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,,§,§
Departments of
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Pharmacology and Physiology,
Pathology, and
Molecular Genetics and Cell Biology and the
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Committee on Immunology, University of Chicago, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to these well-studied events, T cell:APC interactions
result in dramatic changes in T cell shape and cytoarchitecture. Before
APC binding, migrating T cells have an inherent polarity, with a
leading edge that responds readily to an encounter with an APC and with
a trailing uropod that is relatively insensitive to APC contact (10, 11). Upon encountering an APC bearing appropriate peptide-MHC
complexes, T cell migration is arrested, the uropod is retracted, and
APC binding is stabilized by integrin activation (6, 12). These
morphological changes are accompanied by the recruitment to the
cell:cell interface of a number of cell surface receptors such as the
TCR, CD4, and the integrin LFA-1, as well as signaling molecules such
as protein kinase C
(13, 14, 15, 16). T cell cytoskeletal elements also
rearrange in response to APC binding. Filamentous actin (17, 18) and
the actin-binding protein talin (15) accumulate at the T cell:APC
interface, and the T cell microtubule-organizing center
(MTOC)3, which was previously
in the trailing uropod (12), is actively recruited into the portion of
the cytoplasm proximal to the APC (14, 15, 19). These polarization
events facilitate T cell function at several levels. Interactions with
the cortical actin cytoskeleton have been shown to regulate
integrin-mediated adhesion (5, 7), and the recruitment of cell surface
receptors and signaling molecules to the APC-binding site forms a
specialized domain of the T cell surface that may enhance or modulate T
cell signaling. Finally, polarization of the MTOC is important for
effector function because it establishes polarity of the Golgi complex
and associated secretory organelles, thereby directing the secretion of
cytokines (20) and lytic granules (10, 21, 22) toward the
appropriate APC.
Relatively little is known about the signaling events that induce T cell polarization. In T cell:B cell couples, both talin polarization and MTOC reorientation have been shown to be Ag-dependent processes. MTOC reorientation is clearly downstream of TCR signaling, since this reorganization can be induced by cross-linking of the TCR with Ab-coupled beads (23, 24). This process has been shown to involve the immunoreceptor tyrosine-based activation motifs of the TCR, as well as the TCR-proximal tyrosine kinases Lck and ZAP-70 (24). Expression in T cells of a dominant-negative form of the Rho family GTPase Cdc42, a protein that regulates actin remodeling in fibroblasts (25), disrupts both cortical actin polarization and MTOC reorientation (18). This observation suggests that the two processes are mechanistically linked, with cortical cytoskeletal rearrangements a necessary prerequisite for MTOC reorientation. However, the precise series of events that leads from TCR signaling to cytoskeletal polarization has not been elucidated.
In addition to stimulation via the TCR, it is likely that other receptor-ligand interactions contribute to cytoskeletal reorientation. In this report, we specifically address the role of TCR, LFA-1, and CD28 signaling in cytoskeletal polarization by engaging these receptors with their relevant ligands on transfected cell lines. The use of transfected cells for these studies is important because T cells are known to respond differently to Ag receptor signals derived from Ab cross-linking of the TCR vs signals initiated by peptide-MHC complexes (26). We find that talin polarization is largely integrin dependent, while MTOC reorientation is both dependent on and targeted toward the site of TCR signaling. CD28 engagement alone cannot induce either cytoskeletal rearrangement but can contribute to MTOC reorientation directed toward the TCR through signal amplification. Surprisingly, polarization of the MTOC toward the site of TCR engagement can be separated from detectable cortical (actin and talin) polarization in response to isolated receptor:ligand interactions, indicating that these two cytoskeletal changes may not be mechanistically linked.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A20 B cells, 6132 Pro cells transfected with MHC class II alone
(ProAd) or in combination with ICAM-1 (ProAd-ICAM) or B7-1 (ProAd-B7)
have been previously described (27, 28). To generate ProEk-ICAM cells,
6132 Pro cells were transfected with cDNA clones encoding I-Ek (29) and
ICAM-1 (30) by the calcium phosphate method as previously described
(27). Cells coexpressing I-Ek and ICAM-1 were sorted using
anti-Ig-conjugated magnetic beads (Dynal, Great Neck, NY) and were
maintained as a bulk transfected cell line. 6132 Pro cells transfected
with MHC class II covalently linked to OVA peptide (residues 323–339)
(OVA peptide) (ProAd/OVA) alone or in combination with ICAM-1 were
generated in the same manner, and will be described more fully
elsewhere.4 Pro line cells were grown in
DMEM supplemented with 10% FCS, 2 mM glutamine, 50 mM 2-ME, 0.1 mM
nonessential amino acids, 2 mM HEPES buffer, and 40 µg/ml gentamicin
(Life Technologies, Gaithersburg, MD). For transfectants, this medium
was supplemented with 200 µg/ml G418 and/or 250 µg/ml xanthine, 15
µg/ml hypoxanthine, and 6 µg/ml mycophenolic acid. U937 cells,
Jurkat cells, and Jurkat cells transfected with the 2B4 TCR 
- and
ß-chains (JBN-ß.2.8.24) (31) (gift of Dr. R. Germain, National
Institutes of Health, Bethesda, MD) were grown in RPMI supplemented
with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg of
streptomycin. pGL2 and pGL10, Th1 clonal cell lines specific for OVA
peptide 323–339 in the context of I-Ad, were maintained by stimulation
with Ag and irradiated BALB/cJ splenocytes every 2 wk as already
described (27) and rested for 7–14 days before use.
Abs were obtained as follows: rat anti-yeast -tubulin (YL 1/2,
Serotec, Washington, DC); mouse anti-rat -tubulin (Amersham Life
Science, Arlington Heights, IL); hamster anti-rat CD3
, 2C11 (32)
(kind gift of Dr. J. Bluestone, University of Chicago, Chicago, IL);
mouse anti-mouse class I MHC, 28.14.8 (33), (gift of Dr. A. Sperling,
University of Chicago); mouse anti-rat talin, 8d4 (Sigma); mouse
anti-sea urchin acetylated tubulin, 6-11B-1 (Sigma); rat
anti-mouse CD11a, M18/2 (PharMingen, San Diego, CA); mouse
anti-human CD3, OKT3 (Ortho Biotech, Raritan, NJ); and appropriate
fluorochrome-conjugated secondary Abs (Jackson Immunoresearch, West
Grove, PA). BODIPY-conjugated phallacidin was from Molecular
Probes (Eugene, OR). Rhodamine-conjugated phalloidin and Hoechst stain
(No. 33258) were from Sigma. Abs and BODIPY-phallacidin or rhodamine
phalloidin were diluted to working concentration in PBS-saponin-gelatin
(PBS containing 0.05% w/v saponin and 0.25% v/v fish skin gelatin
(both from Sigma).
Cell conjugation for immunofluorescence microscopy
Pro line APCs grown to subconfluence at 37°C in TC flasks were harvested by incubation with trypsin-EGTA (Life Technologies), plated at 35,000 cells/well in Flexiperm 8-well chambers (Heraeus, South Plainfield, NJ) on glass slides, and grown overnight. Pro line APCs (except ProAd/OVA) were pulsed with OVA peptide 323–339 (27) to a final concentration of 2 µg/ml for 2 h at 37°C before conjugation with T cells. T cells were harvested by Ficoll purification at day 7–14 after stimulation and were plated at 200,000 cells/well in APC culture slides. T cells were allowed to interact with APCs for 30 min at 37°C, fixed, and processed for immunofluorescence analysis as described below.
To distinguish A20 B cells from T cells in the immunofluorescence assay, A20 B cells were loaded with the vital dye 7-amino-4-chloromethylcoumarin (CMAC Cell-Tracker Blue; Molecular Probes). To do this, A20 B cells were preincubated in medium or in medium plus 2 µg/ml OVA peptide for 1 h; then A20 cells were incubated in 10 µM CMAC in serum-free medium or in serum-free medium plus 2 µg/ml OVA peptide for 30 min at 37°C, washed, and chased for 30 min in medium or medium plus OVA peptide. A20 B cells (2 x 105) were mixed with an equal number of Ficoll-Paque-purified T cells and centrifuged for 5 min at 200 x g in Falcon 2058 tubes at room temperature, and the conjugates were then gently resuspended using a 1000-µl pipettor and plated onto poly-L-lysine (m.w. 30,000–70,000, from Sigma)-coated slides for 20 min before fixation.
Ab-coated beads were made by incubating 50 x 106 polystyrene, sulfate-coated beads (5 µm, Interfacial Dynamics, Portland, OR) overnight at room temperature with 100 µg of Ab (hamster anti-mouse CD3, 2C11, mouse anti-human CD3, OKT3, or rat anti-mouse Class I MHC, 28-14-8) in 1 ml of 0.2 M carbonate-bicarbonate buffer, pH 9.5. Beads were then washed twice in medium and stored at 4°C for up to 2 wk. To form conjugates, T cells and Ab-coated beads were mixed at a 1:1 ratio in medium and allowed to interact for 5 min at room temperature (RT). Conjugates were then gently resuspended and plated onto poly-L-lysine-coated slides for 20 min before fixation. For three-way (bead:T cell:APC) experiments, T cell:APC conjugates were formed for 20 min as described above, and then 400,000 beads/well were added (2 beads:1 T cell) and allowed to interact for 20 min before fixation.
For conjugation experiments using Jurkat cells, U937 cells (106 cells/ml) were dyed with 10 µM CMAC as described above for A20 cells. U937 cells were then incubated with or without 2 µg/ml OKT3 mAb at 37°C for 30 min, washed in balanced salt solution (BSS, Life Technologies), and resuspended at 106 cells/ml in BSS. Jurkat cells (2 x 105 cells) and an equal number of U937 cells were mixed in BSS, centrifuged at 200 x g for 5 min at RT, and then incubated at RT for 10 min. The cells were gently resuspended and plated onto poly-L-lysine-coated slides for 20 min before fixation. Adherent ProEk-ICAM cells were labeled with 10 µM CMAC and then incubated with or without antigenic peptide (4 µg/ml DASP; moth cytochrome c residues 86–90, 94–103) (34) for 2 h at 37°C. The cells were harvested with trypsin, and ProEk-ICAM cells (2 x 105) were mixed with Jurkat cells (2 x 105) in the presence of antigenic peptide, centrifuged at 200 x g for 5 min at RT, and then incubated at RT for 10 min. The cells were gently resuspended, plated for 10 min at 37°C onto poly-L-lysine-coated slides, and fixed.
Use of inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) in T cell polarization assays
Wortmannin and Ly 294002 were obtained from Calbiochem (San Diego, CA) and stored in DMSO at -20°C. Drugs were diluted in medium before use. T cells were pretreated with the indicated concentration of wortmannin or Ly 294002 for 30 min at 37°C, and the inhibitor was retained at this concentration throughout the experiment. For control experiments, T cells were left untreated while Pro line APCs were pretreated with wortmannin for 30 min, washed, and then allowed to conjugate with T cells. Additional controls were performed where T cells were pretreated with wortmannin and then washed free of drug before conjugation with APCs.
Immunofluorescent labeling of specimens
For all experiments, slides were rinsed in PBS and fixed in 3% (w/v) paraformaldehyde in PBS for 20 min. Specimens were quenched by washing in 50 mM NH4Cl (Sigma) in PBS, permeabilized in 0.3% (vol/vol) Triton X-100 (Sigma) in PBS for 5 min, and then rinsed in PBS and blocked in PBS-saponin-gelatin for 10 min. All subsequent Ab incubations and washes were performed in PBS-saponin-gelatin. Primary and secondary Abs were applied sequentially for 45 min at RT and washed five times after each Ab. In experiments requiring visualization of actin, BODIPY-phallacidin or rhodamine-phalloidin was applied for 15 min at room temperature after application of primary Abs. In adhesion assays, Hoechst stain was applied to samples for 15 min after application of secondary Abs. After labeling, specimens were mounted in Mowiol 4-88 (Hoechst Celanese, Charlotte, NC), with 10% 1,4-diazobicyclo[2.2.2]octane (Sigma) added as antifade. Samples were analyzed using a Zeiss Axioscop microscope equipped with a motorized stage and a Photometrics PXL-cooled CCD camera. Image capture and deconvolution analysis (where appropriate) was performed using Openlab v1.1.7 (Improvision, Coventry, U.K.) software running on a Power Macintosh 9600/300.
Analysis of polarization and conjugation
Conjugates were first identified in differential interference contrast (DIC) images as cases where substantial contact between the T cell and the APC (or bead) occurred; between 50 and 100 events were counted per condition per experiment. T cells were scored as conjugates only if they contacted no more than one APC or Ab-coated bead, and if there were no other T cells touching or near the T cell being scored. For experiments using both Ab-coated beads and APCs, only clear three-way conjugates (bead:cell:APC) were scored for these experiments. To count T cell conjugates with the various APCs, cells were labeled in three colors: mouse anti-rat talin to label both T cells and APCs; rat anti-mouse CD11a to specifically label T cells; and Hoechst stain to label nuclei. The number of T cells adhering to APCs was assessed as the number of interacting T cells per 100 APCs. For talin or actin polarization experiments, conjugates were assessed for polarization by counting the number of conjugates exhibiting notable brightness at the border between the T cell and APC. Conjugates were examined for MTOC reorientation by visually dividing the T cell cytoplasm into thirds: 1) APC-proximal; 2) middle third; and 3) distal third. Only conjugates with MTOCs that were located in the APC-proximal third of the T cell cytoplasm were counted as positive reorientation events. Because not all contacts were rounded but often involved significant flattening of T cell morphology, it was not always possible to divide the T cell into meaningful thirds; therefore, these T cells were divided in half and scored as positive/negative events along the midline. Because the T cell MTOC could theoretically be located anywhere in the cell at the time of fixation, this convention led to a background of 30–50% T cells with the MTOC oriented toward the APC or bead, even in the absence of positive signaling events. Conjugates with T cell MTOCs that could not be located were discarded from the sample.
Statistical analyses
Unless otherwise noted, at least three independent repetitions
were performed of each experimental condition, and the results were
analyzed using post hoc one-way (Fig. 5
C) or two-way (Figs. 3 and 5, A and B) analysis of variance with
Bonferroni correction. The Bonferroni correction is used to raise the
threshold to establish statistical significance, when multiple
statistical comparisons are performed. The Bonferroni correction
establishes a new, more stringent , according to the number of
comparisons that were performed, such that, for n
comparisons, the new = 0.05/n. The new values
obtained by the Bonferroni correction are reported in the figure
legends, where appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell interactions with Ag-specific APCs result in the rearrangement of surface receptors and signaling molecules within the T cell to face the APC, and a corresponding rearrangement of actin and tubulin-based cytoskeletal elements. To test the relative contribution of individual receptor:ligand interactions to the polarization of cytoskeletal elements, we used a panel of cells generated by transfection of the mouse fibrosarcoma cell line 6132 Pro (35) (ProPAR), to express MHC class II alone (ProAd) or in combination with ICAM-1 (ProAd-ICAM) or B7-1 (ProAd-B7). This panel of APCs has been previously characterized for interactions required to induce IL-2 expression, T cell activation, anergy, and clonal expansion in Th1 T cell clones and in naïve T cells (27, 28). Briefly, Ag presentation by ProAd cells does not result in the induction of IL-2 gene expression or proliferation but does engage the TCR, inducing anergy in Th1 clones. Ag presentation by ProAd-ICAM cells results in low levels of IL-2 gene expression but still induces anergy in Th1 clones; ProAd-ICAM induces proliferation, but not clonal expansion, in naïve T cells. Finally, Ag presentation by ProAd-B7 cells results in efficient T cell activation, clonal expansion, and long term survival. We used this well-characterized T cell:APC system to investigate the cytoskeletal rearrangements that take place after conjugate formation.
We began by comparing the ability of the Pro panel APCs to form stable
conjugates with pGL2 T cells under conditions where cytoskeletal
polarization could be studied. Using an assay based on direct
visualization of conjugates, distinct differences were observed in the
ability of the individual Pro line APCs to stably interact with pGL2 T
cells (Fig. 1). While very few T cells
bound to Ag-pulsed ProPAR or ProAd cells, a large number of T cells
bound to Ag-pulsed ProAd-ICAM APCs, reflecting the additional
contribution of LFA-1:ICAM interactions to T cell adhesion. Ag-pulsed
ProAd-B7 cells were intermediate in their ability to form T cell
conjugates, possibly due to the adhesive contacts provided by CD28:B7
interactions (36), or by enhancement of ß1 integrin adhesion through
costimulatory signaling (37). Adhesion of Th1 clones to Pro line APCs
in the absence of Ag followed the same profile as that shown in the
presence of Ag, but fewer conjugates were retained in all cases (data
not shown).
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The actin-binding protein talin has been shown to colocalize with
clustered and occupied integrins, in part via direct interaction with
the ß integrin cytoplasmic tail (38, 39, 40, 41, 42, 43), and talin polarization has
been used as a marker for identifying Ag-specific T cell:B cell
conjugates (15). To determine which receptor:ligand interactions are
responsible for triggering talin polarization, conjugates between pGL2
T cells and individual Pro panel APCs were processed for talin
immunofluorescence (Fig. 2). In the cases
in which talin polarization was observed, it occurred as a bright band
at the cell:cell interface (Fig. 2H). Talin polarization was
quantitated by counting the percentage of T cells engaged in stable
conjugates in which this bright band was observed. T cell interactions
with ProPAR and ProAd cells rarely resulted in talin polarization in
either the presence or the absence of Ag (Fig. 3
A). In contrast, talin
polarization was observed in the vast majority of cells bound to
ProAd-ICAM. This was further enhanced in the presence of Ag, possibly
reflecting the activation of the ß2 integrin LFA-1 by TCR
signaling (44). ProAd-B7 cells generated moderate levels of talin
polarization. It is not clear whether this recruitment of talin is a
direct consequence of CD28 engagement or an indirect effect mediated by
CD28 activation of ß1 integrins. Overall, we find that
the frequency of talin polarization responses toward the individual
APCs mirrors the abilities of these APCs to form stable conjugates
(compare Fig. 3A with Fig. 1). This indicates that talin
reorganization is closely linked to adhesion, as expected if both
processes are integrin dependent.
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To analyze the effects of different receptor:ligand interactions
on the microtubule cytoskeleton, the orientation of the MTOC in T cells
stably interacting with APCs was assessed by immunofluorescent labeling
of tubulin and quantified as described in Materials and Methods.Because the T cell MTOC exists within the cell at all times,
random orientation will juxtapose the MTOC toward the APC in 30–50%
of the cell:cell conjugates, so that a specific response is represented
by reorientation above this background. As shown in Figs. 2 and 3B, none of the Pro line APCs could cause MTOC reorientation
in the absence of Ag, but all the lines transfected with class II
molecules induced MTOC reorientation in the presence of Ag. MTOC
reorientation responses were essentially equivalent following Ag
presentation by ProAd, ProAd-ICAM, and ProAd-B7 cells. Thus, induction
of MTOC reorientation does not require the adhesive and/or
costimulatory signals that are required for complete T cell activation.
That TCR cross-linking alone is sufficient to induce MTOC reorientation
is further demonstrated by the fact that reorientation can be induced
in pGL2 T cells by anti-CD3-coated polystyrene beads, but not by
anti-class I-coated beads (Refs. 23 and 24; see Fig. 5B).
Actin polarization is not required for MTOC reorientation
As previously shown for other T cell:B cell conjugates (17, 18),
polarization of actin in T cells interacting with A20 B cells occurred
in an Ag-dependent manner and was observed as a bright band of
filamentous actin at the site of cell:cell contact (Fig. 4, A and B).
However, actin polarization was not observed in response to Ag-pulsed
ProAd APCs, although MTOC reorientation did occur under these
conditions (Fig. 4, C and D). Even when Ag was
presented by ProAd-ICAM cells, where we consistently observed strong
polarization of talin (Fig. 3), we noted only occasional, Ag-dependent,
actin accumulation (see Fig. 6E, below). These results show
that MTOC reorientation can occur at high frequency under conditions in
which actin polarization was not observed. Therefore, either actin
polarization occurs at levels below our threshold of detection or the
strong actin accumulations are not a necessary consequence of strong
talin accumulations.
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CD28 engagement synergizes with TCR signaling in the pathways that
lead to gene induction and T cell proliferation (3). Although our
results show that CD28 engagement is not required to induce MTOC
reorientation, they do not rule out an effect on the downstream
signaling pathways involved. Because PI 3-kinase has been implicated in
the signaling pathways downstream of both TCR and CD28 (45, 46, 47, 48) and in
cytoskeletal remodeling in other systems (49, 50, 51, 52, 53, 54), we tested the role
of this kinase in MTOC reorientation. pGL2 T cells were pretreated with
the PI 3-kinase inhibitor wortmannin and allowed to interact with the
individual Pro line APCs. Wortmannin inhibited MTOC reorientation by
50% following Ag presentation by ProAd or ProAd-ICAM cells (Fig. 5A). This inhibition was
maximal at 10 nM and was not enhanced by 10-fold higher concentrations
of the drug (data not shown). Because the effects of wortmannin are
irreversible, control experiments were performed by preincubating
either T cells or APCs with wortmannin, but omitting the drug during
the conjugation period. Inhibition of MTOC reorientation was observed
only when T cells were drug treated, indicating that the effects were
specific for the responding T cell (data not shown). A more specific PI
3-kinase inhibitor, Ly 294002, inhibited MTOC reorientation toward
ProAd-ICAM APCs to the same extent as wortmannin (50% inhibition at
500 mM), verifying that the observed effects on reorientation are due
to inhibition of PI 3-kinase.
In contrast to our findings with ProAd and ProAd-ICAM, wortmannin had
no effect on MTOC reorientation toward Ag-loaded ProAd-B7 cells (Fig. 5A) or B7-positive A20 B cells (data not shown). Thus,
costimulation through CD28 overcomes the sensitivity of MTOC
reorientation to wortmannin. It is not clear whether this wortmannin
insensitivity occurs because CD28 coligation initiates a PI
3-kinase-independent signaling pathway, or because CD28 coligation
induces levels of PI 3-kinase activity that are not readily inhibitable
by wortmannin. Nevertheless, these data show that coligation of TCR and
CD28 delivers a cytoskeletal signal that is qualitatively different
from ligation of TCR alone.
The role of PI 3-kinase in MTOC reorientation is to amplify a weak TCR signal
The role of PI 3-kinase in MTOC reorientation has been examined by
others, but with conflicting results: wortmannin inhibited MTOC
reorientation in murine T cells interacting with B cell APCs (18) but
did not affect MTOC reorientation of Jurkat T cells toward
anti-TCR-coated beads (24). The observed differences in wortmannin
sensitivity in response to the Pro line APCs suggested an explanation
for this apparent discrepancy, i.e., that the sensitivity to wortmannin
differs with the signal received. To address this issue, we took
advantage of the fact that MTOC reorientation can be induced in pGL2 T
cells either by peptide-MHC complexes or by anti-CD3-coated
polystyrene beads. While wortmannin was capable of inhibiting MTOC
reorientation toward Ag-loaded ProAd APCs, it failed to inhibit MTOC
reorientation in response to anti-CD3-coated beads (Fig. 5B). Similarly, MTOC reorientation in JBN-ß.2.8.24, a
Jurkat cell line stably transfected with the - and ß-chains of the
murine 2B4 TCR (31), was sensitive to wortmannin when tested in
response to Ag presentation by I-Ek-transfected APCs (Fig. 5C), whereas wild-type Jurkat cells showed no sensitivity to
wortmannin when presented with anti-CD3-coated beads (data not
shown) or anti-CD3-coated U937 cells (Fig. 5C). These
results demonstrate that the differential sensitivity of T cell MTOC
reorientation to wortmannin is not a result of cell type differences
but rather of differences in the TCR signal received.
The inability of wortmannin to inhibit MTOC reorientation in
anti-CD3-stimulated T cells could result from either the high
affinity (low off-rate) of Ab binding or the high number of TCRs
engaged. To address this issue, we used fibroblasts expressing class II
molecules bearing a covalently linked OVA peptide fragment (ProAd/OVA)
(Fig. 5A). ProAd/OVA cells are estimated to express 100-fold
higher TCR ligand density than can ProAd APCs loaded with maximal
exogenous peptide.4 Stimulation of pGL2 T cells with
ProAd/OVA cells induced MTOC reorientation that was not inhibitable by
wortmannin (Fig. 5A). Taken together, our results show that
the requirement for PI 3-kinase can be overcome either by extremely
high ligand density (as with anti-CD3-coated beads or ProAd/OVA
APCs) or by coengagement of costimulatory molecules (i.e., Ag
presentation by ProAd-B7 APCs), suggesting that the role of PI 3-kinase
in MTOC reorientation may be to amplify weak TCR signals.
Polarization of cortical cytoskeletal elements and the MTOC can occur on opposite faces of the T cell
Normally, when the full complement of receptor:ligand interactions takes place together at the T cell:APC interface, both cortical cytoskeletal elements and the MTOC polarize toward this site, and it has been assumed that these processes are mechanistically linked. The differences in the abilities of the individual Pro line APCs to induce talin polarization and MTOC reorientation, however, suggested that this might not be the case. Moreover, although we observed MTOC reorientation toward anti-CD3-coated beads, we detected no talin polarization toward the beads (data not shown). Staining with rhodamine-phalloidin also failed to reveal strong actin accumulation at the site of bead binding, although T cells were sometimes observed to have formed a phagocytic cup around the beads (data not shown).
To address the relationship between cortical cytoskeletal
rearrangements and MTOC reorientation, we tested the effects of
ligating LFA-1 and the TCR on opposite faces of the same T cell. For
this, conjugates were first formed between T cells and ProAd-ICAM cells
in the absence of Ag. Under these conditions, engagement of LFA-1 is
sufficient to recruit talin, but not to reorient the MTOC (see Fig. 3).
A TCR signal was then supplied at a different site on the T cell by
adding anti-CD3-coated beads to the preformed cell:cell conjugates.
We reasoned that if the primary function of TCR signaling is to
transduce a signal that links the microtubule array to the cortical
cytoskeleton, the MTOC should localize toward the ProAd-ICAM cells. In
contrast, if the site of TCR engagement also serves to define the locus
for MTOC reorientation, then the MTOC should localize toward the
anti-CD3-coated beads. We found that the latter possibility was
correct (Fig. 6). In the bead:T cell:APC
conjugates, talin polarization (Fig. 6B) (and occasionally
actin polarization (Fig. 6E)) was detected only at the
contact zone between T cells and ProAd-ICAM APCs, while MTOC
reorientation was observed toward the anti-CD3-coated beads (Fig. 6, C, F, and G). Therefore, MTOC
reorientation can be separated from detectable actin and talin
polarization, suggesting that any mechanistic link that may exist
between these processes is not obligatory.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results show that talin polarization occurs as a direct result of LFA-1 cross-linking by its physiological ligand, ICAM-1, and that talin polarization is not induced by cross-linking of TCR alone. Thus, previous studies showing that talin polarization in T cell:B cell conjugates is an Ag-dependent process (15) probably reflect not the direct involvement of TCR, but rather the requirement for TCR-mediated activation of integrin-based adhesion (44). Our finding that talin polarization occurs toward ProAd-ICAM cells in the absence of Ag may be due to the activation status of the Th1 clones or to the relatively high levels of ICAM presented by ProAd-ICAM cells, since high ligand density can induce integrin activation (55). By analogy with focal adhesions in other cell types, where the cytoplasmic tails of ß1 integrins have been shown to bind talin upon engagement and clustering (39), it is likely that in T cells, ICAM binding initiates direct interaction of talin with the ß2 subunit of LFA-1. This interpretation is also supported by our finding that talin polarizes under conditions in which actin polarization is not observed.
Although we did not detect strong actin polarization in pGL2 cells
responding to Pro cells or to anti-CD3-coated beads, we did observe
filopodia and actin-containing extrusions in bead-stimulated T cells.
We interpret this as the formation of phagocytic cups, since at later
time points beads were partially engulfed (data not shown). These
actin-containing phagocytic structures resemble the actin polarization
events described by Lowin-Kropfe et al. (24), but they bear little
resemblance to the more distinct actin clusters observed in T cell:B
cell couples in the presence of Ag (Fig. 4) (17, 18) or to the strong
actin accumulations that we occasionally observed in response to the
Pro panel APCs (Fig. 6E). Since we were unable to
consistently induce strong actin clustering in response to either Pro
line APCs or anti-CD3-coated beads, we conclude that TCR signaling
alone is insufficient to drive this process and that the
receptor-ligand interactions required are not among those we have
tested thus far.
While we find that talin polarization responds to integrin ligation,
our results show that MTOC reorientation occurs in response to TCR
engagement. Our findings confirm previous studies showing that MTOC
reorientation can be induced by Ab-mediated cross-linking of the TCR
(23, 24) and further show that presentation of Ag by MHC class II
molecules has the same effect. The manner in which the MTOC
reorientation event is linked to TCR signaling is unknown. Recent work
has implicated TCR-associated tyrosine kinases, since Jurkat T cells
lacking Lck fail to reorient their MTOCs and since overexpression of a
dominant-negative ZAP-70 mutant partially inhibits MTOC reorientation
(24). It has been proposed that ZAP-70 phosphorylation of -tubulin
could cause localized microtubule catastrophe at the site of TCR
signaling (24), bringing about MTOC reorientation by shortening
microtubules in the proximity of TCR signaling while microtubules
elsewhere in the cell continue to lengthen. However, since the ZAP-70
mutant only partially inhibits MTOC reorientation, it must be
considered that ZAP-70 might not be solely responsible for this event.
Furthermore, since both Lck and ZAP-70 act early in TCR signaling, it
is possible that their role is indirect; there may be many intervening
steps in between TCR signaling and the effector mechanisms that
actually bring about MTOC reorientation.
PI 3-kinase has been implicated in signaling by both TCR and CD28,
although there is some disagreement in the literature regarding whether
PI 3-kinase activity is essential to CD28 signaling (56, 57, 58). We found
that while CD28:B7 interactions alone could not induce MTOC
reorientation, they could affect the sensitivity of TCR-induced MTOC
reorientation to wortmannin, a specific inhibitor of PI 3-kinase. While
wortmannin inhibited MTOC reorientation in response to ProAd cells by
50%, it had no effect on MTOC reorientation in response to ProAd-B7
APCs. In this context, it is important to note that although Stowers et
al. (18) found that wortmannin did inhibit MTOC reorientation using the
B cell line CH27 as an APC, the T cell they used as a responder is a
hybridoma that is not responsive to costimulatory signals. The effect
of CD28 is probably not due to increases in adhesion mediated by
CD28:B7 interactions because wortmannin inhibits MTOC reorientation
following Ag presentation by ProAd-ICAM cells, where the adhesive
effects are greater. Instead, CD28 engagement could transduce a PI
3-kinase-independent signal that enhances TCR-stimulated MTOC
reorientation or could activate additional PI 3-kinase isoforms that
are insensitive to wortmannin inhibition.
The finding that wortmannin failed to inhibit MTOC reorientation in the presence of costimulatory signaling suggests that the role of PI 3-kinase in MTOC reorientation is one of signal amplification. This conclusion is supported by the observation that wortmannin inhibits MTOC reorientation in response to TCR stimulation by peptide-MHC complexes but is incapable of affecting MTOC reorientation when the TCR is cross-linked by anti-CD3 Ab-coated beads, or when the T cell is presented with exceptionally high levels of peptide-MHC complexes, expressed by ProAd/OVA cells. These observations explain a discrepancy in the literature regarding the role of PI 3-kinase in MTOC reorientation (18, 24). The contradictory observations made by these two groups are likely due to the fact that presentation of the polarizing signal was accomplished by Ab-coated beads in the former case and by peptide-MHC complexes (in the absence of CD28 signaling) in the latter. On the basis of our cumulative results, we conclude that PI 3-kinase likely amplifies suboptimal TCR signaling to produce MTOC reorientation but is not required in the presence of exceptionally strong TCR signaling such as that induced by Ab cross-linking of the TCR.
In many systems, the cortical cytoskeleton and the microtubule cytoskeleton are thought to act in concert to produce the morphological changes that accompany cellular responses to extracellular stimuli. It therefore seemed likely that T cell cortical rearrangements could facilitate MTOC reorientation by creating an anchoring superstructure for the proteins involved. Evidence in favor of an interaction between the cortex and the microtubule cytoskeleton comes from the work of Stowers et al. (18), showing that a dominant negative form of Cdc42 (a member of the Rho family of small GTPases, which are involved in actin remodeling) disrupts both actin polarization and MTOC reorientation in 2B4 T cells. Recent work has demonstrated that PI 3-kinase products and substrates can modify the activity of Vav, an exchange factor for Rho family proteins (59), suggesting that PI 3-kinase may indirectly affect MTOC reorientation via modulation of the cortical cytoskeleton.
Surprisingly, however, we have found that MTOC reorientation, which responds specifically to TCR signaling, can occur in the absence of detectable talin or actin accumulation. Moreover, we found that MTOC reorientation can occur on the opposite face of the cell from detectable cortical polarization events. Although our results do not preclude a contribution by the cortical cytoskeleton to the process of MTOC reorientation, they clearly show that the dramatic accumulations of cortical cytoskeletal elements normally seen during T cell:B cell interactions are not required for TCR-induced MTOC reorientation. TCR engagement alone is capable of defining the site toward which MTOC reorientation occurs. Thus, either TCR signaling itself is capable of recruiting the necessary effector molecules responsible for MTOC reorientation or the cortical cytoskeleton can transmit the TCR signal to the relevant effectors without undergoing obvious reorganization.
The T cell cytoskeleton, far from being a passive observer of signaling events, is directly involved in the mechanisms and consequences of T cell:APC interactions. Therefore, understanding the interplay of these cytoskeletal rearrangements and the signaling events that bring them about is likely to be an area of much interest in the future. The experimental systems described here will provide valuable tools with which to investigate these and related questions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jim Miller, University of Chicago, Department of Molecular Genetics and Cell Biology, 920 E. 58th Street, CLSC 1021, Chicago, IL 60637, E-mail address:
3 Abbreviations used in this paper: MTOC, microtubule-organizing center; PI 3-kinase, phosphatidylinositol 3-kinase; CMAC, 7-amino-4-chloromethylcoumarin; OVA peptide, OVA peptide (residues 323–339); BSS, balanced salt solution; DIC, differential interference contrast; RT, room temperature.
4 C. Abraham, J. Griffith, and J. Miller. The dependence for LFA-1/ICAM-1 interactions in T cell activation cannot be overcome by high density TCR ligand. Submitted for publication.
Received for publication July 30, 1998. Accepted for publication October 19, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or absence (
) of Ag for 30 min before fixation and
immunofluorescent labeling with anti-talin mAb (A)
or anti-tubulin mAb (B). Polarization was scored as
described in Materials and Methods. Data are the means
of four independent experiments; error bars are SD of the mean for each
data set. Talin polarization responds most strongly to integrin
engagement, with significant Ag-dependent enhancement
(p < 0.0083) observed only in T cells interacting
with ProAd-ICAM APCs. In contrast, addition of Ag significantly
enhances of MTOC reorientation in response to ProAd, ProAd-ICAM, and
ProAd-B7 APCs (p < 0.0083). The fraction of
polarization observed reflects the percentage of adherent cells
demonstrating polarized morphology. Therefore, for APCs to which T
cells bound poorly (ProPAR and ProAd), the absolute number of
polarization events per sample was far fewer than for APCs which
induced good adhesion (ProAd-ICAM and ProAd-B7).
