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Department of Pathology,
Cellular and Molecular Biology Graduate Program, and
Geriatrics Center, University of Michigan School of Medicine, Ann Arbor, MI 48109;
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University of Michigan Institute of Gerontology, Ann Arbor, MI 48109; and
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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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among individual cells. LAT
migration to the immunological synapse depends on actin polymerization
as well as on activity of Src family kinases, but aging leads to only a
small change in the percentage of CD4 cells that redistribute F-actin
to the site of APC contact. These results suggest that defects in the
ability of T cells from aged donors to move kinase substrates and
coupling factors, including LAT and Vav, into the T cell/APC contact
region may contribute to the decline with age in NF-ATc-dependent gene
expression, and thus to defects in T cell clonal
expansion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, at this immunological synapse may also play a
key role in discrimination of agonist from nonagonist peptides and the
commitment of the T cell to proliferation and cytokine production
(12, 13).
Aging leads to a decline in the ability to mount strong T cell
responses to new Ags and to previously encountered recall Ags both in
mice and in humans (reviewed in Ref. 14). Research from
several laboratories has shown that T cells from healthy old mice
exhibit multiple defects early in the signal transduction cascade.
These include declines in tyrosine-specific protein phosphorylation,
including phosphorylation of the TCR-associated CD3
chain
(15, 16, 17), calcium signal generation (18, 19),
phosphorylation of multiple substrates in responses initiated by the
PKC activator PMA and by the calcium ionophore ionomycin
(20), Shc-tyrosine phosphorylation (21), and
activation of the extracellular signal-related kinase, RAF, and c-Jun
N-terminal kinase/stress-activated protein kinase pathways (22, 23) in response to stimulation by polyclonal stimuli. All of
these age-related changes are demonstrable within the first 10 min of
the activation process, and most are detectable within 1–2 min. In
contrast, ZAP-70 protein kinase activity, measured directly using an in
vitro kinase assay or indirectly by measurement of tyrosine
phosphorylation of the ZAP-70 protein, has shown no difference between
activated CD4+ T cells from young and old mice
(24). ZAP-70, a key enzyme in the TCR signaling pathway,
is recruited to the TCR complex by doubly phosphorylated immunoreceptor
tyrosine-based activation motifs on CD3, motifs that are
phosphorylated by Lck within seconds of TCR engagement
(25). The fact that ZAP-70 activity is not defective in
cells from old mice suggests the hypothesis that aging might alter
accessibility to ZAP-70 of one or more of its critical substrates.
These substrates include the adapter proteins LAT and SH2 domain
containing leukocyte protein of 76 kDa (SLP-76) and the
guanine-nucleotide exchange factor Vav (26, 27, 28), whose
phosphorylation is crucial for T cell activation (reviewed in Ref.
29). LAT is a linker protein that plays a role in
activation of phospholipase C-
1, phosphatidylinositol 3-kinase, and
Grb2. Vav is a guanine nucleotide exchange factor for the Rho-like
small GTPases. Vav acts downstream to SLP-76 and influences
cytoskeletal reorganization (29, 30).
Monks et al. (12, 13) have shown that clustering of
proteins involved in T cell activation can be detected at the single
cell level using immunofluorescence staining and confocal microscopy.
These studies used T cells from transgenic mice bearing T cells
specific for a peptide derived from pigeon cytochrome c
(PCC), presented to the T cells by the B cell line CH12. To study
polyclonal activation in aging nontransgenic mice, we have used as
surrogate APC the hybridoma cell line 145-2C11, which expresses cell
surface anti-CD3
as well as ICAM-1 and B7 (M. Eisenbraun and
R. A. Miller, unpublished data), and report in this work analyses
of the effect of aging on the activation-induced redistribution of
plasma membrane proteins LAT, PKC-, Vav, actin, and the DNA-binding
factor NF-ATc.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 used were at 4–8 (young) and 18–22 (old) mo of age. Mice were housed for at least 1 wk 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. Breeding pairs of TCR-transgenic mice whose T cells bear receptors specific for PCC were a generous gift from Dr. Susan L. Swain (Trudeau Institute, Sarnac, NY). Transgene-positive mice were used at 4–6 mo of age.
Abs and reagents
Anti-phosphotyrosine (PY-20) and anti-CD45 (Ly-5) used for
Western blot analyses were purchased from Transduction Laboratories
(Lexington, KY) and PharMingen (San Diego, CA), respectively. Primary
detection of proteins for fluorescence microscopy analysis was
performed using either a rabbit polyclonal Ab against CD3 (Dako,
Carpenteria, CA), LAT (Upstate Biotechnology, Waltham, MA), or Vav
(Santa Cruz Biotechnology, Santa Cruz, CA), a goat polyclonal Ab
against PKC- (Santa Cruz), or a mouse mAb against NF-ATc1 (Santa
Cruz). Secondary detection was performed using either a Texas
Red-conjugated goat anti-mouse (Jackson ImmunoResearch, West
Grove, PA), goat anti-rabbit (Jackson), or donkey anti-goat
polyclonal Ab (Polysciences, Warrington, PA). For LAT and PKC- or
NF-ATc double staining, a fluorescein-conjugated donkey anti-rabbit
polyclonal (Jackson) was used in combination with the Texas
Red-conjugated anti-goat or anti-mouse polyclonal for secondary
detection. F-actin was detected using Alexa 488-labeled phalloidin
(Molecular Probes, Eugene, OR). Rabbit anti-mouse IgG Abs were
purchased from ICN (Costa Mesa, CA). Magnetic bead-conjugated goat
anti-rat IgG was obtained from PerSeptive Diagnostics (Cambridge,
MA). Purified ascites to murine CD3 (clone 145-2C11), CD4 (clone
GK1.5), and CD8 (clone 53.6) as well as to DNP (clone UC8) were
produced in our laboratory from cell lines purchased from the American
Type Culture Collection (ATCC, Manassas, VA). Cytochalasin-D was
purchased from Sigma (St. Louis, MO), and
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine
(PP2) and cholera toxin ß-subunit coupled to HRP from Calbiochem (La
Jolla, CA).
Cell culture
The CH12 B cell line and hybridoma lines expressing hamster mAbs
specific for either murine CD3 (clone 145-2C11) or DNP (clone UC8)
were obtained from ATCC. All cell lines were maintained in DMEM with
10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1x
nonessential amino acids, and 55 µM 2-ME at 37°C and 10%
CO2.
Cell preparation
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 Lymphocyte-M (Cedarlane Laboratory, Ontario, Canada), and adherent cells and B cells were depleted by panning with rabbit anti-mouse IgG Ab-coated plates. CD4+ cells were further purified by negative immunomagnetic selection using anti-CD8 ascites and goat anti-rat IgG magnetic beads to remove CD8+ T cells. Flow-cytometric analysis of a series of typical preparations showed the recovered cells to be 85–95% positive for both CD3 and CD4 and to contain fewer than 5% CD8+ cells.
For each experiment, 145-2C11 and UC8 hybridoma cells in log-phase growth were harvested, washed twice with prewarmed (37°C) HBSS-BSA, and then resuspended at 2.5 x 106 cells/ml in prewarmed RPMI 1640 supplemented with 10% heat-inactivated FCS. In experiments using CH12 B cells, the CH12 cells were incubated in fresh medium at concentration of 2.5 x 105 cells/ml overnight before use and then pulsed with 2 µM agonist or nonagonist peptide 2 h before harvesting, as described above. The agonist peptide sequence was derived from aa 88 to 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) (31).
Slide preparation and microscopy
A total of 1.25 x 105 purified normal or TCR-transgenic CD4+ T cells (resuspended at 5 x 106 cells/ml in RPMI 1640 + 10% FBS) was combined with 6.25 x 104 145-2C11, UC8, or peptide-pulsed CH12 cell (resuspended at 2.5 x 106 cells/ml) to achieve a 2:1 cell ratio, respectively. Cell mixtures were incubated at 37°C for 15 min and then gently resuspended and spread onto prewarmed poly(L-lysine)-coated slides (Sigma). Slides were incubated for another 15 min at 37°C to promote cell attachment, fixed in freshly prepared 3.7% formaldehyde/PBS for 20 min, and then finally washed three times in PBS. Slides to be used for immunofluorescence detection of NF-ATc were further permeabilized with 0.2% Triton X-100/PBS for 5 min, followed by three washes in PBS. All slides were placed in blocking solution (1% BSA/0.1% NaN3 in PBS) and stored at 4°C for at least 24 h.
Slides were stained with appropriate primary Abs (or phalloidin)
diluted in blocking solution (anti-CD3, anti-Vav,
anti-PKC-, and anti-NF-ATc, each used at 10 µg/ml;
anti-LAT used at 20 µg/ml; phalloidin used at a 1/1000 dilution)
for 2 h at room temperature in a humidified chamber. With the
exception of phalloidin-stained samples, slides were washed three times
in PBS and a final time in blocking solution, and then counterstained
for 90 min in the dark, as above, using appropriate secondary Abs
diluted to a concentration of 7–15 µg/ml in blocking solution. All
slides were washed three times in PBS before mounting coverslips with
ProLong antifade reagent (Molecular Probes). After drying overnight at
room temperature, slides were coded for blind analysis and stored at
4°C protected from light. Single- and two-color confocal 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). Fluorescence analysis of NF-ATc localization was
performed at x100 magnification on a Zeiss Axioskop microscope
equipped with a low light CCD camera (MicroImage Video Systems,
Boyertown, PA). Randomly selected T cell-APC conjugates were
analyzed by confocal or fluorescence microscopy only if the
following criteria were first met under light-microscopy
conditions: 1) tightly formed cell-to-cell contact, 2) T cell
in contact with only one APC, 3) both cells in the same relative
z-axis plane, and 4) no significant overlap of cell
membranes at contact interface. At least 50, and in some cases 100
acceptable conjugates on each slide were visualized by confocal or
fluorescence microscopy 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
without knowledge of the age of the T cell donor.
Analysis of LAT tyrosine phosphorylation
For TCR stimulation and LAT immunoprecipitation, we followed a
protocol described previously (15). Briefly, 5 x
106 freshly isolated CD4+ T
cells were incubated with 5 µg/ml of hamster anti-CD3 plus 5
µg/ml rat anti-CD4 or instead with anti-DNP as control, for
30 min at 4°C, followed by 5 min cross-linking with goat anti-rat
IgG (cross-reacts with hamster Ig) at 37°C. Cells were washed with
cold PBS and lysed with a buffer containing 1% Nonidet P-40, 50 mM
Tris-HCl, pH 8, 120 mM NaCl, 50 mM NaF, 10 µg/ml aprotinin, 100
µg/ml leupeptin, 10 mM PMSF, and 0.1 mM sodium orthovanadate for 30
min at 4°C. After centrifugation at 13,000 x g, LAT
was immunoprecipitated from each supernatant by overnight incubation
with 2 µg of anti-LAT precoupled to protein G-Sepharose beads
(Amersham Pharmacia Biotech, Piscataway, NJ) at 4°C. The beads were
washed four times with PBS, 0.1% Tween-20, and proteins were then
resolved by SDS-PAGE and transferred to polyvinylidene difluoride
(PVDF) membranes (Millipore, Bedford, MA). The membranes were probed
with anti-phosphotyrosine or anti-LAT, and the bands visualized
by chemifluorescence on a PhosphorImager (Storm 840; Molecular
Dynamics, Sunnyvale, CA) using an alkaline phosphatase-conjugated
secondary Ab (Sigma) and enhanced chemifluorescence substrate solution
(Amersham); the digital images were quantitated using ImageQuant
software (Molecular Dynamics).
Purification and analysis of membrane rafts
Raft microdomains were purified from control or stimulated CD4+ T cells, as described by Montixi et al. (5) with modification. Briefly, CD4 T cells were lysed in 300 µl of lysis buffer (1% Brij-58, 100 mM NaCl, 2 mM EDTA, 10 µg/ml aprotinin, 100 µg/ml leupeptin, 10 mM PMSF, 0.2 mM sodium orthovanadate, 25 mM HEPES, pH 6.9) for 30 min on ice. The lysed cells were gently mixed with 300 µl of 85% sucrose (w/v), placed in the bottom of a ultracentrifuge tube, and then overlaid with 1 ml of 35% sucrose and 300 µl of 5% sucrose (w/v). After ultracentrifugation at 200,000 x g for 16 h at 4°C, 600 µl ml from the top (low density raft fraction) and 1 ml from the bottom (soluble fraction) were collected. For the analysis of GM-1, aliquots of the rafts and soluble fractions were resolved by 15% SDS-PAGE and transferred to PVDF (0.2 µm) membranes, and the GM-1 analyzed by Western blotting with cholera toxin ß-subunit coupled to HRP. The bands are visualized by chemiluminescence and quantified, as described by Garcia et al. (15). For the analysis of LAT and CD45 distribution, rafts and soluble fractions were dialyzed and concentrated, and the proteins were resolved in 10% SDS-PAGE, transferred to PVDF membrane, and detected by Western blots with specific Abs.
Statistical analyses
Data are presented in text and figures as means ± SEM, and
N indicates the number of individual mice tested in the set of
experiments. Between-group comparisons of the proportion of cells
showing protein redistribution used the Student’s t test;
data from the two color experiments were analyzed using a
2 test for independence.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Engagement of the TCR induces a series of biochemical events that
culminate in T cell differentiation and proliferation. These events
include a general migration of proteins and lipids on the surface of T
cells toward the TCR/APC interaction synapse (32),
including migration of the protein kinases Lck, Fyn, and PKC- to the
same area (12, 13). Preliminary experiments were performed
to determine whether the adapter protein LAT and the nucleotide
exchange factor Vav also migrate to this synapse region. We compared
two systems for T cell stimulation: 1) 145-2C11 (2C11) anti-CD3
hybridoma cells as a polyclonal activator of T cells from normal mice
(using UC8 anti-DNP hybridoma cells as a negative control), and 2)
peptide-pulsed CH12 cells as a stimulator of PCC-specific transgenic T
cells (with a nonagonist peptide as a negative control). Fig. 1
shows examples of LAT and Vav
redistribution to the APC synapse in 2C11-stimulated (top
row) and in peptide-stimulated cells (third
row), as well as examples of T cells conjugated to negative
control stimulators, in which redistribution did not occur
(second and fourth rows). Kinetic experiments
(not shown) established that LAT and Vav redistribution was detectable
15 min after the addition of APC, the earliest time point examined.
The proportion of conjugates showing redistribution increased only
slightly at the 30-min time point, and then remained stable for at
least another 30 min (data not shown). Additional controls tested the
specificity of the fluorochrome-labeled secondary Ab, using slides
stained without primary Ab or with purified rabbit IgG as an isotype
control. We did not detect any significant staining in the absence of a
primary Ab, but did observe nonspecific staining at the T cell/APC
interface in a small proportion of 2C11 (18%) and peptide-CH12 (24%)
conjugates when normal rabbit serum was used as an isotype control in
the place of the primary LAT or Vav Ab, as indicated in the legend to
Table I. The data in Table I (and
subsequent figures) are presented as the crude percentage of conjugates
showing protein redistribution at the 30-min time point, i.e., without
subtraction for nonspecific staining background.
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70% of the conjugated cells, a value similar to that previously
reported (12) for synaptic localization of protein kinases
in similar pseudo-monoclonal systems. Using 2C11 cells to stimulate
nontransgenic T cells induces relocalization of LAT or Vav in 46% or
61% of the conjugated CD4 cells, respectively. These somewhat lower
responses in the polyclonal system are unlikely to represent a failure
of TCR engagement per se, in that relocalization of CD3 itself is
induced in 71% of the conjugated CD4 cells. The much smaller
proportion of PCC-CH12 conjugates that exhibits dramatic redistribution
of CD3 (16%, equivalent to negative controls) is consistent with
published data from other laboratories (12). The disparity
between the two systems in CD3 translocation reflects the much
higher avidity of the 2C11 Ab for CD3 compared with the avidity of
peptide-TCR complexes. It is noteworthy that CH12 cells bearing
nonagonist peptides induce LAT and Vav redistribution at rates not
higher than that of negative staining controls, supporting the idea
that redistribution of these two molecules requires specific forms of
interaction between TCR and complexes on stimulator cells. LAT and Vav redistribution decline with age in CD4+ T lymphocytes
Fig. 2 shows the results of a series
of four experiments comparing LAT and Vav redistribution in CD4 T cells
from young or old mice after conjugation to 2C11 stimulators. About
one-half of the CD4+ T cells from young mice
exhibited redistribution of LAT into the APC contact area, but the
proportion of conjugated CD4+ T cells with
redistributed LAT was lower for old mice (p =
0.011). A similar age effect was seen for Vav: the frequency of
activated T cells with redistributed Vav dropped significantly
(p = 0.006) from 61 ± 3.3% in cells from
young mice to 34 ± 5.5% in cells from old mice. These
age-associated declines in LAT and Vav redistribution did not appear to
result from changes in the ability of TCRs (CD3) to relocalize to
the APC interface (young = 71.5 ± 3.5% compared with
old = 65 ± 2%, n = 4, data not shown).
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The tyrosine phosphorylation of LAT after TCR activation
facilitates its association with several molecules involved in T cell
activation (26). To see whether the age-related defect in
LAT redistribution was associated with parallel changes in LAT
phosphorylation, we determined levels of tyrosine-phosphorylated LAT by
immunoprecipitation and immunoblotting in lysates prepared 5 min after
activation of T cells by CD3/CD4 cross-linking. Fig. 3A shows a representative
experiment: LAT phosphorylation is strongly induced in young, but not
in old CD4 cells. Fig. 3B shows a statistical summary of
replicate experiments involving six young and five old mice. The basal
levels of phosphorylated LAT in unstimulated CD4+
T cells from young and old mice were similar. Stimulation of CD4 cells
from young mice leads to a 7.8-fold increase in LAT phosphorylation,
significantly greater (p = 0.011) than the
3.4-fold stimulation seen in cells from old mice. This difference
cannot be attributed to a change with age in the levels of LAT: Western
blot analyses conducted on stripped membranes used for the
phosphotyrosine detection showed that LAT levels in CD4 cells from old
mice were 96% ± 0.07% (mean ± SEM) of those in young mice
assessed in parallel. Fig. 3C shows an experiment in which
CD4 cells were tested for LAT phosphorylation 5, 10, or 15 min after
CD3/CD4 cross-linking. CD4 cells from young and old mice show similar
kinetics of LAT phosphorylation, with peak effects at 5 min and
diminishing phosphorylation thereafter. The responses of CD4 T cells
from aged mice are therefore diminished, rather than merely delayed.
The time course after stimulation with anti-CD3 and anti-CD4
Abs is more rapid than when cells are stimulated with 2C11 cells,
presumably because the latter system requires time for cell/cell
conjugation before signal initiation, and does not produce the
synchronized start produced using purified Abs. The decline in total
levels of detectable LAT has been noted in each of three replicate
experiments, and could reflect internalization and degradation of this
protein.
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Wulfing and Davis (32) recently reported evidence
that implicated actin filament (F-actin) assembly in the migration of
proteins and lipids in the T cell membrane toward the APC interface
after conjugation. We therefore investigated the role F-actin assembly
might play in LAT redistribution and whether an age-related defect in
actin assembly might contribute to impaired LAT translocation in T
cells from older mice. Using fluorochrome-labeled phalloidin, a protein
that specifically binds to F-actin, we detected F-actin accumulation at
the APC interface (Fig. 4A) in
a high percentage of CD4+ T cells from young mice
after formation of conjugates with 145-2C11 cells (86 ± 2.7%,
n = 4); actin redistribution was seen only very rarely
when UC8 hybridoma cells were used as negative controls. To determine
whether F-actin assembly was required for LAT redistribution,
CD4+ T cells from young mice were pretreated with
2 µM cytochalasin D, an inhibitor of F-actin assembly, before
stimulation with APC. We found that this treatment, as expected,
completely prevented F-actin reorganization (Fig. 4B), and
that it also diminished to background levels the frequency of cells
able to undergo LAT redistribution. In two-color analyses in which
F-actin distribution was examined together with LAT or Vav
redistribution, we found no cells that showed redistribution of LAT and
Vav in the absence of F-actin cap formation (data not shown). In
further tests, we added PP2, an inhibitor of Src family tyrosine
kinases (33), to the T cell-APC culture at the start of
the incubation period to determine its effect on actin and LAT
reorganization. As shown in Fig. 4B, 2 µM PP2
treatment completely inhibited both F-actin assembly and LAT
redistribution. This finding is consistent with previous studies that
have shown that F-actin assembly is dependent on tyrosine kinase
activity in T cells (34, 35). CD3 movement to the
interface with 2C11 cells was not altered either by cytochalasin D nor
by PP2 pretreatment.
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NF-ATc translocation to the nucleus is impaired by aging in stimulated CD4+ T cells
One of the first events induced by TCR engagement is a rise in
intracellular Ca2+ concentration. One result of
this Ca2+ influx is the activation of
calcineurin, which in turn dephosphorylates NF-ATc
(36, 37, 38). Dephosphorylated NF-ATc subsequently enters the
nucleus, where it cooperates with other transcription factors to
activate genes important for T cell activation, including IL-2
(36). T cells deficient in LAT, and those in which a LAT
mutation prevents its constitutive localization in GEMs, fail to
trigger Ca2+ influx and nuclear translocation of
NF-ATc after TCR activation (39, 40). To determine whether
age-related defects in LAT redistribution and phosphorylation are
accompanied by defects in signaling events downstream of LAT, we
examined the influence of age on NF-ATc nuclear translocation in
activated CD4+ T cells from young and old mice.
Unconjugated CD4+ T cells, and those stimulated
with UC8 (negative control) cells, showed a homogeneous distribution of
NF-ATc throughout the cytoplasm (Fig. 5).
In contrast, NF-ATc became localized in the nucleus of many, although
not all, T cells that formed conjugates with 2C11 stimulators. The
lower panel of Fig. 5 shows a summary of four replicate
experiments comparing translocation of NF-ATc between 2C11-activated
CD4+ T cells from young and old mice. Similar to
the data on LAT and Vav redistribution, NF-ATc translocation to the
nucleus occurred significantly more frequently
(p = 0.014) in conjugated
CD4+ T cells from young (57 ± 7.8%) than
from old mice (29 ± 2.6%).
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, and NF-ATc in single
CD4 cells
These results shown above are consistent with the hypothesis that
the age-related defects in reorganization of LAT, Vav, and (in a
previous publication) PKC- (41) occur in the same cells
that fail to induce nuclear migration of NF-ATc, but do not exclude the
possibility that these defects instead characterize different, or
overlapping, populations of CD4 cells. We therefore conducted a series
of experiments in which 2C11-activated CD4 cells were stained for LAT
as well as for either PKC- or NF-ATc. The left-hand
panels of Fig. 6 show that PKC-
redistribution was seen in all CD4 cells that exhibited LAT
redistribution (i.e., 44% of the conjugated cells); in addition, a
small fraction of cells (14%) showed PKC- relocalization without
associated LAT. We found no cells that distributed LAT without PKC-.
We cannot tell from this approach whether those cells whose synapses
apparently contained PKC- without LAT reflect technical artifacts or
asynchrony—perhaps some of the cells might have rearranged LAT at
later time points?—or whether they differ in biologically meaningful
ways from cells in which both LAT and PKC- are present at the
synapse. For NF-ATc (Fig. 6, right-hand panels), the
majority of the conjugates (50%) exhibited both LAT redistribution and
NF-ATc nuclear translocation. A small fraction of the conjugates
apparently had either LAT (6%) or NF-ATc (14%) translocation alone.
2 analysis showed that the proportion of cells
showing both LAT and NF-ATc reorganization was much greater than that
expected by coincidence (p < 0.001), showing
that LAT relocalization and NF-ATc migration were closely associated
with one another in individual cells.
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Alterations with age in phosphorylation of LAT and in its ability
to migrate to the site of interaction with APCs could in principle
result from changes in the degree to which this palmitoylated protein
is associated with low density membrane microdomains (membrane rafts).
To assess this idea, we examined LAT levels in membrane fractions
enriched or depleted in these low density raft domains before and after
stimulation by anti-CD3/anti-CD4 cross-linking. The
glycosphingolipid GM-1, shown by others to be restricted to the raft
domains, and CD45, excluded from rafts, were used as internal controls
for the separation procedure (6, 7, 8). Fig. 7A, a typical experiment,
shows that LAT is restricted to the raft fraction, in agreement with
results from other laboratories, and that, as expected, GM-1 and CD45
were restricted to rafts or to soluble fractions, respectively.
Stimulation of the cells did not alter these distributions, nor was
there any obvious effect of donor age. Fig. 7B shows a
statistical summary of four such experiments. There is no evidence for
an effect of age or stimulation on the amount of GM-1 or LAT associated
with the membrane rafts of CD4 T cells. However, these data do not rule
out the idea that intact T cells from aged donors may have changes in
raft size or in the ability of rafts to move to the immune synapse area
after contact with APCs. These possibilities will require investigation
using other approaches, including microscopic methods.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(12) in
the group of signaling molecules, whose movement to the synapse is
correlated with commitment to proliferation. Our findings, together
with studies that have shown LAT and Vav are localized in GEMs after
activation (6, 43), lend support to the idea that GEMs act
to organize groups of interaction molecules at the site of T cell-APC
contact. The protein kinases Lck and Fyn have also been found to become
localized in GEMs following activation, and at the APC interface
(13), where they are likely to interact with LAT and
Vav.
T cells from healthy old mice exhibit multiple defects early in the
signaling cascade initiated by TCR stimulation, including defects in
calcium signal generation (18, 19) and in activation of
the downstream kinases Raf-1, MEK, and extracellular signal-related
kinase (22, 23). We therefore asked whether aging might
also affect the migration of GEM-associated proteins to the T cell-APC
contact area. We used 145-2C11 hybridoma cells as a polyclonal source
of APC stimulation so that we could study the effect of aging in
heterogeneous (nonclonal) populations of T cells from normal, i.e.,
nontransgenic mice. We found that aging led to a decline in the
proportion of CD4+ T cells that redistribute LAT
and Vav to the T cell-APC synapse after conjugation; the age decline is
between 39% and 64% for LAT, depending on whether the false positive
background is subtracted, and 45% to 64% for Vav. Previous work from
this laboratory (41) has documented a similar decline with
age in the proportion of 2C11-stimulated CD4 cells that relocalize
PKC-. There was, however, no change with age in the fraction of
cells that could respond to 2C11 conjugation by recruitment of CD3 to
the synapse area (data not shown), and no change in the proportion of
CD4 cells that form conjugates with 2C11 stimulators (41).
Moreover, flow-cytometric analyses also revealed that T cells from
young and old donors expressed similar levels of TCR, CD28, CD2, and
LFA-1 (A. Tamir and R. A. Miller, data not shown), suggesting that
the differences in LAT and Vav clustering are unlikely to be due to
changes in the levels of TCR or accessory molecules involved in T cell
activation.
Phosphorylation of LAT allows it to bind to, and presumably help to
concentrate and orient, a number of SH2-containing proteins, including
Grb2 and phospholipase C-, that play a role in propagation of
downstream signals in the early phases of T cell activation (26, 27). We found that the decline with age in the percentage of
CD4+ T cells that could move LAT to the
kinase-containing synapse region was accompanied by a proportionate
decline in induction of LAT tyrosine phosphorylation induced by
cross-linking cell surface CD3 and CD4 molecules on CD4 cells. We used
Abs to CD3 and CD4, rather than 2C11 cells, for the test of LAT
phosphorylation, because LAT phosphorylation is transient, returning to
basal level within minutes after stimulation, and its quantitation thus
requires greater synchrony than can be obtained using cell conjugation
to initiate T cell signals. Aggregation of TCR by anti-CD3 Ab
cross-linking causes a similar coaggregation of GEM-associated
proteins, although, which in turn leads to activation of tyrosine
kinases within the GEMs (44). Because LAT molecules are
located in the GEMs of murine CD4+ T cells
(39) (G. Garcia and R. A. Miller, unpublished data),
the decline with age in LAT phosphorylation seems likely to result from
the parallel decline in LAT relocalization to the kinase-rich synapse
region.
To investigate whether the age-associated defects in LAT
redistribution might lead to a decline in LAT-dependent events
downstream in the signaling process, we conducted a series of
experiments using NF-ATc translocation. The transcription factor
NF-ATc plays an essential role in T cell activation, and previous work
has shown that LAT must be located in GEMs in order for NF-ATc
translocation to occur (39, 40). Our data show that aging
leads to a decline in the fraction of
CD4+ T cells that exhibit NF-ATc migration to the
nucleus after activation by anti-CD3 hybridoma cells, and also that
those cells unable to translocate NF-ATc also characteristically fail
to show LAT translocation to the synapse. Similarly, PKC-
redistribution, shown previously to be age sensitive (41),
was in our work shown to occur in virtually all CD4 cells that
relocalize LAT, and conversely to occur only rarely in T cells that
failed to undergo LAT migration to the synapse. The strong
correlations, among individual cells, between LAT and PKC-
redistribution, and between LAT migration and NF-ATc translocation,
together with the ability of LAT, Vav, and PKC- responses to
discriminate between agonist and nonagonist peptides in the TCR
transgenic models, suggest strongly that the age-related decline in
translocation of these plasma membrane proteins to the synapse may
account for much of the decline with age in NF-ATc translocation and
subsequent downstream changes in IL-2 gene expression. We have also
found that a subset of CD4 memory T cells characterized by poor
proliferation, low cytokine production, poor calcium signal generation,
and high expression of P-glycoprotein (45, 46) also
exhibits diminished relocalization of LAT and PKC-, and diminished
nuclear translocation of NF-ATc, in response to 2C11
conjugation,4 lending
further support to the idea that altered migration of proteins to the
synapse is associated with, and probably contributes to, deficits in
downstream signals and functional responsiveness.
TCR-mediated stimulation leads to formation of an F-actin cap at the T
cell-APC contact zone, and organization of supramolecular activation
clusters (SMACs) (10). The central area of the SMAC has
been shown to contain TCR, CD3, Lck, Fyn, and PKC- molecules, with
the adhesion protein LFA-1 and the actin-binding protein talin
localized at the periphery of the SMAC (13). Formation of
SMACs requires actin polymerization (32, 47), while
F-actin cap formation in turn requires phosphorylation of
immunoreceptor tyrosine-based activation motifs in the CD3 complex as
well as functional Lck and Vav (34, 35, 48, 49). Our data
presented in Fig. 4
B show that redistribution of LAT to the
synapse zone is disrupted by cytochalasin D, an inhibitor of actin
polymerization, and by PP2, a potent and selective inhibitor that
targets Src family tyrosine kinases such as Lck and Fyn
(33). PP2 also prevented F-actin capping in
CD4+ T cells, although neither agent interfered
with the clustering of CD3 at the region of contact with the APC.
Thus, our results, together with the published data, are consistent
with models in which kinase-dependent events induce LAT migration via
actin-dependent transport. F-actin assembly may also play a role in the
prolonged maintenance of TCR signaling by directing the redistribution
of LAT and other proteins to the T cell-APC contact. The proportion of
T cells that exhibit F-actin relocalization after 2C11 stimulation
declines 22% with age (Fig. 4), and it seems possible that this change
could explain a portion, although not all, of the larger age-dependent
change in the proportion of CD4 cells showing LAT, Vav, and NF-ATc
responses. Others have previously noted a decline with age in F-actin
polymerization in T cells activated by Con A (50). Even in
old mice, however, 67% of the CD4+ cells are
able to assemble F-actin cap structures upon 2C11 conjugation. Although
elucidation of the biochemical differences between young and old T
cells that interfere with assembly of immunological synapses will
require additional experimentation, our results indicate that
alterations in the movement of signaling molecules into specific
regions of the plasma membrane are likely to diminish T cell activation
and thus the protective immune response in aged mice.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. R. A. Miller, University of Michigan, 5316 CCGC, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0940.
3 Abbreviations used in this paper: GEM, glycolipid-enriched membrane; LAT, linker for activation of T cells; NF-ATc, cytoplasmic NF-AT; PCC, pigeon cytochrome c; PKC, protein kinase C; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PVDF, polyvinylidene difluoride; SMAC, supramolecular activation cluster.
4 M. D. Eisenbraun, A. Tamir, and R. A. Miller. Altered composition of the immunological synapse in an anergic, age-dependent memory T cell subset. Submitted for publication.
Received for publication January 5, 2000. Accepted for publication May 15, 2000.
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redistribution in
CD4+ cells activated with different APC
