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* Department of Pathology, School of Medicine, and
Institute of Gerontology, University of Michigan, Ann Arbor, MI 48109; and
Ann Arbor Department of Veterans’ 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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in response to CD3/CD4/CD28 cross-linking is much
stronger in young than in old T cells. Thus, defects in cytoskeletal
reorganization triggered by initial contact between TCR and
peptide-bearing APC precede, and presumably contribute to, defective
activation of protein kinase-mediated signals in the first few minutes
of the activation cascade in T cells from aged
mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD4+ T cells from aged mice show defects in TCR
signal transduction that include deficits in serine/threonine and
tyrosine phosphorylation, diminished activation of Raf-1, c-Jun
N-terminal kinase, and extracellular signal-regulated kinase, and
diminished elevation of intracellular Ca+, among
other problems (for comprehensive review, see Ref. 8). In
contrast, our experiments (9) have shown no effect of
aging on the level of activation of the TCR-associated tyrosine kinase
Zap-70 after CD3-CD4 stimulation in freshly isolated mouse
CD4+ T cells. The contrast between the
age-insensitive activation of Zap-70 and the age sensitivity of
multiple downstream events led us to postulate that age-related defects
in TCR-mediated activation signals are related to a lack of
accessibility of the TCR-associated kinases to their substrates.
Experiments using two-color confocal microscopy (10, 11, 12, 13)
supported this hypothesis by showing age-dependent defects in migration
of multiple substrates, including the linker for activation of T cells
(LAT), Vav, Grb2, and phospholipase C (PLC)-
, to the site of T
cell/APC contact in peptide-specific and CD3-dependent conjugates. In
this study, we describe a series of experiments to explore the
hypothesis that age-dependent alterations in T cell cytokeletal
organization and lamellopodia formation might contribute to the decline
with age in the proportion of T cells that can form functional immune
synapses when conjugated to stimulatory APC.
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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 pigeon cytochrome c (PCC), were a generous gift from S. Swain (Trudeau Institute, Saranac Lake, NY). Transgene-positive mice were aged in a specific pathogen-free colony at the University of Michigan. Specific pathogen-free male (BALB/c x C57BL/6)F1 (CB6F1) mice were purchased from the National Aging Institute contract colonies at the Charles River Laboratories (Kingston, NJ) and at Harlan (Indianapolis, IN). Mice were given free access to food and water. Sentinel animals were examined quarterly for serological evidence of viral infection; all such tests were negative during the course of these studies. 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) or 18–20 (old) mo of age. The CH-12 B cell line (expressing the I-Ak MHC-II and high levels of ICAM and B-7.1) was obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI with 10% FCS and 2 mM L-glutamine at 37°C and 10% CO2.
Abs and reagents
Rabbit polyclonals anti-PLC-, c-Cbl, and CD3 (clone
6B10) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Rabbit anti-Lck was purchased from Upstate Biotechnology (Lake
Placid, NY). Anti-talin (clone 8, day 4) and anti-vinculin (clone
hVIN-1) were purchased from Sigma-Aldrich (St. Louis, MO). Alexa-488
phalloidin was purchased from Molecular Probes (Eugene, OR).
Anti-paxillin was purchased from Transduction Labs (Lexington, KY). The
GST-Grb2 construct was a donation of G. Koretzky (University of
Pennsylvania, Philadelphia, PA). The GST-band III construct was a gift
from A. Weiss (Howard Hughes Medical Institute, San Francisco, CA). A
construct encoding the cytoplasmic tail of CD3 was a gift of M.
Exley (Beth Israel Deaconess Medical Center, Boston, MA). The GST-Grb2,
GST-band III, and GST-CD3 fusion proteins were purified
(14) and biotinylated in our lab, as described
(15). Rabbit anti-CD3 (recognizing all
phosphorylated and nonphosphorylated forms of CD3) was developed in
our lab using the full cytoplasmic domain of -chain.
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 antagonist peptide had the same sequence, with the exception of a single amino acid substitution (K to N) at position 99, marked in bold (16).
Cell preparation
CD4+ T cells were obtained and stimulated from transgenic or CB6F1 mice using the negative selection methods previously described (12, 17). Flow cytometry analysis of a typical preparation showed it to be 90–95% positive for both CD3 and CD4.
TCR-APC conjugation, spreading assay, and confocal microscopy
CH-12 cells pulsed with 20 µg/ml of agonist or antagonist
peptide were conjugated with CD4+ T cells, as
described (11). For the two-color analyses, we first used
specific rabbit or mouse Abs to cytokeletal proteins (at 1 µg/ml in
PBS containing 0.2% BSA), or in some cases Alexa-488 phalloidin (at 2
U/ml in PBS/BSA), followed by biotin-GST-Grb2 (0.5 µg/ml in PBS/BSA).
The slides were developed by incubation for 1 h with goat
anti-rabbit or anti-mouse Fc Abs coupled to Alexa-488
(except for phalloidin) and then streptavidin Alexa-594 (Molecular
Probes), all at 1 µg/ml in PBS/BSA.
For the spreading assays, polylysine slides were incubated with anti-DNP (clone UC-8), anti-CD28 (clone 37.51), or anti-CD3 (clone 145-2C11) in PBS for 2 h at 37°C using the concentrations indicated in the text. The slides were then washed several times with PBS and prewarmed at 37°C in PBS before use. CD4+ T cells at 2 x 106 cells/ml in RPMI 1640 were then gently deposited on the Ab-coated slide, and incubated in a humidified chamber at 37°C for the times indicated in the text. The slides were fixed, permeabilized with 1% saponin in PBS for 10 min at 4°C, and stained with Alexa-488 phalloidin. After extensive washing with PBS, the slides are mounted, as described (11).
The two-color analyses were performed at x100 magnification on a Nikon Diaphot microscope equipped with a Bio-Rad MRC 600 confocal laser imaging system (Bio-Rad, Hercules, CA), as described (10). For the spreading assay, digital images for at least 100 cells/slide were recorded. The digital images were converted to TIFF files, and the area of individual cells was measured using Scion Image software (www.scioncorp.com). All slides were coded and scored blind, i.e., without knowledge of the age of the T cell donor.
Cytoskeleton purification, immunoprecipitation, and Western blots
Approximately 10 x 106
CD4+ T cells were stimulated for 5 min by
cross-linking CD3
with CD4 and CD28, as described previously
(17). The cells were lysed in 1 ml of 1% Brij-58, PBS, pH
7.4, 10 µg/ml aprotinin, 100 µg/ml leupeptin, 10 mM PMSF, and 0.1
mM sodium orthovanadate for 30 min on ice. The lysates were
ultracentrifuged at 100,000 x g for 1 h at 4°C.
The pellet (cytoskeleton fraction) was washed once with lysis buffer,
centrifuged at 100,000 x g for 10 min, resuspended in
50 µl of SDS sample buffer, and boiled for 15 min. The CD3
contained in the supernatant (soluble fraction) was immunoprecipitated
overnight with rabbit anti-CD3 chain precoupled to protein
G-Sepharose. The beads were washed and then boiled for 5 min with 50
µl of SDS sample buffer. Aliquots from cytoskeleton and the CD3
soluble fraction were resolved in 12% polyacrylamide gel, transferred
to polyvinylidene difluoride, and incubated with anti-CD3 mouse
monoclonal (clone 6B10); the bands were visualized by chemifluorescence
and quantified, as previously described (9).
Statistical analyses
Unless otherwise indicated, results are presented as means ± SD of at least six young and three old subjects tested in at least three separate experiments. Statistical significance was assessed using a Mann-Whitney test at p = 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously used transgenic mice, whose naive
CD4+ T cells are specific for a peptide derived
from PCC, to show age-associated decline in T cell immunosynapse
formation (11). We have now extended these studies to
document the usefulness of biotin-GST-Grb2 as a single cell marker for
immunosynapse formation. We chose biotin-Grb2 for this purpose based on
previous demonstrations that it interacts with specific domains of
Slp-76 (18), Cbl, and LAT (19), proteins that
are themselves components of the SMAC. The use of biotin-GST-Grb2 also
provides a technical advantage as a second color stain for confocal
microscopy, in that it avoids the problems of cross-reactivity between
secondary Abs used to localize other intracellular molecules of
interest. Fig. 1
A shows
digital images of T cells, conjugated with peptide-bearing CH-12 cells
and then stained for GST-Grb2 as well as for PLC-, Lck, or the
cytoskeletal proteins paxillin, vinculin, and F-actin. The top
panel shows a T cell/CH-12 conjugate in which neither Grb2 nor
PLC- has moved to the immunosynapse; double-negative cells of this
kind are seen, at varying proportions as discussed below, for all pairs
of stains used. The next two rows of Fig. 1A show
double-positive immunosynapses in which PLC- (or Lck) and
biotin-GST-Grb2 become localized to the area of T cell-APC contact. We
found very few single-positive cells, i.e., cells in which the synapse
area contained either Grb2 alone or PLC- (or Lck) alone. Results
using Abs to LAT or c-Cbl together with GST-Grb2 were similar to those
for PLC- and Lck (not shown). For controls of the GST-Grb2
specificity, we use a biotin-GST-band III fusion protein (for a
description of this construct, see Ref. 9). Under our
experimental conditions, we find that the GST-band III has no
detectable binding of any protein from the CD4+ T
cell. These results indicate that the biotin-GST-Grb2 construct can be
used to identify CD4+ T cells that have formed
immunosynapses containing PLC-, Lck, LAT, and c-Cbl. During
immunosynapse formation, there is a reorganization of the cytoskeleton
characterized by polymerization of actin into F-actin filaments and
accumulation of actin-related protein 2–3 (Arp2–3) at the
immunosynapse (20). We therefore also examined
translocation of paxillin, vinculin, and F-actin, in relation to
GST-Grb2 localization. We found that all three of these molecules can
move to the GST-Grb2-containing immunosynapses; results with F-actin
and paxillin are shown in the bottom rows of Fig. 1A.
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, Lck,
F-actin, vinculin, and paxillin in that it has been reported to move to
the area of APC-T cell interaction even when antagonist peptides are
used in place of agonists for stimulation (6). This
translocation requires TCR/MHC interactions, but does not result in the
formation of a SMAC (6). To confirm these results in our
own transgenic system, we studied redistribution of talin using CH-12
cells pulsed with an antagonist peptide. Fig. 1
B shows
typical results using Abs to talin along with GST-Grb2. We noted
double-negative conjugates in which neither protein had moved to the
synapse area (Fig. 1B, top), others in which
talin, but not GST-Grb2 had been redistributed (Fig. 1B,
middle), and still others (Fig. 1B,
bottom) in which both molecules had translocated to the
immunosynapse. Conjugates in which only GST-Grb2 had moved to the
synapse without talin redistribution were very rare (not shown).
Using this methodology, we then quantified the percentage of transgenic
CD4+ T cells from young and old mice that can
produce F-actin fibrils and can redistribute focal adhesion proteins to
the immunosynapses using GST-Grb2 as our indicator for synapse
formation. Table I shows mean values for
double-negative (-/-), single-positive (i.e., positive for the
cytoskeletal proteins, but not for Grb2) (+/-), and double-positive
(+/+) cells from three experiments involving six young and three old
mice. Conjugates in which only GST-Grb2 had relocated, without
relocation of the cytoskeletal element, represented fewer than 1% of
the total in nearly all experiments and are therefore not shown in
Table I. Antagonist peptide, as expected, induced formation of synapses
containing both talin and GST-Grb2 in only a small percentage (3–11%)
of the conjugates, consistent with previous reports that antagonist
peptides do not induce the formation of functional synapses containing
Grb2-binding proteins. When the antagonist peptide was used, 51% of
the CD4+ T cells from young mice showed talin
redistribution without GST-Grb2 translocation, consistent with models
in which antagonist peptides induce early, but not late stages in
immunosynapse formation. In CD4+ T cells from old
mice, however, only 12% of the conjugates showed talin redistribution
by itself. This age effect is statistically significant at
p < 0.001, and shows that T cells from old mice have a
defect at a very early stage of the interaction with APCs, i.e., before
the discrimination by the TCR between agonist and antagonist peptides,
which leads to formation of the SMAC. Table I also shows that neither
paxillin, vinculin, nor F-actin moved to the area of T cell-APC contact
in responses to antagonist peptides, even in CD4+
T cells from young mice. The results suggest that the translocation of
these focal adhesion proteins and detectable polymerization of actin
are, unlike talin translocation, dependent on SMAC formation, or on
processes triggered by agonist peptides that lead to SMAC
formation.
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, demonstrated that
during immunosynapse formation, 61–75% of the conjugates from young
donors show translocation of paxillin and vinculin and polymerization
of actin in the T cell-APC contact area. In the same
experiments, only 15% of the T cells from young mice are single
positive for talin, and fewer than 2% are single positive for F-actin
or for the other three focal adhesion proteins; thus, nearly all of the
young CD4 cells that are able to relocalize the focal adhesion proteins
to the synapse proceed to the stage of Grb2 relocalization as well.
Consistent with our previous studies of immunosynapse formation
(11), there is a far smaller proportion of
CD4+ T cells from aged transgenic mice that can
form immunosynapses in response to agonist-pulsed APC, ranging from 19
to 25%, depending on the specific cytoskeletal protein examined along
with GST-Grb2. These age-related declines in cytoskeleton protein
relocalization are statistically significant at p <
0.05. These results show that, with the exception of talin,
translocation of cytoskeletal proteins to the T cell-APC contact area
requires agonist peptide. Thus, the age-related defects in T cell
activation include changes at early stages of TCR-APC interaction,
changes that precede recruitment of other cytoskeletal elements, as
well as proteins that bind to Grb2, to the APC interaction
site. Age-related decline in lamellopodia formation by CD4+ T cells
T cells form lamellopodia, a flattening of the contact area with
the APC with a partial reorganization of the cytoskeleton, before the
formation of a complete functional immune synapse
(21, 22, 23). To test whether formation of lamellopodia is
affected by aging, we developed an assay to study the spreading of
CD4+ T cells, purified from
CB6F1 mice, on slides covered with anti-CD3
Ab; slides covered with anti-CD28 or anti-DNP are used as
negative controls. The upper panel of Fig. 2A shows an example of a young
CD4+ T cell incubated for 10 min on an
anti-CD28-coated slide. The figure compiles images of the different
Z planes of a single cell, stained with phalloidin to show F-actin. In
this cell, F-actin is distributed uniformly around the cell periphery
from the top of the cell (left part of the series) to
the area in which the cell is in contact with the anti-CD28 on the
surface of the glass slide. Identical results are found using
anti-DNP-coated slides (not shown). In contrast, cells incubated on
slides covered with anti-CD3 typically spread and acquire a conical
shape, as illustrated in the bottom panel of Fig. 2A. The Z plane of contact with the anti-CD3 (the
rightmost Z plane section in the series) shows high levels
of polymerized actin localized as a ring at the edge of the cell, with
a central area containing lower levels of F-actin. The T cell shape and
F-actin redistribution and cytoskeleton reorganization are similar to
alterations previously seen in T cells conjugated to APC (24, 25), although the SMAC is not formed in such slide-adherence
cultures (24). Fig. 2B shows typical digital
images of CD4+ cells from young or old mice
obtained from the contact zone using anti-DNP-, anti-CD28-, or
anti-CD3-coated slides. As expected, CD4+ T
cells from young mice generate contact zones that are round and
relatively small when the slides are coated with anti-DNP or
anti-CD28. Contact zones of CD4+ T cells from
old mice are similarly round and homogeneous, but for unknown reasons
are typically somewhat larger than those produced by T cells from young
donors. Incubation on anti-CD3-coated slides induces in young CD4
cells a larger contact zone characterized by a bright ring and an inner
zone of diminished phalloidin-staining intensity.
CD4+ T cells from old mice, incubated on
anti-CD3 slides, fall into two classes. Some cells show with no
detectable spreading (similar to images seen on anti-CD28), but
others closely resemble T cells from young mice on anti-CD3 (see
arrows in Fig. 2B for contrasting examples).
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C shows a
representative result, comparing cells from one young and one old
CB6F1 mouse. T cells from the old mice incubated
on anti-CD28 slides (
) have slightly larger contact zones
compared with T cells from young donors (
), i.e., 1.8 arbitrary
units (SD = 0.8). When T cells from young mice are incubated with
anti-CD3 (
), the mean area increases from 1 to 3.2 ± 0.7.
The mean value for old T cells on anti-CD3 slides increases only
slightly, i.e., from 1.8 to 2.2 ± 0.9 U (•). The distribution
of cell areas for the anti-CD3-treated old
CD4+ cells is much broader than that seen for
young CD4+ cells, and there is substantial
overlap with the distribution of areas seen in the anti-CD28
control condition. We interpreted this distribution of old CD4 cells as
a composite of two overlapping populations, one that responds slightly,
if at all, to anti-CD3 contact, and a second that can spread nearly
as well as T cells from young mice. We also performed this spreading assay using CD4+ T cells from the transgenic mice. The anti-CD3 induced a 3.5 ± 0.2 (mean ± SD)-fold induction of spreading in T cells from the young mice, but only a 2.1 ± 0.2-fold induction in T cells from the old mice. We also noted a bimodal distribution, including some responsive and some nonresponsive cells, in T cells from the aged transgenic mice, similar to that seen in experiments with CB6F1 mice (data not shown).
It has been reported that PMA-induced increases in the mobility of membrane LFA-1 reflect partial cytoskeleton reorganization and release of LFA-1 from the cytoskeleton matrix as a result of protein kinase C activation or increases in membrane fluidity (26). To test the hypothesis that age-related defects in cytoskeleton reorganization can be overridden by pharmacological agents, we treated T cells from young and old with 5 nM of PMA for 10 min and then performed the spreading assay, as described above. The results of three separate experiments showed no significant differences in the frequency distribution between young and old after PMA treatment (data not shown). The result suggests that cytoskeleton rearrangement in the old CD4+ T can be triggered by CD3 in the presence of low doses of PMA.
Table II shows the results of a more
comprehensive analysis, using two concentrations of anti-CD28 or
anti-CD3 (20 and 2 µg/ml) and two different incubation times (10
and 20 min) for CB6F1 mice, and using a single
dose and time for cells from transgenic mice. Because before
stimulation the cells from the old CB6F1 mice are
larger than those of young donors, for these mice we used two criteria
for a positive response, one set at a level exceeded by only 5% in the
first of the two young mice used on CD28 slides in each experiment, and
the other, higher, criterion set at a level exceeded by only 5% of the
old cells on CD28 slides in the same experiment. As suggested by Fig. 2, CD4+ T cells from the old
CB6F1 mice were indeed slightly larger than young
cells on control slides with anti-CD28; for the 10-min incubation
at high Ab dose, for example, 42% of the old cells exceeded the cutoff
area value, compared with only 7% of the cells from young mice. A
10-min incubation on anti-CD3-coated slides induced 98% of the
young cells to spread out to an area above the criterion (7%
background), but only 76% of the old cells (despite their higher, 42%
background value). On the control slides coated with anti-CD28,
there was no difference in spreading between young and old transgenic
mice (with 5% of cells about threshold for both genotypes), consistent
with the idea that the difference in the size of unstimulated
CB6F1 cells might reflect the age-dependent
increase in memory T cells, which is much less dramatic in transgenic
mice. A total of 96–97% of the cells from young transgenic mice
spread onto anti-CD3-coated slides, compared with only 49–52% of
cells from old transgenic mice, showing that the age-dependent decline
in spreading responses is not limited to CB6F1
animals.
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show
that 70–98% of the CD4+ T cells from young mice
can produce lamellopodia and spread out onto anti-CD3-coated
slides, but that only 17–30% of the T cells from old mice can trigger
a detectable membrane flattening when compared with the responses of
old cells to control slides coated with anti-CD28 in the same
experiments. The lack of T cell response cannot be attributed to the
shift between the naive to memory phenotype, because the T cells from
old transgenic mice remain in naive state (11) and show
similar decline in the spreading assay.
Age-related decline in translocation of CD3 from the plasma
membrane to cytoskeleton
The lack of talin redistribution and lamellopodia formation in
CD4+ T cells from the old mice could be the
result of age-related changes in the interactions between cytoskeleton
and the TCR complex. Age-related changes in CD3 association with the
cytoskeleton could limit the initial cytoskeleton reorganization and
decrease the amount of TCR translocated to the immunosynapse. To test
this hypothesis, we stimulated CD4+ T cells
purified from spleen of CB6F1 mice, by
cross-linking the TCR with CD4 and CD28, and then separated the
proteins associated with the cytoskeleton matrix (cytoskeletal
fraction) from the proteins in the detergent-soluble fraction, which
includes proteins from both membrane and cytosolic compartments. The
purity of the cytoskeletal fraction was evaluated by Western blots
showing that Raf-1 and ribosomal S6 protein kinase were found in the
soluble fraction, but not in the cytoskeleton preparation (data not
shown). Fig. 3 shows CD3 Western blots
for the cytoskeleton and soluble fractions purified from young and old
CD4+ T cells. Three predominant forms can be
detected; p16 and p21, both in the cytoskeleton and soluble
fractions, and p23, detected only in the cytoskeleton fractions.
These results are similar to previous observations using T cell lines
(4). Fig. 3 illustrates effects of donor age and of
stimulation, which we quantitated and show in Table III as mean values from five individual
mice of each age.
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molecules: a shift from the p16 form to the p21 form in the soluble
fraction. CD4+ T cells from old mice have 2-fold
lower levels of p16 (p = 0.02) and almost
2-fold higher levels of p21 in the soluble fraction
(p = 0.04), compared with T cells from young
donors.
Activation induces changes in the distribution of all three forms of
CD3 in T cells of young mice. After 5 min of activation, T cells
from young mice show increases in the amount of p16, p21, and
p23 associated with the cytoskeleton, and corresponding declines in
the levels of p16 and p21 in the soluble fraction; each of these
changes is statistically significant at p < 0.05.
These translocations from the soluble fraction to the cytoskeleton
were, however, not detected after activation of
CD4+ T cells from old mice. These results show
that aging inhibits the translocation of CD3 from the
detergent-soluble fraction to the cytoskeletal matrix. Such a change
might constrain the movement of TCR complexes to the immunosynapse by
interfering with the ability of TCR complexes to flow within plasma
membrane domains to the site of APC contact.
Two experiments of this kind were conducted using purified CD4+ T cells from four young and two old transgenic mice (data not shown). The CD4+ T cells from the young transgenic mice showed a 2.5-fold induction in p16 translocation to the cytoskeleton (mean 1.0 ± 0.2 SD in resting cells, compared with 2.5 ± 0.5 after CD3 stimulation), with a corresponding decline in p16 content in the soluble fraction (2.1 ± 0.3 in resting cells compared with 1.1 ± 0.3 after CD3 stimulation). In contrast, there were no significant changes in distribution of p16 in CD4+ T cells from the old transgenic donors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and other
signal-coupling elements. Our current results are consistent with this
model, in that both antagonist and agonist peptides induced the
translocation of talin to the T cell-APC contact area (see Fig. 1B), while only agonist peptide can trigger the
translocation of several TCR-associated proteins and formation of the
SMAC (11). To study the relation between the immunosynapse
and proteins belonging to the focal adhesion family, we have analyzed
the localization of paxillin and vinculin with respect to SMAC
formation. We selected paxillin and vinculin because these have been
reported to associate, respectively, with talin and LFA-1 (for a
review, see Ref. 27). In addition, paxillin can associate
to Src homology 2 domains of the Src family of tyrosine kinases
(27). As shown in Table I, agonist peptides can trigger
the translocation of these proteins to the immunosynapse in 60–75% of
the young naive CD4+ T cells conjugated to
agonist-bearing CH-12 APC. The results suggest that these focal
adhesion molecules play a role in immunosynapse formation, although
further studies will be needed to determine whether blocking of their
translocation abrogates the formation of SMACs. When we analyzed naive
CD4+ T cells from old mice, only 
20% of the
cells show this pattern of translocation of cytoskeletal proteins to
the APC contact area, consistent with our previous work
(11) showing age-related defects in movement of many
kinases and substrates to the SMAC in aged T cells. Our observations of talin translocation suggest that age-related changes in reorganization of the cytoskeleton may be a cause, rather than merely a consequence, of defects in SMAC formation. When redistribution was analyzed using an antagonist peptide, 62% of young CD4 cells, but only 15% of old CD4 cells, were able to relocate talin to the area of APC contact. Thus, the lack of talin translocation in the naive CD4+ T cells from old mice suggests age-related defects in the initial TCR-APC interactions before recruitment of other elements of the SMAC.
The kinetic segregation model of TCR signal transduction suggests
several possibilities that could explain defects early in the
interaction between TCR and APC, including not only alterations in
cytoskeleton reorganization, but also potentially changes in TCR-MHC-II
interaction caused by negative regulators such as CD43
(28) or other large glycoproteins (29), or a
decline in the calcium signal in the initial T cell-APC contact area.
To evaluate these ideas, we used a simplified system in which the APC
surface was replaced by glass slides coated with stimulatory
(anti-CD3) or nonstimulatory (anti-CD28 or anti-DNP) Abs,
and in which T cell cytoskeletal changes were monitored by evaluating
lamellopodia formation. This system has been shown previously to mimic,
in part, the initial TCR-APC interaction by inducing a TCR-dependent
cytoskeleton reorganization, localized calcium flux, and flattening of
the T cell membrane without formation of immunosynapses
(24). Using this system (Fig. 2 and Table II), we noted
that anti-CD3 induces spreading of CD4+ T
cells, which acquire a conical shape that resembles T cells adhering to
authentic APC (see Fig. 1 for examples), and triggers a limited
reorganization of the cytoskeleton including an F-actin ring localized
in the area of the T cell periphery that is in contact with the slide
(Fig. 2). Anti-CD3 induces this spreading response in 70–90% of the
CD4+ T cells from young mice, but only 17–31%
of the CD4+ T cells from old
CB6F1 mice. The response of transgenic T cells in
the spreading system also declines by 50% in old age.
We hypothesized that this age-related change in cytoskeletal
responsiveness might reflect underlying changes in interactions between
proteins of the TCR/CD3 complex and proteins responsible for
cytoskeletal structure. Previous work has shown that different CD3
forms are associated with different fractions of the T cell membrane
(4). Some, including p23, are anchored to the
cytoskeletal matrix, while other forms of CD3 remain relatively free
of cytoskeleton interaction in resting cells. After TCR engagement,
however, all isoforms of CD3 are translocated from the soluble
fraction to the cytoskeletal matrix, as illustrated for young CD4 cells
in Fig. 3. Our data show that aging alters the pattern of CD3
distribution in both resting and activated cells. In the resting cells,
there seems with age to be a replacement of p16 by p21 in the
soluble fraction, and after activation there is an age-related decline
in the extent to which all three forms of CD3 translocate to the
cytoskeletal fraction. These shifts could affect the migration of the T
cell protein kinase Zap-70 to the SMAC, because there is evidence that
Zap-70 does not bind to p16 (4), but does associate
with p21 and p23 (30, 31, 32, 33). This differential Zap-70
association, along with the age-related increase in p21 levels,
could potentially explain our previous observation of higher levels of
Zap-70 associated to TCR complex in CD4+ T cells
from old mice (9). The age-related decline in
TCR-stimulated translocation to the cytoskeleton of all forms of CD3
is consistent with, and may prove to account for, our previous report
of defects in translocation of Lck and c-Cbl to the SMAC in CD4 T cells
of age TCR transgenic mice (11). In addition, we have
unpublished data to show age-related defects in the translocation of
Lck and c-Cbl from the soluble to the cytoskeletal fraction in
activated CD4 cells (not shown). All three approaches, synapse
analysis, spreading, and immunoprecipitation, reveal age-related
changes in T cells from TCR transgenic mice, making it unlikely that
our results can be explained by the age-related shift from naive
to memory T cells, which is greatly inhibited in the transgenic
model. Thus, each of these results supports the notion of an
age-related change in interaction of the cytoskeleton with components
of the TCR complex, at least in naive CD4+ T
cells.
We do not know whether the age-related changes in the association of
CD3 isoforms with the cytoskeleton are the sole explanation for the
signal transduction defects in aged naive CD4 cells. The TCR
segregation model suggests other possibilities, including potentially
alterations in interaction of TCR or other T cell surface molecules
with APC proteins caused by interferences mediated by large proteins
such as CD43 and CD45. Further studies will need to be performed to
differentiate among these and other mechanistic hypotheses.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard A. Miller, 5316 CCGC, 1500 E. Medical Center Drive, Ann Arbor, MI 48109-0940. E-mail address: millerr{at}umich.edu
3 Abbreviations used in this paper: SMAC, supramolecular activation cluster; LAT, linker for activation of T cells; PCC, pigeon cytochrome c; PLC, phospholipase C.
Received for publication June 11, 2002. Accepted for publication September 5, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain and displays a unique activation-dependent phosphorylation pattern. J. Biol. Chem. 271:20705.
2 cytoplasmic tail: activation-dependent regulation of associations with talin and 
-actinin. J. Biol. Chem. 273:33588. in CD4 T cells from old mice. Cell. Immunol. 190:91.[Medline]
during murine T cell activation: effect of age. Cell. Immunol. 195:28.[Medline]
chain dimers in mouse CD4 T lymphocytes: effect of age. Cell. Immunol. 175:51.[Medline]
chain tyrosine-based activation motif. J. Biol. Chem. 270:13981. chain tyrosine-based activation motif. Eur. J. Immunol. 25:2863.[Medline]
in murine thymocytes and lymph node T cells. Immunity 1:675.[Medline]
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