments using c-Cbl and PLC markers are
also significant at p < 0.04.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Zap-70, and LAT relocalized to the central
domain of the synapse, similar to the c-SMAC pattern previously
documented in other systems (2). The distribution of
PLC, Grb-2, Vav, c-Cbl, and Fyn resembles that seen for cytoskeletal
proteins, such as talin, which localize to the p-SMAC of the synapse
(2, 4).
However, not all naive CD4 T cells, freshly isolated from mouse
spleens, are able to form synapses of this composition after
conjugation to peptide-loaded APC. When the T cells are derived from
young donors, only 50% of those forming tight conjugates with APC
translocate any of the listed proteins to the synapse (Table I). These
values are consistent with those noted in previous studies of LAT and
PKC translocation in T cells activated by exposure to hybridoma
cells bearing cell surface Ab to mouse CD3 (28, 30, 31). In the current study, the proportion of T cells showing
translocation of CD3 was higher than in our previous work
(28) and in reports from other groups (2),
perhaps because our current system uses higher concentrations of
agonist peptide (20 µM, instead of 2 µM). The distribution of Lck
also deserves comment. In our studies we found Lck to be distributed
throughout the area of APC-T cell contact (presumably within the
p-SMAC). Consistent with our findings, Krummel et al. (38)
found that CD4/Lck complexes, initially present within the central area
of the synapse, migrated within minutes of synapse formation to more
peripheral areas of the contact zone.
The one-color data summarized in Table I show that relatively few naive
CD4 T cells from old mice are able to generate synapses that include
any of the tested molecules: c-Cbl, PLC, Lck, Zap-70, Fyn, LAT,
Grb-2, and Vav. In each case the proportion of responsive naive CD4
cells from aged donors is about half that of cells from young donors.
These results are similar to those seen in our previous studies of
unseparated CD4 cells from young and old nontransgenic donors
responding to anti-CD3 stimulation, as monitored by translocation
of LAT and Vav (28). The new data show that aging affects
T cell responses to peptide Ags in addition to responses triggered by
high affinity anti-receptor Abs, show that the changes affect all
eight of the tested components of the synapse, and show that the aging
effect is seen in naive T cells, and thus not due simply to the
accumulation of memory CD4 T cells in old age. The two-color
experiments of Table II show that cells, from young or old donors,
which exhibit defects in any one of the four proteins tested (c-Cbl,
LAT, PLC, and CD3) usually show defects in all of them; in this
sense the changes are "all or nothing" at the single-cell level. We
have not examined the effects of aging on the time course of
translocation of these proteins to the synapse in transgenic T cells,
and, therefore, it is possible that differences in response to
peptide-APC conjugates may be slowed, rather than absent, in T cells
from old donors, even though our previous studies using an anti-CD3
hybridoma cell line as a polyclonal stimulator found no evidence for an
effect of aging on the time course of synapse assembly in CD4 or CD8
cells (31). Although not all of the eight molecules
summarized in Table I have been examined in two-color experiments, the
excellent agreement among them in the proportions of responsive cells
in mice of either age are consistent with the idea that they, too, show
parallel responses, all on, or all off, within individual cells.
Although many of these proteins are either constitutively associated
with high viscosity raft domains in the T cell membrane, or can become
raft-associated by binding to LAT (13, 14), our previous
work (28) found no evidence for an age effect on
distribution of LAT, RAFT-constitutive GM-1, or raft-excluded CD45 in
resting or anti-CD3-activated T cells. Thus defects in immune
synapse formation probably cannot be attributed with changes in the
initial composition of the raft microdomains (28).
However, it is possible that further studies of raft-associated
proteins may provide clues to the mechanism of the alterations in
complex formation we have documented. It is also plausible that age
effects on formation of functional immune synapses might be due to
changes in cytoskeleton reorganization during responses to TCR
stimulation. In this context it is noteworthy that at least one of the
cytoskeletal proteins, talin, can apparently become relocalized to the
immune synapse in T cells exposed to nonagonist peptides (1, 2), whereas other proteins, such as PKC, migrate only in
response to agonist peptides. Biochemical and microscopic techniques
will help to sort out the ways in which aging might alter association
of signaling proteins with cytoskeletal proteins before and during
responses to agonist peptides.
Two-color experiments can also test the linkages between events
immediately tied to TCR stimulation, and those down-stream events, such
as migration of transcription factors and induction of new gene
expression, that are triggered by kinase-dependent cascades. The data
presented in Table III show that T cells from aged mice have, over and
above the problems in synapse formation summarized in Table II, a
diminished ability to translocate NF-ATc to the nucleus. Among young
CD4 T cells that show LAT migration, for example, 77% proceed to
NF-ATc migration, but this figure falls to 31% for cells from aged
donors, with very similar changes seen when c-Cbl or PLC is used as
the index of functional synapse formation. Thus there seem to be three
classes of CD4 T cells that can be discriminated in responses to
peptide-bearing APCs: 1) those that form synapses and induce NF-ATc
migration; 2) those that form synapses but do not undergo NF-ATc
migration; and 3) those that do neither, with the latter two classes
increasing as a function of age. T cells from aged donors also show
defects in activation of the Raf-1 and JNK-dependent protein kinase
pathways (19, 25), the latter of which depends upon
CD28-mediated signaling. It is possible that alterations in CD28/JNK
signals or in PLC-independent generation of calcium signals
(39) might contribute to derangements in the postsynaptic
processes required for NF-ATc translocation and induction of IL-2 gene
expression. Further work should now be able to define additional
biochemical and functional differences among these three classes of
cells, and may help to develop a more detailed picture of the ways in
which T cells discriminate between agonist and nonagonist peptides, and
of the changes that impair activation of T cells in old mice.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard A. Miller, 5316 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0940.
3 Abbreviations used in this paper: SMAC, supramolecular activation cluster; LAT, linker for activation of T cells; NF-ATc, cytoplasmic NF-AT; PCC, pigeon cytochrome-C; PKC, protein kinase C; Vav, p95vav; c-SMAC, central area; p-SMAC, peripheral area.
Received for publication September 28, 2000. Accepted for publication December 28, 2000.
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L. Cicin-Sain, I. Messaoudi, B. Park, N. Currier, S. Planer, M. Fischer, S. Tackitt, D. Nikolich-Zugich, A. Legasse, M. K. Axthelm, et al. Dramatic increase in naive T cell turnover is linked to loss of naive T cells from old primates PNAS, December 11, 2007; 104(50): 19960 - 19965. [Abstract] [Full Text] [PDF]
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G. G. Garcia, A. A. S. Akha, and R. A. Miller Age-Related Defects in Moesin/Ezrin Cytoskeletal Signals in Mouse CD4 T Cells J. Immunol., November 15, 2007; 179(10): 6403 - 6409. [Abstract] [Full Text] [PDF]
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K. Clise-Dwyer, G. E. Huston, A. L. Buck, D. K. Duso, and S. L. Swain Environmental and Intrinsic Factors Lead to Antigen Unresponsiveness in CD4+ Recent Thymic Emigrants from Aged Mice J. Immunol., February 1, 2007; 178(3): 1321 - 1331. [Abstract] [Full Text] [PDF]
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M. G. Marko, T. Ahmed, S. C. Bunnell, D. Wu, H. Chung, B. T. Huber, and S. N. Meydani Age-Associated Decline in Effective Immune Synapse Formation of CD4+ T Cells Is Reversed by Vitamin E Supplementation J. Immunol., February 1, 2007; 178(3): 1443 - 1449. [Abstract] [Full Text] [PDF]
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S. N. Han, O. Adolfsson, C.-K. Lee, T. A. Prolla, J. Ordovas, and S. N. Meydani Age and Vitamin E-Induced Changes in Gene Expression Profiles of T Cells J. Immunol., November 1, 2006; 177(9): 6052 - 6061. [Abstract] [Full Text] [PDF]
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D. E. Faunce, J. L. Palmer, K. K. Paskowicz, P. L. Witte, and E. J. Kovacs CD1d-Restricted NKT Cells Contribute to the Age-Associated Decline of T Cell Immunity J. Immunol., September 1, 2005; 175(5): 3102 - 3109. [Abstract] [Full Text] [PDF]
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S. Davanture, J. Leignadier, P. Milani, P. Soubeyran, B. Malissen, M. Malissen, A.-M. Schmitt-Verhulst, and C. Boyer Selective Defect in Antigen-Induced TCR Internalization at the Immune Synapse of CD8 T Cells Bearing the ZAP-70(Y292F) Mutation J. Immunol., September 1, 2005; 175(5): 3140 - 3149. [Abstract] [Full Text] [PDF]
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 S. B. Berger, A. A. S. Akha, and R. A. Miller A glycoprotein endopeptidase enhances calcium influx and cytokine production by CD4+ T cells of old and young mice Int. Immunol., August 1, 2005; 17(8): 983 - 991. [Abstract] [Full Text] [PDF]
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W. Ise, K. Nakamura, N. Shimizu, H. Goto, K. Fujimoto, S. Kaminogawa, and S. Hachimura Orally Tolerized T Cells Can Form Conjugates with APCs but Are Defective in Immunological Synapse Formation J. Immunol., July 15, 2005; 175(2): 829 - 838. [Abstract] [Full Text] [PDF]
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 L. Haynes, S. M. Eaton, E. M. Burns, T. D. Randall, and S. L. Swain Newly generated CD4 T cells in aged animals do not exhibit age-related defects in response to antigen J. Exp. Med., March 21, 2005; 201(6): 845 - 851. [Abstract] [Full Text] [PDF]
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I. Messaoudi, J. LeMaoult, J. A. Guevara-Patino, B. M. Metzner, and J. Nikolich-Zugich Age-related CD8 T Cell Clonal Expansions Constrict CD8 T Cell Repertoire and Have the Potential to Impair Immune Defense J. Exp. Med., November 15, 2004; 200(10): 1347 - 1358. [Abstract] [Full Text] [PDF]
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L. A. Norian and P. M. Allen No Intrinsic Deficiencies in CD8+ T Cell-Mediated Antitumor Immunity with Aging J. Immunol., July 15, 2004; 173(2): 835 - 844. [Abstract] [Full Text] [PDF]
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 A. Ortiz-Suarez and R. A. Miller Antigen-Independent Expansion of CD28hi CD8 Cells From Aged Mice: Cytokine Requirements and Signal Transduction Pathways J. Gerontol. A Biol. Sci. Med. Sci., December 1, 2003; 58(12): B1063 - 1073. [Abstract] [Full Text] [PDF]
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 V. Jankovic, I. Messaoudi, and J. Nikolich-Zugich Phenotypic and functional T-cell aging in rhesus macaques (Macaca mulatta): differential behavior of CD4 and CD8 subsets Blood, November 1, 2003; 102(9): 3244 - 3251. [Abstract] [Full Text] [PDF]
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G. G. Garcia and R. A. Miller Age-Dependent Defects in TCR-Triggered Cytoskeletal Rearrangement in CD4+ T Cells J. Immunol., November 1, 2002; 169(9): 5021 - 5027. [Abstract] [Full Text] [PDF]
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Y. M. Vyas, K. M. Mehta, M. Morgan, H. Maniar, L. Butros, S. Jung, J. K. Burkhardt, and B. Dupont Spatial Organization of Signal Transduction Molecules in the NK Cell Immune Synapses During MHC Class I-Regulated Noncytolytic and Cytolytic Interactions J. Immunol., October 15, 2001; 167(8): 4358 - 4367. [Abstract] [Full Text] [PDF]
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O. Adolfsson, B. T. Huber, and S. N. Meydani 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., October 1, 2001; 167(7): 3809 - 3817. [Abstract] [Full Text] [PDF]
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