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,
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Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Jerusalem, Israel;
Department of Pathology, Case Western Reserve University, Cleveland, OH 44106;
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104
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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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T cells interacting with APCs undergo rearrangement of critical surface receptors, signaling molecules, and cytoskeletal elements, so that these components face the zone of contact with APC (9). This polarization process forms a specialized domain on the T cell surface, also known as an "immune synapse," which serves to increase the amplitude and duration of TCR signaling (10). Following activation, some cell surface molecules are enriched in the central zone of the immune synapse, while large, highly glycosylated molecules and certain phosphatases (such as CD43 and CD45) migrate away from this zone (11, 12, 13).
These dynamic changes in surface molecular topology are thought to be important in sustaining T cell activation after the initial TCR triggering event. However, because this clustering phenomenon is general and involves many molecules linked to cytoskeletal actin (10), it is plausible that certain negative regulators of T cell activation might also localize to contact sites. In view of our previous demonstration that PP14 functions in a unique way at an early step of T cell activation to elevate the TCR activation threshold (8), we hypothesized that PP14 might achieve this inhibitory effect by modulating TCR signaling components within activation clusters. Such an immunoregulatory mechanism, based upon localized interference with TCR signaling within immune synapses, would be distinct from the alternative mechanism that entails negative signaling through independent, dispersed inhibitory receptors.
In this study, we use immunofluorescence microscopy to show that when APC and T cells form conjugates, PP14 translocates to the sites of cell contact. Significantly, the data further indicate that the inhibitory activity of PP14 depends upon its access to the triggered TCR, and leads to decreased stability of TCR-induced phosphorylated proteins. Taken together, these results support a model wherein PP14 attenuates TCR signaling within the signaling unit. Thus, PP14 may represent a new type of soluble regulatory protein that dampens T cell responses by its physical presence in the contact site at the time of TCR triggering.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were purified from the venous blood of healthy donors by density gradient centrifugation as described (7). As a source of APC, monocytes were isolated from mononuclear cell populations by adherence to plastic. PBMC (2 x 106 cells/ml) were incubated in serum-free medium for 1 h at 37°C. The adherent cells were washed extensively with medium to remove any residual nonadherent cells, and fresh complete medium was added. Following a 24-h incubation period, adherent monocytes were removed by gentle scraping with a plastic cell scraper. Cell viability was >80% as determined by trypan blue exclusion. Cells were washed with medium, resuspended at 1 x 107 cells/ml, and then pulsed with the superantigen staphylococcal enterotoxin B (SEB) at the indicated concentrations at 4°C for 2 h. The SEB-pulsed cells were then washed three times with medium, diluted to 1 x 106 cells/ml, and incubated in culture medium at 37°C for 20 min before adding them to cultures.
CD4+ T cells were isolated from the PBMC pool by first depleting monocytes by adherence to tissue culture flasks, as described above, and then further purifying the nonadherent T cells with a magnetic cell isolation system (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were maintained in RPMI medium (Biological Indrustries, Beit-Haemek, Israel) supplemented with 10% heat-inactivated FCS (Biological Industries), 2 mM glutamine, and penicillin/streptomycin. The Jurkat cell line was obtained from the American Type Cell Culture Collection (Manassas, VA).
AF samples and PP14 immunoabsorption
Discarded human AF samples were obtained from the Center for Human Genetics Laboratory at University Hospitals of Cleveland (Cleveland, OH) and stored at -80°C. Samples obtained from several patients (collected at 14–16 wk of gestation) were pooled and filter-sterilized before use. Anti-PP14 polyclonal Ab (4) were coupled to protein A-Sepharose beads (Sigma Aldrich, St. Louis, MO) to generate an immunoabsorbent. Immunoabsorption was conducted by adding AF to Ab-coupled beads and incubating the mixture overnight at 4°C with gentle rotation. The beads were pelleted by centrifugation, and the supernatant was filtered and used in assays as described. The presence of PP14 was verified by Western blotting.
Production of PP14 · Fc
1
The coding sequence for full-length human PP14 (4),
including its signal sequence, was fused to the 5'-end of the coding
sequences for the hinge, CH1 and CH2 domains of human IgG1. This
chimeric sequence was inserted into the EBV episomal expression vector
pIgG/REP7
(14), generating
pPP14 · Fc1/REP7. Stable 293 cell
(ATCC) transfectants secreting PP14 · Fc1
were grown in Ultraculture (BioWhittaker, Walkersville, MD)
supplemented with hygromycin B (200 µg/ml; Calbiochem, La Jolla, CA)
at 37°C with 5% CO2. To purify the protein,
conditioned media were mixed with equal volume of binding buffer (3.0 M
sodium chloride/1.5 M glycine, pH 8.6). Protein A-Sepharose (Sigma
Aldrich) was added, and the suspension was mixed overnight at 4°C.
The matrix was collected and washed with 10-column volumes of 100 mM
citrate, pH 5.0. PP14 · Fc1 was eluted
with 100 mM citrate, pH 3.0 into 0.4 volumes of 0.2 M sodium phosphate
(dibasic). The purified protein was concentrated and buffer-exchanged
into PBS using Centricon 10 (Millipore, Bedford, MA).
Flow cytometry
T cells were incubated with 2 µg/ml of either
PP14 · Fc1 or
CTLA-4 · Fc1 (R&D Systems, Minneapolis,
MN) for 30 min at 37°C, and then methanol fixed. Fixed cells were
labeled with PE-conjugated F(ab)2 fragment goat
anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove,
PA). A total of 1 x 104 cells/sample were
analyzed on a FACSCaliber flow cytometer (BD Biosciences, Mountain
View, CA) using CellQuest software.
Conjugate formation and immunofluorescence microscopy
CD4+ T cells were mixed at a 1:1 cell
ratio with autologous monocytes that had been pulsed with SEB (1
µg/ml) in the presence of 2 µg/ml
PP14 · Fc1. This relatively low
concentration of PP14 · Fc1 was chosen to
optimize the visualization of PP14 at the cell surface as a ring of
fluorescence. Of note, significantly higher concentrations (20–40
µg/ml) of PP14 are generally needed for inhibiting T cells in
standard assays, including decreasing conjugate formation (data not
shown). After centrifugation for 5 min at 100 x g, the
cells were incubated for an additional 5–90 min at 37°C. Conjugates
were then resuspended and fixed with methanol. Following rehydration,
PP14 · Fc1 was detected using
Cy5-conjugated F(ab)2 fragment goat
anti-human IgG (Jackson ImmunoResearch Laboratories). Images were
obtained using an LSM confocal laser scanning system attached to a
Zeiss Axiovert 135 M inverted microscope (Zeiss, Oberkochen, Germany)
with 100/1.3 plan-Neofluor lens, with emission spectra at 633-nm line
and RG655 emission filter.
Cell stimulation and measurement of cytokine production
Jurkat cells (5 x 105/well in 1 ml
volume) were cultured in individual wells of 24-well culture plates,
and were stimulated with PHA or immobilized anti-CD3 plus soluble
anti-CD28 mAb for 20 h.
PP14 · Fc1 was added either in soluble
form, or immobilized on protein A beads, or on beads previously coated
with anti-CD3 mAb. Butanedione monoxide (BDM; Calbiochem) was added
to a final concentration of 20 µM, because this concentration does
not significantly inhibit IL-2 secretion by Jurkat cells. IL-2 levels
in the conditioned media were assayed by ELISA (Genzyme,
Cambridge, MA).
Cell stimulation and assessment of tyrosine phosphorylation
For tyrosine phosphorylation analysis, 2 x 106 Jurkat cells were stimulated with anti-CD3 mAb (2 µg/ml) for 5 min at 37°C in 0.5 ml culture medium. In other experiments, protein A-Sepharose beads were sequentially coated with anti-CD3 mAb (OKT3 at 10 µg/ml) followed by anti-CD28 mAb (clone 9.3 at 10 µg/ml). These beads were combined with Jurkat cells and centrifuged, incubated for 5 min at 37°C, and then the selective inhibitor of the src family tyrosine kinases PP2 (10 µM; Calbiochem) was added for varying incubation periods. Harvested cells were lysed for 30 min on ice in Nonidet P-40 lysis buffer (15). Western immunoblotting was performed as described (7) using the antiphosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 that
was produced in a glycosylated form by a mammalian expression system.
The binding of PP14 · Fc1 to the T cell
surface was detected by flow cytometry using anti-human Ig.
PP14 · Fc1, but not
CTLA-4 · Fc1 (as a negative control for
nonspecific Fc-mediated interactions), bound to purified
CD4+ T cells, establishing the presence of a
surface receptor for PP14. This binding was specific, because it could
be abrogated by preincubating the cells with AF as a rich source of
native PP14 for competitive blockade (Fig. 1
A, left). Similar
results were obtained with Jurkat cells (Fig. 1A,
right); receptor binding was also documented for these cells
using radiolabeled 125I-labeled
PP14 · Fc1 as a probe (data not shown).
Thus, specific binding of PP14 to T cells can be detected.
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Bb), it was markedly enriched within the interaction
sites of APC-T cell conjugates (Fig. 1Bd). A high density of
PP14 epitopes was observed in the APC-T cell contact sites of 67% ±
11.6 (±SD; from three separate experiments) of those T cells forming
conjugates, which comprised 10–20% of the total T cells added to the
assay. Interestingly, the enrichment of PP14 in contact sites was
observed as early as 5 min following conjugate formation. In the
absence of Ag, few cell conjugates were formed; none of them clustered
talin (data not shown; Refs. 12 and 18), and
PP14 staining was uniformly distributed on the T cell surface, as seen
in the unbound T cells in Fig. 1Bb. These data demonstrate
that in conjunction with APC-T cell conjugate formation, cell surface
PP14 is polarized toward the contact site.
In parallel experiments, we tested whether PP14 can inhibit T cell
activation when the T cells are stimulated under the conditions that
allowed demonstration of the translocation of PP14 to contact sites. T
cells were induced to secrete IL-2 by mixing them with SEB-pulsed
monocytes, as in Fig. 1B, in the absence or presence of
PP14 · Fc1 or first-trimester AF, which
was used as a natural source of PP14. Both
PP14 · Fc1 and AF inhibited IL-2 secretion
by the T cells (Fig. 2A). The
AF inhibitory activity was PP14-dependent because it could be abrogated
by depleting the AF of its PP14. Similar results were obtained when T
cells or Jurkats were stimulated with PHA instead of SEB (Fig. 2B).
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; Refs. 7 and 8). For this experiment,
AF was either combined with PHA in the same chamber, or alternatively,
the AF and PHA were added on opposite sides of the Jurkat-sealed
membranes. AF-mediated inhibition of IL-2 secretion by Jurkat cells was
evident only when the AF and PHA (at concentration of both 1 and 2
µg/ml) were added to the same chamber (Fig. 3A). Hence, under activation
conditions that maximize the generation of T cell polarity, the PP14
inhibitor and the PHA activator must be in physical proximity to each
other.
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1
fusion protein as inhibitor, to either the same or separate protein A
precoated beads. Significantly, potent T cell inhibitory activity, as
measured by decreases in IL-2 secretion from Jurkats, was observed
when anti-CD3 mAb and PP14 · Fc1 were
colocalized on the same beads (Fig. 3B). The level of
inhibition associated with the immobilized
PP14 · Fc1 was considerably greater than
that for the soluble form. In contrast, no inhibition was observed when
anti-CD3 mAb and PP14 · Fc1 were
anchored on separate beads (Fig. 3B), conditions under which
PP14 · Fc1 cannot readily access triggered
TCRs. These findings provide additional support for the dependence of
PP14-mediated T cell inhibition on the proximity of PP14 to the
triggered TCR.
Clustering of receptors at contact sites is actively driven by the T
cell cytoskeleton and depends upon myosin motor proteins
(10). Although the inhibition of the myosin motors with
BDM interferes with the activation of naive T cells (Ref.
10 and data not shown), in our hands, a low concentration
of BDM (20 µM) did not significantly inhibit the capacity of Jurkat
cells to secrete IL-2. However, this concentration of BDM significantly
interfered with AF-mediated inhibition of IL-2 secretion from Jurkats
triggered with either PHA or immobilized anti-CD3 (Fig. 3C). Several additional BDM findings supported the notion
that the inhibitory effect of PP14 requires cytoskeleton-dependent PP14
transit to surface sites where TCR triggering transpires. First, BDM’s
interference with AF-mediated inhibition could be attenuated by adding
more AF (data not shown). Second, as expected, BDM treatment had no
effect on inhibitory activity when
PP14 · Fc1 and anti-CD3 mAb were
coimmobilized on the same beads (Fig. 3C). In this setting,
the need for myosin motor proteins to drive colocalization is obviated.
Third, similar results were obtained when taxol, an antitumor agent
that binds -tubulin and locks the microtubule apparatus, was
substituted for BDM (Fig. 3D). Thus, our data indicate that
PP14 requires access to the triggered TCR site to implement its
inhibitory effects on T cell activation.
Active accumulation of molecules at the interface of the T cell and the
APC serves to increase the overall amplitude and sustain T cell
signaling (10). Given the evidence that PP14 targets
proximal events during TCR signaling (7), the accumulation
of PP14 in the contact site could result in reduced levels of receptor
signaling. Therefore, the effect of PP14 on tyrosine phosphorylation,
an essential element in early T cell activation, was evaluated. Jurkats
were stimulated with soluble anti-CD3 mAb, and the impact of AF on
the induction of tyrosine phosphorylation events was assessed using
antiphosphotyrosine mAb. Although AF did not significantly perturb the
overall pattern of tyrosine phosphorylation in these cells, some
differences in band intensities were noted (Fig. 4A).
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B). After the addition
of PP2, tyrosine phosphorylation persisted for >1 min, as previously
reported (20). In contrast, in the presence of AF,
accelerated substrate dephosphorylation was elicited for multiple
molecular species (Fig. 4, B and C). There is
some degree of specificity in this dephosphorylation effect, given that
phosphorylation of the 70-kDa band is retained despite AF treatment
(Fig. 4B). Interestingly, this same 70-kDa band appears to
be the only one that persists when resting T cells are triggered with
anti-CD3 mAb in the absence of costimulation (20). In
aggregate, these results suggest that PP14 functions within sites of
TCR triggering, where it reduces the half-life of TCR-induced tyrosine
phosphates by augmenting their dephosphorylation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The confocal microscopic analysis, focusing on PP14’s transit to the APC-T cell interface, as well as the dual-chamber bead coimmobilization and cytoskeletal inhibitor experiments, together raise the intriguing possibility that an exogenously added soluble inhibitor (such as PP14) may function by transiting to the immune synapse. Hence, not only proteins known to promote activation, but also inhibitory proteins may segregate into functional sites at the APC-T cell interface. This broader effect on multiple membrane proteins is a consequence of the nonspecific nature of cytoskeleton-driven molecular clustering at the T cell surface (10). It is likely that PP14’s translocation to the contact site is an early event during activation of the T cell because a significant PP14 accumulation in the contact site is already seen by 5 min following stimulation.
The explanation for the inhibitory effect of PP14 on TCR signaling is supported by our demonstration that it reduces the half-life of TCR-induced tyrosine phosphates via augmenting their dephosphorylation. In view of these findings, it is tempting to speculate that PP14 exerts its immunoregulatory effects on T cells by entering contact sites and altering the organization of the immune synapse. Activation of T cells by APC is restricted to their site of contact, where receptors on the T cells engage their counter receptors on the APCs. The formation of the immune synapse serves to facilitate and stabilize TCR signaling (10, 20), probably by excluding phosphatases from the contact site. The expected outcome of PP14’s recruitment to the contact site might be to physically disrupt this functional site and to alter the balance between protein tyrosine kinases and phosphatases within them, thereby negatively regulating proximal signaling processes. This type of contact site-directed inhibitory mechanism may be relevant to other negative T cell regulators as well, such as galectin-3 (21).
This model unifies positive and negative signals within a single mechanistic framework, whereby the immune synapse functions to dynamically integrate positive and negative inputs into a finely tuned T cell response. Systematic confocal microscopic analysis of the effect of PP14 on the segregation of various molecules during contact site formation, as well as its effect on the duration of TCR-evoked signaling, should serve to substantiate the proposed model for the mode of action of PP14.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark L. Tykocinski, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 6 Gates Building, 3400 Spruce Street, Philadelphia, PA 19104-4283. E-mail address: mlt4{at}mail.med.upenn.edu
3 Abbreviations used in this paper: PP14, placental protein 14; AF, amniotic fluid; SEB, staphylococcal enterotoxin B; BDM, butanedione monoxide.
Received for publication June 6, 2001. Accepted for publication January 14, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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); AF-treated cells (
).