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,
Departments of
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Pathology and
Medicine, and
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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2 integrin LFA-1 binds to the intracellular
adhesion molecules ICAM-1, ICAM-2, and ICAM-3 (1, 2, 3, 4). In
contrast, T cell adhesion to VCAM-1 and extracellular matrix proteins
such as fibronectin is mediated by the 1
integrin subfamily (5). LFA-1 and CD2 participate in T cell adhesion in distinct ways. The adhesion mediated by CD2 interaction with LFA-3 is present in resting T cells, but becomes more active over a period of hours following T cell activation (4). CD2 ligation can mediate or enhance T cell activation (4), and triggering through CD2 results in the persistent activation of LFA-1 (6). In contrast, LFA-1 binding to ICAMs is transiently up-regulated within minutes of TCR stimulation (2, 3, 4). It has been suggested that the CD2 pathway plays a critical role in initiating cell-cell interactions before the engagement of the TCR, and that LFA-1 strengthens the T cell-APC contact following TCR engagement (7).
Activation of both LFA-1 and the 1 integrins
in T cells has been the focus of numerous studies; adhesion mediated by
both integrins is quickly up-regulated following TCR cross-linking or
activation with phorbol esters (2, 6, 8, 9, 10). The
stimulation through cell surface receptors is thought to produce
intracellular second messengers that alter the adhesive state of
integrins, a process termed inside-out signaling. This rapid modulation
of adhesion is due to an increase in avidity brought about by
qualitative rather than quantitative changes in the cell surface
expression of integrins (3, 4, 8, 11, 12, 13). Evidence
suggests that rather than conformational changes being involved,
increased adhesion results from the transient release of the integrins
from the actin cytoskeleton, allowing them to move more freely in the
membrane to find their ligands and to cluster at the cell-cell contact
site, where they reassociate with F-actin in an organized adhesion
complex (14, 15, 16).
Numerous studies have attempted to identify and characterize the inside-out signaling pathways that regulate integrin avidity in T cells and other cell types (reviewed in Refs. 17, 18). Although the results of these studies are somewhat variable, they have established that signaling through protein kinase C (PKC)4 (2) plays an important role in the activation of adhesion (17). Other late signaling molecules have also been implicated in TCR-induced, LFA-1-mediated adhesion; roles for the Ras/mitogen-activated protein kinase (MAPK) pathway, phosphatidylinositol-3 (PI-3) kinase, calpain, and cytohesin-1 have all been reported (19, 20, 21, 22, 23). By comparison with these later signaling requirements, relatively little is known about the proximal signaling events that regulate TCR-activated integrin adhesion.
Many studies of integrin adhesion have used as an experimental model the binding of previously activated T cells to purified ligands immobilized on plastic. Others have examined the interaction between T cells and B cells following T cell activation with PMA or CD3 cross-linking. Because it is now recognized that there are significant differences in T cell responses to Abs and immobilized ligands as opposed to bona fide APCs, we tested the role of early signaling molecules in T:B adhesion without prior activation of the T cells. We developed an assay to study superantigen-dependent conjugation of Jurkat cells to EBV-B cells, and used the Lck-deficient mutant JCaM1 and the ZAP-70-deficient mutant P116 to ask about the proximal signaling events involved. Our results show that adhesion is largely mediated by LFA-1, and requires Lck and downstream signaling molecules, but is independent of ZAP-70 activity. At least part of the role of Lck in adhesion is to organize actin and LFA-1 at the cell-cell contact site in response to TCR engagement.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-CD3 mAb OKT3 was purchased from Orthobiotech
(Raritan, NJ), and anti-LFA-1 
-chain TS1/22 was obtained from
Endogen (Woburn, MA). The anti-LFA-1 -chain TS1/18,
anti-LFA-1 -chain TS2/4, and anti-class I W6/32 hybridomas
were obtained from American Type Culture Collection (Manassas, VA);
anti-CD2 Ab 95-5-49 was a gift from R. Gress (National Cancer
Institute, Bethesda MD); and anti-CD29 Ab Lial1/2 was purchased
from Biodesign International (Saco, ME). The anti-TCR chain c305
has been described (24), and the rabbit
anti-mitogen-activated protein/extracellular signal-regulated
kinase (ERK) kinase 1 (MEK1) was a gift from M. Rosner (University of
Chicago, Chicago, IL). Rabbit polyclonal anti-phospho-MEK and
anti-phospho-ERK were obtained from New England Biolabs (Beverley,
MA), and anti-phosphotyrosine mAb 4G10 was purchased from Upstate
Biotechnology (Lake Placid, NY). Purified rICAM-1-Fc protein was
supplied by P. Hoffman (ICOS, Bothell, WA). Fc
-specific goat
anti-human IgG was purchased from ICN (Costa Mesa, CA).
HRP-conjugated secondary Abs were Obtained from Jackson
ImmunoResearch (West Grove, PA). Alexa-488-conjugated
secondary Abs were purchased from Molecular Probes (Eugene, OR).
Partially purified staphylococcal enterotoxin E (SEE) was
obtained from Toxin Technology (Sarasota, FL). The p-EGFP actin plasmid
was purchased from CLONTECH Laboratories (Palo Alto, CA).
Wortmannin, LY924002, U73122, PD98059, calphostin C, calpeptin, and SB202190 were purchased from Calbiochem (San Diego, CA). Hydroethidine (HE; dihydroethidium), PKH26, poly-L-lysine, paraformaldehyde, and fish skin gelatin were obtained from Sigma-Aldrich (St. Louis, MO). Calcein-AM, 7-amino-4-chloromethylcoumarin (CMAC), and CFSE were purchased from Molecular Probes. Pefabloc was obtained from Roche Diagnostics (Indianapolis, IN), and other protease inhibitors were purchased from Sigma-Aldrich. Mowiol 4-88 was obtained from Hoechst Celanese (Charlotte, NC).
Cell lines and plasmids
Human cell lines were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, glutamine, penicillin, and streptomycin (all from Life Technologies, Grand Island, NY) at 37°C and 5% CO2. The EBV-transformed human B cell line, EBV-B, was a gift from H. Ochs (University of Washington, Seattle, WA), and the Jurkat-derived ZAP-70-deficient cell line, P116 (25), was a gift from R. Abraham (Duke University, Durham, NC). The B cell line BBN (gift from M. Dustin, Washington University, St. Louis, MO) is from a human patient (patient 1) with leukocyte adhesion deficiency (LAD) and expresses no surface LFA-1 (26). The Lck-deficient derivative of Jurkat, JCaM1, has been characterized (27), along with JCaM1 transfectants expressing Lck R154K (28), LckW97A, and wild-type Lck (29). JCaM1 cells expressing Fyn at levels equivalent to Lck have been described (30). The Lck substitution mutants, G2A and K273A, have been described previously (31, 32). Substitutions at these positions block Lck myristoylation or catalytic activity (33, 34). LckG2A and K273A were subcloned into pBJ1-neo (35) and pBP1 (29), respectively, and transfected into JCaM1 by electroporation. Antibiotic-resistant clones that expressed Lck at levels equivalent to the parental Jurkat E6 subclone were selected for further analysis. Biochemical studies confirmed that LckG2A was mislocalized to the cytosol, and that LckK273A lacked in vitro kinase activity (M. Denny and D. Straus, unpublished data). Clones expressing either LckG2A or LckK273A failed to initiate signaling in response to TCR stimulation. All Jurkat cell lines were analyzed by fluorescence flow cytometry every 4–6 wk to ensure that they expressed equivalent levels of TCR and LFA-1.
Murine primary T cell blasts specific for OVA peptide residues 323–339 (OVA323–339) presented by I-Ad were prepared from DO-11.10 TCR-transgenic (Tg) mice. Lymph node cells from DO-11.10 mice (2 x 105/well) were expanded by coculture with irradiated splenocytes (2500 rad, 6 x 106/well) and 0.15 µg/ml OVA323–339 peptide in complete medium (DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.29 mM L-glutamine, nonessential amino acids, 10 mM HEPES, and 5 x 10-5 M 2-ME), at 10% CO2. T cells were harvested and live cells recovered by Ficoll-Hypaque gradient separation 7–14 days after activation, by which time they were rested, as judged by small size and requirement for additional stimulation to induce significant proliferation. A20 B cells were cultured in complete DMEM, supplemented as above.
FACS-based conjugation assay
EBV-B cells to be used as the APCs were stained with HE or PKH26. For HE staining, EBV-B cells were incubated in RPMI 1640 containing 3 µg/ml HE at 37°C for 30 min, washed twice with RPMI 1640, then resuspended in RPMI 1640 with or without 2 µg/ml SEE and incubated at 37°C for 1.5 h. B cells were then washed again and resuspended in serum-free RPMI 1640 (SF-RPMI) at 1 x 106 cells/ml. For PKH26 staining, EBV-B cells were washed with SF-RPMI, resuspended in diluent C (Sigma-Aldrich; provided with PKH26) at 1 x 107 cells/ml, then mixed with an equal volume of 6 µM PKH26 and incubated at room temperature for 10 min. After an equal volume of FBS was added to quench the staining, cells were washed with RPMI 1640 and incubated with or without SEE, as described above. Just before conjugation, EBV-B cells were pelleted and resuspended in SF-RPMI at 1 x 106 cells/ml. Jurkat cells were incubated in RPMI 1640 containing 0.15 µg/ml calcein-AM for 30 min at 37°C, then washed twice with RPMI 1640 and resuspended in SF-RPMI at 1 x 106 cells/ml.
For conjugation, 0.5 ml of EBV-B cells were combined with 0.5 ml of Jurkat cells in 6-ml polystyrene tubes, and pelleted at 500 rpm for 5 min at room temperature. A total of 0.5 ml supernatant was aspirated from each tube, and the pellets were incubated at 37°C for 10–15 min. The tubes were vortexed for 5–10 s to resuspend the cells, then fixed by the addition of 0.5 ml of PBS/6% paraformaldehyde. The relative proportion of red, green, and red/green events in each tube was determined by two-color flow cytometric analysis using a BD Biosciences (Franklin Lakes, NJ) FACScan flow cytometer with an excitation wavelength of 492 nm and emission filters for green (FL1, 520 ± 15 nm) and red (FL2, 585 ± 21 nm). For each experiment, gates were set to exclude crenated and unstained cells and to optimize the separation of the two fluorescent signals. The number of gated events counted per sample was at least 15,000. Percentage of conjugation was calculated as the number of dual-labeled (red/green) events divided by the sum of the dual-labeled events and the unconjugated Jurkat cells (green events), as described previously (36). Conjugation of DO-11.10 T cells and A20 B cells was performed similarly, except that A20 cells were pulsed for 1–2 h with 2 µg/ml OVA323–339, in place of SEE.
Ab-blocking and drug inhibition experiments
For Ab-blocking experiments, the number of APCs and Jurkat cells used in the conjugations was halved, which did not significantly alter the percentage of conjugation. Following labeling with calcein-AM, Jurkat cells were preincubated with blocking Abs at 20 µg/ml for 10 min at room temperature, before combining with an equal volume of APCs to give a final Ab concentration of 10 µg/ml. Since B cells also express some LFA-1 (37), the APCs were also preincubated with anti-LFA-1 Abs in the experiments using TS1/18 and TS1/22.
For experiments using wortmannin, PD98059, SB202190, U73122, calphostin C, and calpeptin, T cells were first labeled with calcein-AM, washed, resuspended in SF-RPMI containing the appropriate concentration of drug, and incubated at 37°C for 30 min. For LY294002 experiments, D0–11.10 T cells were treated as described above, while Jurkat cells were incubated in RPMI 1640 containing drug for 6 h at 37°C, then labeled by adding calcein-AM to a final concentration of 0.125 µg/ml and incubating another 30 min at 37°C. Cells were then washed and resuspended in SF-RPMI containing the appropriate concentration of LY294002. Unless otherwise indicated, drug concentrations were adjusted upon addition of APCs to maintain the indicated concentrations throughout the conjugation. Where indicated, T cells or B cells were treated with drugs for 30 min before conjugation, washed, and added to conjugation reactions in the absence of additional drug.
Immunoblotting of whole cell lysates
Jurkat cell suspensions (1 x 107 cells/ml in PBS or RPMI 1640) were unstimulated, stimulated for 2 min at 37°C with c305, or stimulated for 5 min at 37°C with OKT3. Alternatively, cells were stimulated by conjugation with SEE-coated (or control) EBV-B cells. Cell suspensions of Jurkat and EBV-B cells (2 x 106 cells/ml in RPMI 1640) were combined, pelleted at 500 rpm (room temperature), and incubated for 10 min at 37°C. In each case, cells were lysed at 4°C in 1% (v/v) Nonidet P-40, 10 mM Tris (pH 7.8), 150 mM NaCl, 1 mM pefabloc, 1 mM sodium orthovanadate, 10 mM NaF, 0.5 µg/ml leupeptin, 4 µg/ml aprotinin, 0.5 µg/ml N-tosyl-L-phenylalanine chloromethyl ketone, and 1 µg/ml pepstatin. Debris was removed by centrifugation at 12,500 x g for 10 min at 4°C. Lysates were resolved by SDS-PAGE and transferred to nitrocellulose. Filters were probed with 4G10 anti-phosphotyrosine, anti-phospho-ERK, anti-phospho-MEK, or anti-MEK1. Bound primary Abs were detected using HRP-conjugated secondary Abs and chemiluminescence. For quantitation, blots were scanned using a FluorChem imager (Alpha Innotech, San Leandro, CA). The peak area for each band was determined, and only measurements falling within the linear response range were used. Phospho-MEK and ERK values were corrected for protein loading based on total MEK values, and expressed as percentage of the positive control response for each set of conditions.
Adhesion to ICAM-1-Fc
Nunc Maxi-Sorp 96-well plates (Naperville, IN) were coated with
0.5 µg/well goat anti-human IgG (Fc specific) overnight at
4°C, blocked with 1% BSA in PBS, then incubated with 0.3 µg/well
ICAM-1-Fc at room temperature for 90 min. Wells were rinsed with PBS
and blocked with PBS/1% BSA for 1 h before use. Jurkat cells were
labeled with 5 µM CFSE in SF-RPMI for 20 min at 37°C. The reaction
was quenched with FBS, then the cells were washed twice in RPMI 1640
and resuspended in SF-RPMI. A total of 2 x
105 cells was added per well, followed by the
addition of either PBS, the anti-TCR Ab c305 to a final
concentration of 5 µg/ml, PMA to a final concentration of 20 ng/ml,
or MgCl2/EGTA to a final concentration of 2 mM
Mg/5 mM EGTA. Cells were allowed to adhere for 1 h at 37°C, then
the wells were carefully washed three times with 0.2 ml HBSS to remove
unbound cells. Bound cells were fixed by the addition of 0.l ml 3%
paraformaldehyde in PBS to each well and assayed using a Cyto Fluor II
Fluorescence Multiwell Plate Reader (PerSeptive Biosystems,
Framingham, MA).
PMA and Mg2+ conjugation experiments
EBV-B cells were stained with PKH26, and Jurkat cells were stained in calcein-AM, as described above. For Mg/EGTA conjugations, the EBV-B and Jurkat cells were resuspended in serum-free, Mg/Ca-free RPMI 1640 at 1 x 106 cells/ml. Mg/EGTA was added to a final concentration of 2 mM Mg/5 mM EGTA, then the conjugation was conducted as described above. For PMA experiments, Jurkat cells were resuspended in SF-RPMI at 1 x 106 cells/ml. PMA was added to 20 ng/ml, and the cells were incubated at 37°C for 15 min. Conjugation with EBV-B cells was conducted as previously described. PMA levels were adjusted upon the addition of the APCs to maintain a concentration of 20 ng/ml during the conjugation.
Transient transfection of Jurkat cells with p-EGFP actin
Wild-type and mutant Jurkat cells were seeded in 100 ml of RPMI
1640 in 150-cm2 flasks at 1.5 x
104 cells/ml and grown at 37°C until cells were
4 x 105 ml (typically 3–4 days). Cells
were washed with RPMI 1640 and resuspended in RPMI 1640 at 8 x
107 cells/ml. A total of 2 x
107 cells was combined with 20 of µg p-EGFP
actin in an electroporation cuvette and mixed gently. The cells were
pulsed at 240 V, 1500 µF using a Bio-Rad (Richmond, CA) Gene Pulser
II apparatus, then placed on ice for 8 min. The cells were then
transferred into 15 ml RPMI and incubated overnight at 37°C.
Conjugation and immunofluorescence labeling of T:B conjugates for microscopy
EBV-B cells were used as the APCs for conjugation with green fluorescent protein (GFP) actin-transfected Jurkat cells, and the LAD B cell line BBN was used as the APCs for LFA-1 immunofluorescence. B cells were stained by incubation in SF-RPMI containing 10 µM CMAC cell tracker blue for 30 min at 37°C, washed, and incubated in RPMI 1640 with or without 2 µg/ml SEE at 37°C for 1.5 h. The cells were then washed and resuspended in SF-RPMI at 106 cells/ml. Before use, live GFP actin-transfected Jurkat cells were separated from dead cells by passage over Ficoll, washed, and resuspended in SF-RPMI at 106 cells/ml. Conjugations were performed as above, except that after vortexing, cells were pipetted onto poly-L-lysine-coated slides and incubated at 37°C for 5–10 min. Slides were rinsed briefly in PBS and fixed in 3% paraformaldehyde/PBS for 20 min at room temperature. For the GFP actin transfectants, slides were dipped in water and mounted (see below).
For LFA-1 immunofluorescence, slides were quenched in 50 mM ammonium chloride, then blocked in PBS/0.25% fish skin gelatin (PBS/gel) for 15 min at room temperature. A total of 20 µl of primary Ab diluted in PBS/gel was added to each well, and the slides were incubated for 45 min at room temperature. Slides were washed in PBS/gel and incubated with Alexa-488-conjugated secondary Ab diluted in PBS/gel for 45 min at room temperature. The slides were then washed, dipped in water, and mounted in Mowial 4-88 (Hoechst Celanese) with 10% 1,4 diazobicyclo-[2,2,2]octane (Sigma-Aldrich) as an antifade. Slides were viewed using a Zeiss Axioplan microscope equipped with a Photometrics PXL-cooled CCD camera. Image capture and deconvolution analysis was performed using Openlab version 2.06 (Improvision, Coventry, U.K.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the course of studying cytoskeletal responses in a
microscopy-based assay (38), we observed that the
Lck-deficient T cell line JCaM1 was poor at forming conjugates with
APCs, but that the ZAP-70-deficient cell line P116 seemed to conjugate
normally. To explore this observation, we adapted a FACS-based assay
(36) to measure the ability of Jurkat cells to form
conjugates with EBV-B cells. B cells were labeled with the red dyes HE
or PKH26, and Jurkat cells were labeled with the green dye calcein-AM.
Used in this way, these dyes have no effect on conjugation or effector
function (36, 39, 40, 41). B cells were then incubated with or
without SEE and pelleted together with T cells in a 1:1 ratio. After
incubation at 37°C, pellets were vortexed, fixed in suspension, and
analyzed by flow cytometry. Fig. 1
shows
a typical assay. The percentage of T cells in conjugates is increased
3- to 5-fold in the presence of SEE. The conjugates are quite stable;
they can be submitted to pipetting and vigorous vortexing without
dissociation (data not shown). Moreover, the percentage of conjugation
was remarkably consistent from experiment to experiment, provided that
the input T:B ratio was maintained at 1:1.
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, 
) was
unaffected by control Abs reactive to MHC class I and
1 integrins, but was inhibited by
anti-LFA-1 and anti-CD2 Abs. This indicates that both LFA-1 and
CD2 contribute to the basal superantigen-independent adhesion. In the
presence of SEE (Fig. 2, 
), anti-LFA-1- and
-chain-specific Abs inhibited conjugation to almost background
levels, alone or in combination, whereas anti-class I,
anti-1, and anti-CD2 Abs had no effect. When anti-CD2
Abs were combined with anti-LFA-1 Abs, binding was decreased
slightly more than with anti-LFA-1 Abs alone, but the difference
was not significant (data not shown). Taken together, these results
show that the SEE-dependent adhesion is largely mediated by LFA-1.
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Wild-type Jurkat cells (E6), or Jurkat lines lacking Lck (JCaM1)
or ZAP-70 (P116) were tested for their ability to form stable
conjugates with SEE-pulsed B cells. The level of conjugation observed
for each of the mutant cell lines in the absence of SEE was not
significantly different from that observed for E6 cells (data not
shown). In the presence of SEE (Fig. 3A), JCaM1 cells failed to
form conjugates above the background levels observed in the absence of
SEE. This was specifically due to the absence of Lck, since conjugation
was restored in JCaM1 cells stably transfected with wild-type Lck. In
contrast, the ZAP-70-deficient cell line P116 formed conjugates as well
as wild-type cells. This indicates that the increased adhesion mediated
by LFA-1 in response to SEE requires the activity of Lck, but not
ZAP-70.
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20% of normal levels, a transfectant expressing similar levels
of wild-type Lck (28) was included as the matched control
for this mutant. As shown in Fig. 3
B, only wild-type Lck
could restore normal conjugation to JCaM1 cells. Even the expression of
wild-type Lck at only 20% of normal levels was sufficient to restore a
significant level of conjugation. In contrast, none of the altered
forms of Lck could rescue the JCaM1 conjugation phenotype. For the Lck
myristoylation, SH2 domain, and kinase-dead mutants, the defect in
adhesion can be attributed to the failure of these cells to initiate
TCR signaling. However, cells expressing the Lck SH3 domain mutant
recruit and activate ZAP-70, and flux calcium normally
(29), but these cells still failed to form conjugates.
Furthermore, although the expression of Fyn in Lck-deficient T cells
has been reported in other systems to partially restore TCR signaling
(30), we found that Fyn could not compensate for Lck in
our conjugation assay. Taken together, these results indicate that
adhesion to APCs via LFA-1 depends upon signaling through Lck, but not
ZAP-70. Conjugation does not require MEK activation, but is sensitive to PKC and phospholipase C (PLC) inhibitors
Since cells expressing the Lck SH3 domain mutant are defective in
MEK activation (29), one possibility was that signaling
through MEK might be required for conjugation. In keeping with this, it
has been reported that inhibition of MEK activity partially inhibits
the TCR-mediated adhesion of murine T cells to immobilized ICAM-1
(21). We therefore looked for evidence that the MAPK
pathway functions downstream of Lck in the SEE-induced activation of
LFA-1-dependent conjugation. When Jurkat E6 cells pretreated with the
MEK inhibitor PD98059 were tested, no inhibition of conjugation was
observed (Fig. 4). The inhibition of MEK
phosphorylation was confirmed by Western blotting with
anti-phospho-MEK (data not shown). Thus, if the defect in
conjugation requires signaling in the Ras-MAPK pathway, the relevant
molecules must lie upstream of MEK.
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). An inhibitor of p38 MAPK
(42) failed to inhibit conjugation. The PI-3 kinase
inhibitors wortmannin and LY294002, which have been reported to inhibit
T cell adhesion to immobilized ICAM-1 (19), did not
inhibit conjugation, even when the Jurkat cells were treated for
extended periods of time. Similarly, the calpain inhibitor calpeptin,
which also inhibits binding to immobilized ICAM-1 (23),
had no effect.
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); however, we found that the PKC
inhibitor calphostin C profoundly inhibited SEE-dependent conjugation
(Table I). Two interpretations of these findings are possible.
Activation of PKC may be necessary, but not sufficient, to induce
conjugation. Alternatively, conjugation may require the localized
activation of PKC at the cell-cell contact site, which cannot be
adequately mimicked by treatment with soluble PMA. The other inhibitor
that affected conjugation was the general PLC inhibitor U73122. This
drug was recently reported to inhibit IL-3-induced integrin activation
in Baf3 cells (43). Importantly, calphostin C and U73122
had similar effects on both wild-type Jurkat T cells, and Jurkat cells
lacking a functional ZAP-70 (Fig. 5, A and B). This suggests that the PLC isoform
required for activation-induced conjugation is not dependent on ZAP-70.
To verify that calphostin C and U73122 inhibit conjugation by affecting
T cell signaling rather than APC-dependent processes, control
experiments were conducted in which T cells or B cells were
preincubated with the inhibitors, which were then removed during
conjugate formation. Inhibition was observed only when T cells were
preincubated with drug (Fig. 5C), showing that the relevant
PKC- and PLC-dependent events occur in the T
cell.
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D, treatment of the murine T cells with PD98059 had
little effect on conjugate formation, and LY294002 inhibited
conjugation only partially. By contrast, both U73122 and calphostin C
completely blocked conjugation, indicating that the requirement for
signaling through PKC and PLC extends to this system as well. U73122 inhibits the MAPK signaling pathway
Since the Lck SH3 domain mutation and the PLC inhibitor U73122
both inhibited conjugation, we looked for evidence that they affect the
same signaling pathways. Initially, Jurkat cells were pretreated with
U73122 and stimulated with anti-TCR Abs. Western blots of whole
cell lysates were then probed with Abs that recognize activated MEK and
activated ERK. Under these conditions, inhibition of PLC induced only a
partial reduction in MEK phosphorylation, with no observable effect on
ERK phosphorylation (Fig. 6A).
To mimic the conjugation assay conditions, we tested the effects of
U73122 on MEK and ERK activation in response to SEE-pulsed B cells.
Under these conditions, we observed a dramatic reduction in MEK
activation and a significant reduction in ERK activation in the
presence of U73122 (Fig. 6B). Quantitative analysis of the
blots revealed that when T cells were stimulated with anti-TCR Ab,
treatment with U73122 inhibited MEK phosphorylation by only 35% (Fig. 6C, ), and had no effect on ERK phosphorylation (Fig. 6C, ). By contrast, when T cells were stimulated with
SEE-pulsed APCs, MEK phosphorylation was inhibited to near-basal levels
(85% inhibition relative to the levels obtained in the absence of
drug; Fig. 6D, ). Under these conditions, ERK
phosphorylation was inhibited by 52% relative to the levels obtained
in the absence of drug (Fig. 6D, ). Although it is not
clear whether this partial reduction in ERK phosphorylation would
impact gene expression, these results show that activation by
superantigen-coated APCs presents a qualitatively different signal to T
cells than activation by anti-TCR Ab, and that a PLC activity
functions upstream of MEK activation under conjugation conditions.
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Adhesion involves avidity changes and protein tyrosine kinase (PTK)-dependent cytoskeletal remodeling
In studies of LFA-1-dependent binding of preactivated T cells to
immobilized ICAM-1, it has been shown that changes in either avidity or
affinity can lead to increased binding. In keeping with this, we find
that binding of unactivated wild-type Jurkat cells to immobilized
ICAM-1 can be activated by either incubation with Mg-EGTA, under
conditions that induce the high-affinity conformation of LFA-1
(44), or by treatment with PMA, which results in enhanced
avidity (16). Binding under either of these conditions
consistently exceeded that of stimulation with anti-TCR (Fig. 7A). We then asked whether we
could bypass the requirement for TCR signaling in the conjugation assay
in each of these ways. As shown in Fig. 7B, PMA treatment
failed to activate conjugate formation in the absence of SEE, while
incubation with Mg-EGTA induced only a 2-fold increase in conjugation.
Varying the conditions under which these experiments were done, for
example altering the timing of PMA stimulation or stimulating with PMA
+ ionomycin, failed to induce additional conjugate formation (data not
shown). Although we saw no enhancement of conjugation by PMA treatment,
this could be explained by a requirement for the localized activation
of PKCs at the cell-cell contact site that cannot be adequately
mimicked by treatment with soluble PMA. However, the finding that
induction of the high affinity form of LFA-1 by incubation with Mg-EGTA
could not account for the large increase induced by SEE-dependent
signaling suggests that adhesion to APCs involves a significant
component of increased avidity. These results indicate that the
requirements for plate-bound adhesion and conjugate formation differ,
and must be assayed in different ways.
It seemed likely that the T cell mutants that fail to conjugate
properly might be defective in actin remodeling and recruitment of
LFA-1 to the cell-cell contact site. To evaluate the remodeling of
actin in the T cell only, without visualizing actin in the bound B
cell, wild-type and mutant T cells were transiently transfected with
GFP actin and conjugates were analyzed by fluorescence microscopy.
Wild-type T cells typically formed a slightly cupped contact with an
accumulation of actin severalfold brighter than in the rest of the T
cell (Fig. 8, A and
B). The length of the contact site (approximately equal to
one T cell in diameter) was quite uniform among conjugates. As
expected, JCaM1 T cells formed very few conjugates. In those that did
form, the contact site morphology was relatively normal, although on
average it was narrower than in wild-type cells, and no actin response
was observed (Fig. 8C). JCaM1 T cells expressing either the
Lck SH3 domain mutant (Fig. 8D) or Fyn (Fig. 8E)
behaved similarly; few conjugates were formed, and little or no actin
remodeling was observed. The contact site was less than one T cell
diameter, and the cells often formed elongated necklike structures
reaching toward the bound B cell (Fig. 8D). The
ZAP-70-deficient T cells formed conjugates with high frequency;
however, the phenotype of these conjugates was strikingly different
from those formed by either the wild-type E6 cells or the Lck mutants.
The size and shape of the contact site were quite variable, and the T
cells often formed "arms" that reached as much as halfway around
the APC (Fig. 8E). The accumulation of actin was generally
less dramatic than in E6 cells, and more variable in structure. In some
conjugates, normal actin organization was observed; in others, actin
accumulated primarily at the tips of the "arms." Sometimes very
little actin response was observed, even in conjugates in which
extensive cupping occurred.
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A). The
length of the LFA-1 band was typically the same as the length of the
contact site. Although Lck-deficient T cells expressed levels of LFA-1
similar to wild-type cells and were equally bright when visualized
alone, in many of the conjugates formed using these T cells, no LFA-1
band was observed (Fig. 9B). Similar results were obtained
with cells expressing the Lck SH3 domain mutant (Fig. 9, C
and D). In both Lck-deficient T cells and cells expressing
the SH3 domain mutant, a very dim band could sometimes be detected, but
this was also observed in wild-type cells contacting B cells in the
absence of SEE (data not shown), and probably represents
activation-independent accumulation of LFA-1. Interestingly, some
conjugates formed using the Lck mutants showed a relatively bright band
of LFA-1, which was quite short in comparison with control conjugates
(Fig. 9D). This appears to represent a limited recruitment
of LFA-1 to a small contact site, and can apparently occur in the
absence of gross actin remodeling. As observed in the actin studies,
conjugates made using the ZAP-70-deficient T cells were more
heterogeneous than conjugates made using either wild-type T cells or
the Lck mutants. In roughly half of the ZAP-70-deficient T cells,
accumulation of LFA-1 at the contact site was apparent (Fig. 9E), although in many of these labeling was dimmer or more
disorganized than in wild-type conjugates (Fig. 9F). In the
remainder, very little LFA-1 accumulated at the contact site (Fig. 9G), or a short, bright band was formed (Fig. 9H). In general, the actin and LFA-1 phenotype of the Lck
mutants was much more severe than the ZAP-70-deficient cells. This
finding is consistent with the conjugation data, and suggests that Lck
plays a role in LFA-1-dependent adhesion that is somehow distinct from
that played by ZAP-70.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that Lck activity is required for conjugate formation is not surprising, since signaling through Lck is required to initiate all aspects of TCR-mediated signaling (27). However, our results show that activation of ZAP-70, the protein normally thought to lie directly downstream of Lck in the TCR signaling pathway, is dispensable for conjugation. Two pieces of evidence support this conclusion. First, we find that ZAP-70-deficient T cells conjugate as efficiently as wild-type T cells. Second, we find that JCaM1 cells expressing an SH3 domain mutant of Lck are deficient in conjugate formation, even though these cells have been shown to activate ZAP-70 normally (29). These results indicate that Lck participates in LFA-1-dependent conjugate formation by a pathway distinct from simple activation of ZAP-70.
Since binding to plate-bound ligands has been used extensively to analyze later events in the inside-out signaling pathway, we attempted to directly measure the adhesion of the Lck and ZAP-70 mutant T cells to immobilized ICAM-1. Unfortunately, the results were inconclusive because of high variability of the mutants in the ICAM-1-binding assay (data not shown). However, with wild-type cells in which the two assays could be performed in parallel, we found that binding to immobilized ICAM-1 differs significantly from superantigen-induced conjugation to APCs. Specifically, we found that inhibitors of calpain, PI-3 kinase, and MEK, all of which have been reported to inhibit binding to immobilized ICAM-1 (19, 21, 23), had no effect on superantigen-induced conjugation. These findings indicate that T:B conjugate formation is regulated by signaling pathways that are distinct from those involved in adhesion to immobilized ligands. It seems likely that the interaction of multiple receptor-ligand pairs results in the generation of redundancies in key signaling pathways that are not observed in a more simplified assay system. In addition, the generation of adhesive strength during T:B conjugation is likely to involve substantial changes in cytoarchitecture that are qualitatively and/or quantitatively different from those that occur in response to immobilized ligands. For example, we have observed that the T:B interface labels much more strongly for F-actin than does the interface with anti-TCR-coated beads, even though both interactions induce activation of CDC42 (38, 48). This is most likely due to the synergistic effect of multiple receptor-ligand interactions in sculpting the cortical cytoskeleton. Finally, the redistribution of ligand-bound receptors within the immunological synapse, which cannot take place in contacts with immobilized ligands, is likely to contribute to optimal adhesion to APCs. Thus, while the more reductionist approach of studying adhesion to immobilized ligands has been very informative, a full understanding of the factors that contribute to adhesive strength during conjugate formation will require additional analysis in model systems like the one described in this work.
Although analysis of conjugate formation introduces complexities into analyzing the signaling pathways involved, it also has benefits. In particular, it is illuminating to view the morphology of the interacting cells, and the distribution of proteins involved in the mechanics of adhesion. It is well established that signaling-dependent increases in avidity involve the recruitment of LFA-1, tethered to the cortical actin cytoskeleton, to the T cell-APC binding site (2, 15, 16, 49). Our results show that Lck-deficient T cells exhibit dramatic defects in actin remodeling and LFA-1 recruitment in response to B cell binding. In most conjugates formed with Lck-deficient T cells, little or no actin accumulates at the cell-cell contact site, consistent with a failure to activate localized actin polymerization. LFA-1 recruitment also fails to occur. These defects almost certainly affect the avidity of T:B binding, and are likely to account for (or at least contribute to) the adhesion defects in these cells. Importantly, similar defects are also observed in cells expressing the Lck SH3 domain mutant, suggesting that a ZAP-70-independent process is involved. Although cytoskeletal defects were also observed in the ZAP-70-deficient T cells, some remodeling was initiated in most of these cells, and many more achieved a wild-type phenotype. Remarkably, ZAP-70-deficient T cells actually tended to form an abnormally large contact site with the APC, suggesting that the structure of the cortical cytoskeleton was aberrantly organized. Although the nature of the defect in these cells is still unclear, the remodeling that does occur is apparently adequate to allow conjugation at normal levels.
We have begun to characterize the signaling pathways downstream of Lck
that ultimately result in the actin and LFA-1 remodeling required for
conjugate formation. Clues about what pathways may be involved come
from the known defects in T cells expressing the Lck SH3 domain mutant.
Exactly how this mutant perturbs TCR signaling is an area of active
investigation. Many proteins have been shown to interact with the SH3
domain, and the mutation is expected to disrupt these interactions. In
addition, however, Lck activity is regulated by conformational changes,
so that the mutation may perturb enzymatic activity through complex
effects on tertiary structure. One known defect in cells expressing
this mutant is in the activation of MEK (29). Since the
MAPK pathway has been shown to be required for murine T cell adhesion
to immobilized ICAM-1 (21), this pathway was a good
candidate. Using the MEK inhibitor PD98059, we show that MEK and ERK
are unlikely to be involved in the activation of LFA-1; however, this
does not rule out a role for proteins upstream of MAPK. Indeed, our
inhibitor studies suggest that conjugation depends on the activity of
members of the PLC and PKC families. Classical models of TCR signaling
pathways include ZAP-70-dependent activation of PLC1, which leads to
downstream activation of PKC, and PKC has been implicated in integrin
activation in many cell systems (49, 50, 51, 52, 53, 54). We find that
conjugation is independent of ZAP-70 activity, and that conjugation of
ZAP-70-deficient T cells is as sensitive as wild-type cells to the PLC
inhibitor U73122. Thus, the relevant PLC may not be PLC1, but rather
another isoform such as PLC
, which has been shown to have
Ras/guanine exchange factor (GEF) activity and to interact with Ras and
its antagonist, Rap-1 (55, 56). Alternatively, PLC1 may
be activated in a ZAP-70-independent manner under conjugation
conditions. If PLC1 is the relevant isoform, it could influence
adhesion either by activation of PKC, or by activation of the Ras
pathway (57, 58, 59, 60). In support of the latter possibility, we
find that the effects of the PLC inhibitor U73122 on MAPK activation
are much more pronounced when APCs are used to stimulate the T cells.
In addition to showing defects in MAPK signaling, T cells expressing
the Lck SH3 domain mutant are defective in costimulation through CD28
(61). Interactions of proline-rich sequences in CD28 with
the Lck SH3 domain influence Ras activity via the Ras antagonist Rap-1,
and play a role in Vav-1-dependent NF-AT activation (62, 63). Thus, defective cytoskeletal remodeling and poor conjugate
formation could occur as a result of aberrant activation of Vav-1, a
GEF for Rac-1. Intriguingly, overexpression of constitutively active
mutants of both Rap-1 and Rac-1 strongly activates the adhesion of T
cells to immobilized ICAM-1 (64).
Taken together, our results show that Lck plays a key role in regulating cortical cytoskeletal rearrangements and LFA-1-dependent conjugate formation. Our findings indicate that Lck activity is crucial to initiate actin polymerization and remodeling. To the extent that ZAP-70 is also required, it appears to play a role in fine- tuning the organization of actin at the cell-cell contact site. Although additional work will be needed to elucidate the relevant signaling pathways involved, it seems clear that small GTP-binding proteins will play a crucial role. In particular, the activation of Rho family members is likely to direct actin remodeling via effectors such as p21-activated protein kinase and Wiskott-Aldrich syndrome protein. Activation of Rho family members and their effectors in response to TCR engagement has been shown to depend on Lck activity (65, 66, 67), and cells in which Rho proteins are inhibited (68, 69, 70), lacking the Rho GEF Vav (71, 72) or the Rho effector Wiskott-Aldrich syndrome protein (73, 74), all show defects in actin-dependent aspects of T cell function. Although we still know relatively little about the regulation of Rho protein activity under conjugation conditions, our group has found that CDC42 is activated in a Lck-dependent fashion at the site of APC engagement, and that interfering with CDC42 activity perturbs conjugate formation (48). Although it will be interesting to pursue the specific role of these proteins, this analysis will be challenging since these proteins function in complex cascades, and their function is highly location specific. Future analysis of signaling pathways that regulate cytoskeletal remodeling will be important for understanding T cell function, but will require new experimental approaches taking into account the complexities of actual cell-cell interactions.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Environmental Health, Boston University School of Public Health, Boston, MA 02118.
3 Address correspondence and reprint requests to Dr. Janis K. Burkhardt, Department of Pathology, University of Chicago, MC1089, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address: jburkhar{at}uchicago.edu
4 Abbreviations used in this paper: PKC, protein kinase C; CMAC, 7-amino-4-chloromethylcoumarin; ERK, extracellular signal-regulated kinase; GEF, guanine exchange factor; GFP, green fluorescent protein; HE, hydroethidine; LAD, leukocyte adhesion deficiency; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein/ERK kinase; PI-3, phosphatidylinositol-3; PLC, phospholipase C; PTK, protein tyrosine kinase; SEE, staphylococcal enterotoxin E; SF-RPMI, serum-free RPMI 1640; SH, Src homology; Tg, transgenic.
Received for publication January 3, 2001. Accepted for publication September 14, 2001.
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References |
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1 Integrin-mediated lymphocyte adherence to extracellular matrix is enhanced by phorbol ester treatment. Eur. J. Immunol. 21:517.[Medline]
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subunit common to the LFA-1, Mac-1, and p150,95 glycoproteins cause leukocyte adhesion deficiency. Cell 50:193.[Medline]
) or 200 nM calphostin C
(
