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Originally published In Press as doi:10.1074/jbc.M211716200 on January 29, 2003

J. Biol. Chem., Vol. 278, Issue 16, 14059-14065, April 18, 2003
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Negative Regulation of T Cell Activation by Placental Protein 14 Is Mediated by the Tyrosine Phosphatase Receptor CD45*

Jacob RachmilewitzDagger §, Zipora BorovskyDagger , Gregory J. Riely, Robin Miller, and Mark L. Tykocinski||

From the Dagger  Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Jerusalem 91120, Israel, the  Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, and the || Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication, November 18, 2002, and in revised form, January 27, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CD45 is the major protein tyrosine phosphatase receptor on T cell surfaces that functions as both a positive and a negative regulator of T cell receptor (TCR) signaling. Although CD45 is required for the activation of TCR-associated Src family kinases, it also dephosphorylates phosphoproteins involved in the TCR-signaling cascade. This study links CD45 to the inhibitory activity of placental protein 14 (PP14), a major soluble protein of pregnancy that is now known to be a direct modulator of T cells and to function by desensitizing TCR signaling. PP14 and CD45 co-capped with each other, pointing to a physical linkage between the two. Interestingly, however, the binding of PP14 to T cell surfaces was not restricted to CD45 alone, with evidence showing that PP14 binds to other surface molecules in a carbohydrate-dependent fashion. Notwithstanding the broader molecular binding potential of PP14, its interaction with CD45 appeared to have special functional significance. Using transfected derivatives of the HPB.ALL mutant T cell line that differ in CD45 expression, we established that the inhibitory effects of PP14 are dependent upon the expression of intact CD45 on T cell surfaces. Based upon these findings, we propose a new immunoregulatory model for PP14, wherein one of its surface molecular targets, CD45, mediates its T cell inhibitory activity, accounting for the intriguing capacity of PP14 to elevate TCR activation thresholds and thereby down-regulate T cell activation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CD45, the major tyrosine phosphatase on T cell surfaces, promotes T cell activation by maintaining the tyrosine kinase Lck in its active form (1). However, CD45's effects on the T cell activation process are, in fact, more complex, because it may also tonically inhibit T cell receptor (TCR)1 triggering by dephosphorylating key tyrosine substrates in the TCR activation pathway. This bi-functional view of CD45 is supported by the following series of findings. 1) CD45 associates with the TCR and its signaling apparatus in resting T cells (2-5). 2) TCR occupancy leads to a progressive inhibition of CD4-associated CD45 phosphatase activity and an increase in CD4-associated Lck activity (6). 3) The tyrosine phosphatase inhibitor pervanadate activates TCR signal transduction on its own in the absence of direct TCR triggering (7, 8). 4) Ab-mediated co-cross-linking of CD45 with various surface T cell molecules, such as CD3, CD4, and CD2, can alternatively enhance or inhibit TCR-triggered T cell activation (9-15). 5) CD45 negatively regulates cytokine receptor signaling by dephosphorylating the Janus kinase (JAK) (16, 17); and 6) CD45 is excluded from the TCR-signaling area in stimulated T cells (18, 19). Together, these various findings support a two-phase model for CD45 function wherein it is essential for T cell activation early on, but, subsequently, it is sequestered away from some of its tyrosine substrates within the antigen-presenting cell, T cell contact site, or immune synapse in order to enable sustained TCR signaling (10, 20).

CD45 exists in multiple isoforms, which arise through alternative mRNA splicing (21, 22) and differential glycosylation (23). The CD45 cytoplasmic domain, which is shared among them, mediates the protein's phosphatase activity. This domain can be regulated by artificial means like, for example, using Ab directed against various extracellular CD45 epitopes (24) or replacing the extracellular and transmembrane domains of CD45 with those of the epidermal growth factor (EGF) receptor (EGFR) and then stimulating the resulting chimeric protein with the cognate ligand, EGF (25). Although uncertainty continues to surround the precise identities and roles of the natural ligands of CD45, two especially interesting candidates have emerged. The human B cell adhesion molecule CD22, a sialic acid-binding lectin, binds to the CD45RO isoform (26) and modulates early TCR signaling through ligation of the CD45 extracellular domain (27). Galectin-1, another lectin, also binds to CD45 and, in so doing, induces apoptosis of Jurkat T cells (28-30). Interestingly, both CD22 and galectin-1, as lectins, bind to multiple other non-CD45 glycoproteins as well.

Placental protein 14 (PP14; progesterone-associated endometrial protein; glycodelin) is a 28-kDa glycoprotein of the lipocalin structural superfamily with documented immunoinhibitory properties (31-34). This glycoprotein is expressed by cells of the female and male reproductive tracts (35, 36) as well as by platelets (34), and it is present at high levels in amniotic fluid (AF) and maternal serum (35). We have reported that PP14 directly inhibits human T cells and accounts for the T cell inhibitory activity of AF (37). Our findings further suggested that PP14 targets early events during TCR signal transduction (37), facilitates the dephosphorylation of TCR-induced phosphoproteins, (38), and has the intriguing capacity to elevate TCR activation thresholds (39). This latter finding points to an unusual immunoregulatory mechanism for PP14 that is distinct from that of other better characterized T cell suppressive factors (such as cyclosporin A).

Considering the possibility that CD45 might explain, at least in part, the effects of PP14 on the dephosphorylation of TCR-induced phosphoproteins and TCR activation thresholds, we proposed a connection between PP14 and CD45. Our data now establish the existence of both physical and functional links between the two, with a clear demonstration that CD45 is required for PP14-mediated immunoinhibition. Furthermore, this study offers an unexpected insight into the nature of PP14 binding to T cells, suggesting that PP14 does not interact with a single discrete surface receptor on T cells but rather with multiple surface molecules in a lectin-like fashion. Among these receptors, the abundant CD45 molecule appears to be, at least from the immunological standpoint, one of the most functionally significant targets of PP14. In this context, an interesting parallel emerges between PP14 and the lectins CD22 and galectin-1, whose immunoregulatory activities have been similarly linked to CD45. Based on our findings and the galectin-1 parallel in particular, we propose a T cell regulation model wherein PP14 interferes with the usual post-triggering sequestration of the CD45 phosphatase away from its critical substrates and thereby attenuates TCR signaling via an alteration of the local balance between tyrosine kinases and phosphatases.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells-- Peripheral blood mononuclear cells (PBMCs) were purified from the venous blood of healthy donors by density gradient centrifugation, as described (37). CD4+ T cells were isolated from the PBMC pool by first depleting monocytes via adherence to tissue culture flasks and then further purifying the non-adherent T cells with a magnetic cell isolation system (Milteny Biotec, Bergisch, Germany). The cells were maintained in RPMI 1640 medium (Biological Industries, Beit-Haemek, Israel) and supplemented with 10% heat-inactivated fetal calf serum (Biological Industries), 2 mM glutamine, and penicillin/ streptomycin.

The Jurkat and the derivative J45 cell lines were obtained from the American Type Cell Culture Collection (ATCC, Manassas, VA). The cells were maintained in the above medium. H45L13 and H45XL2 transfectants were provided by Dr. A. Weiss (University of California, San Francisco, CA) and maintained in the above medium supplemented with 2 mg/ml Geneticin (Invitrogen).

AF Samples and PP14 Immunoabsorption-- Discarded human AF samples were obtained from the Center for Human Genetics Laboratory at Hadassah Hospital and stored at -80 °C. Samples obtained from several patients (collected at 14-16 weeks of gestation) were pooled and filter sterilized before use. Anti-PP14 polyclonal Abs (34) were coupled to protein A-Sepharose beads (Sigma) to generate an immunoabsorbent. Immunoabsorption was carried out 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·Fcgamma 1-- The coding sequence for full-length human PP14 (34), including its signal sequence, was fused in-frame to the coding sequences for the hinge, i.e. the CH1 and CH2 domains of human IgG1. This chimeric sequence was inserted into the Epstein-Barr virus episomal expression vector pIgG/REP7beta (40), generating pPP14·Fcgamma 1/REP7beta . Stable 293 cell (ATCC) transfectants secreting PP14·Fcgamma 1 were grown in Ultraculture (Whittaker Bioproducts, 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 an equal volume of binding buffer (3.0 M sodium chloride/1.5 M glycine, pH 8.6). Protein A-Sepharose (Sigma) was added, and the suspension was mixed overnight at 4 °C. The matrix was collected and washed with 10-column volumes of 100 mM Tris/150 mM NaCl, pH 9.0. PP14·Fcgamma 1 was eluted with 100 mM Tris/150 mM NaCl, pH 12.0 into 1.4 volumes of 300 mM citric acid, pH 6.0. The purified protein was concentrated and buffer exchanged into phosphate-buffered saline using Centricon-10 filters (Millipore, Bedford, MA).

Fluorescence Co-localization-- Co-localization of PP14 and CD45 was assessed in co-capping experiments. CD4+ T cells were incubated with either 2 µg/ml PP14·Fcgamma 1 or 2 µg/ml CTLA-4·Fcgamma 1 (R&D Systems, Minneapolis, MN) in the presence or absence of 10 µg/ml F(ab)2 fragment goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA). The cells were then fixed with methanol and, following rehydration, CD45 was detected with pan-specific anti-CD45 mAb (GAP8.3; ATCC) and Alexa-488-conjugated F(ab)2 fragment goat anti-mouse IgG (Molecular Probes); PP14·Fcgamma 1 was detected using Cy5-conjugated goat anti-human IgG (Jackson ImmunoResearch). Control cells (labeled with Alexa-488- or Cy5-conjugated Ab alone) were always prepared simultaneously for each experiment. Images were obtained using an LSM confocal laser scanning system attached to a Zeiss Axiovert 135M inverted microscope with a 100/1.3 plan-Neofluor lens. Cells were scanned by dual excitation of Alexa-488 (green) and Cy5 (red) fluorescence, with green and red overlapping fluorescence detected as a yellow signal.

T Cell Staining with PP14·Fcgamma 1-- T cells were incubated with 2 µg/ml of either PP14·Fcgamma 1 or CTLA-4·Fcgamma 1 (R&D Systems) for 30 min at 37 °C, and the cells were then methanol fixed. After rehydration, the fixed cells were labeled with phosphatidylethanolamine-conjugated F(ab)2 fragment goat anti-human IgG (Jackson ImmunoResearch). 1 × 104 cells per sample were analyzed on a FACScalibur flow cytometer (BD Biosciences) using Cell Quest software.

Measurement of Intracellular Calcium-- Each cell type was loaded with 3 mM Indo-1 (Molecular Probes, Eugene, OR), according to the manufacturer's protocol. Cells (3 × 106 per ml) were stimulated with anti-CD3 mAb (OKT3; 1:1000 dilution of ascites) at 37 °C. Fluorescence emission was measured by a fluorescence-activated cell sorter (Beckman Coulter, Miami, FL).

Flow Cytometry-- H45L13 and H45XL2 cells were stimulated for 48 h with anti-CD3 mAb immobilized on protein A-Sepharose beads in combination with soluble anti-CD28 mAb in the absence or presence of either AF or PP14·Fcgamma 1. CD69 and CD40L expression were measured by direct immunofluorescence using fluorescein isothiocyanate-conjugated anti-CD69 and anti-CD40L mAb (Pharmingen), respectively, and the immunostained cells (1 × 104 cells/sample) were analyzed on a FACScan flow cytometer (BD Biosciences) using Cell Quest software. The data were calculated as the percentage of positive cells in the cell populations.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Costimulation through T cell surface CD28 increases the stability of phosphorylated proteins (41), thereby amplifying TCR signaling and facilitating T cell activation. In light of our findings that B7-1-driven, CD28-mediated costimulation counteracts PP14 inhibition (39) and that PP14 promotes the dephosphorylation of TCR-induced phosphotyrosines (38), we hypothesized that PP14 may function by promoting the activity of one or more protein tyrosine phosphatases. CD45 was a transmembrane protein tyrosine phosphatase of special interest in this regard, given that it contributes up to 80% of the tyrosine phosphatase activity in T cell membranes (25).

As a first step, we performed fluorescence co-capping experiments to look for CD45 surface aggregation in response to the cross-linking of bound PP14·Fcgamma 1. Purified CD4+ T cells were sequentially treated with PP14·Fcgamma 1 followed by goat anti-human Ig as a cross-linking agent, and, in turn, CD45 epitopes were visualized by indirect immunofluorescence and confocal microscopy, using anti-CD45 mAb as primary Ab. Representative co-capping data are shown in Fig. 1. Whereas CD45 was uniformly distributed over the surfaces of T cells not exposed to Fcgamma 1 fusion proteins and cross-linking Ab, it segregated into large aggregates, co-localizing with PP14 epitopes on T cells pre-treated with the PP14·Fcgamma 1/cross-linking Ab combination. Because an irrelevant Fcgamma 1 fusion protein control, CTLA-4·Fcgamma 1, was substituted for PP14·Fcgamma 1 in this instance, an aggregation of surface-associated CTLA-4·Fcgamma 1 (presumably bound via Fc receptors) could be visualized (albeit requiring a significantly higher red fluorescence gain for detection than that used for PP14·Fcgamma 1), but, importantly, no co-aggregation of CD45 was evident. Thus, the co-capping approach provided evidence for an association between CD45 and PP14 at the T cell surface, although we do not rule out the possibility of an indirect association between the two.


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  		Fig. 1.   CD45 co-caps with PP14·Fcgamma 1. Either PP14·Fcgamma 1 (top panels), CTLA-4·Fcgamma 1 (middle panels), or no Fc fusion protein (bottom panels) was combined with purified CD4+ T cells, and the resultant combination was induced to cap using anti-human IgG for cross-linking. Association with CD45 was demonstrated using a pan-specific anti-CD45 mAb. Dual color fluorescent analysis was performed with detection by confocal microscopy, with green representing CD45, red representing the Fcgamma 1 fusion protein, and yellow indicating areas of overlap. On the left panels, a single cell middle section (for each pre-treatment) is shown. Arrows indicate CD45 aggregates. On the right panels, three-dimensional reconstructions of four representative pseudocolor images for each pre-treatment condition are shown.

Following up on these co-capping data, we turned to flow cytometry as a more quantitative tool for tracking PP14 binding events. First, we documented PP14·Fcgamma 1 binding to purified CD4+ T cells using phosphatidylethanolamine-conjugated anti-human Ig as a detecting reagent (Fig. 2A). PP14·Fcgamma 1 binding was substantially greater than that for CTLA-4·Fcgamma 1, which served as a negative control for nonspecific receptor-dependent Fcgamma 1 binding. The specificity of this binding was established by further showing that it could be competitively blocked by pre-incubating the cells with AF, used as a rich source of native PP14 (Fig. 2A).


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  		Fig. 2.   PP14·Fcgamma 1 binds to Jurkat and HPB.ALL cells and their CD45-deficient variants J45 and H45 cells. A, purified CD4+ T cells were incubated with either PP14·Fcgamma 1 or CTLA-4·Fcgamma 1 (for nonspecific Fc receptor binding) for 30 min at 37 °C. For purposes of competitive inhibition of PP14·Fcgamma 1 binding, cells were pre-incubated with AF (50%, v/v), for 20 min at 37 °C prior to the addition of PP14·Fcgamma 1. Cells were prepared and labeled as described under "Experimental Procedures," and 2 × 104 cells were analyzed by flow cytometry to detect bound protein. B, Jurkat (left panels) or J45 (right panels) cells were incubated with either PP14·Fcgamma 1 (solid lines) or CTLA-4·Fcgamma 1 (dashed lines) for 30 min at 37 °C and then processed for detection of bound PP14·Fcgamma 1 (upper panels) as described for panel A. The levels of cell surface CD45 expression on Jurkat and J45 cells were verified with pan-specific anti-CD45 mAb (GAP8.3) (bottom panels, solid lines); the dashed lines represent staining with control reagent. C, Jurkat or J45 cells were incubated with either 125I-PP14·Fcgamma 1 (open bars) or 125I-CTLA-4·Fcgamma 1 (solid bars) for 3 h at 37 °C. For purposes of competitive inhibition, cells were pre-incubated with AF (30%, v/v) for 3 h at 37 °C prior to the addition of 125I-PP14·Fcgamma 1. Data shown represent the mean values for three experiments. D, the levels of PP14·Fcgamma 1 binding (top panels) and CD45 expression (bottom panels) in HPB.ALL (left panels) or H45 (right panels) cells were determined as for panel B; the dashed lines represent staining with control reagent.

Building upon this ability to visualize bound PP14·Fcgamma 1 by flow cytometry, we next compared the binding of PP14·Fcgamma 1 to Jurkat T cells, which endogenously express CD45, and J45, a CD45-deficient derivative cell line. Substantial binding of PP14·Fcgamma 1 (as compared with CTLA-4·Fcgamma 1) to Jurkat cells was readily detected (Fig. 2B, top left panel). Surprisingly, there was comparable binding of PP14·Fcgamma 1 to the J45-derivative cells (Fig. 2B, top right panel), despite the cells expressing substantially lower levels of surface CD45 (Fig. 2B, bottom panels). As was the case with purified CD4+ T cells, pre-incubating either of the cell lines with AF competitively blocked the binding of PP14·Fcgamma 1.2

Similar results were obtained when radiolabeled 125I-PP14·Fcgamma 1 was used as a more sensitive probe, with comparable binding to both Jurkat and J45 cells (Fig. 2C). Because no chemical cross-linking was required for detecting bound PP14·Fcgamma 1, this sensitive experimental approach additionally served to rule out any artifacts that could arise from chemical cross-linking. Thus, PP14 clearly binds to more than CD45. We further confirmed these results by comparing the binding of PP14·Fcgamma 1 to the HPB.ALL T cell line and its derivative H45, which were used as true CD45-negative cells (Fig. 2D). Once more, similar levels of bound PP14·Fcgamma 1 were detected in HPB.ALL and H45 cells (Fig. 2D, top panel) despite the absence of CD45 expression in the latter (Fig. 2D, bottom panel).

The observation that PP14 binds to more than CD45 on T cell surfaces suggested a parallel to two other immunomodulatory proteins, CD22 and galectin-1, that mediate their functional effects through CD45, and, additionally, as lectins, bind to multiple surface glycoproteins. Therefore, we asked whether PP14 binding to T cell surfaces may be similarly dependent upon carbohydrate interactions. To this end, we tested the ability of several free carbohydrates to competitively block the interaction of PP14·Fcgamma 1 with T cells. The addition of 1 mg/ml asialofetuin, which contains terminal non-reducing Gal residues (42), abrogated PP14·Fcgamma 1 binding to T cells (Fig. 3) as well as to J45 and H45 cell lines that are deficient in CD45 expression.2 In contrast, lactose and cellobiose (Glcbeta 1-4Glc) (which is identical to lactose except for the equatorial orientation of the 4-hydroxyl groups on the non-reducing Glc), both at a concentration of 60 mM, had substantially less effect on the binding of PP14·Fcgamma 1 to T cells (Fig. 3). This competitive inhibition with free carbohydrates is consistent with a carbohydrate-dependent interaction between PP14 and various glycoproteins at the cell surface, including CD45.


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  		Fig. 3.   PP14·Fcgamma 1 binding to T cells can be competitively inhibited with oligosaccharides. Purified CD4+ T cells were incubated with either PP14·Fcgamma 1 or CTLA-4·Fcgamma 1 for 30 min at 37 °C. For purposes of competitive inhibition, cells were pre-incubated with either lactose (60 mM), cellobiose (60 mM) or Asialofetuin (1 mg/ml) prior to the addition of PP14·Fcgamma 1. Cell preparation and labeling was as described in the Fig. 2 legend.

With evidence in hand that PP14 binds to both CD45 and other non-CD45 cell surface molecules, we next turned to function, assessing whether CD45 has a special role in mediating the T cell inhibitory activities of PP14. Although CD45 is usually thought of in terms of its contributions to initial events in T cell activation, some have suggested, based upon its phosphatase activity and expected steric effects, that sustained co-localization of CD45 with the TCR complex may in fact be inhibitory to TCR signal transduction (20). Supporting this idea is the finding that Ab-mediated co-cross-linking of CD45 and TCR suppresses receptor signaling and T cell activation (11, 12, 15). Furthermore, because the extracellular and transmembrane domains of CD45 are known to regulate the phosphatase activity of its intracellular domain (25), we reasoned that the former two CD45 domains might play a role in PP14-mediated inhibition of TCR signaling.

To test this hypothesis, we employed two transfected derivatives of the CD45-deficient HPB.ALL mutant T cell line, i.e. H45L13 (expressing wild-type CD45) and H45XL2 (expressing a recombinant EGFR·CD45 fusion protein composed of the extracellular and transmembrane domains of EGFR and the CD45 intracellular domain) (25). This paired set of H45L13/H45XL2 transfectants has been used previously to demonstrate that a chimeric EGFR·CD45 retains the capacity of intact CD45 to restore TCR-mediated signaling in CD45-deficient cells (25). We first examined the differential effects of PP14 on calcium fluxes for this cellular pair. TCR stimulation induces protein tyrosine kinase activity followed by an increase in intracellular free calcium, both of which are dependent upon the surface expression of CD45 (43, 44). TCR stimulation of both transfectants with anti-CD3 mAb resulted in an increase in intracellular free calcium (Fig. 4A). Whereas anti-CD45 mAb (GAP8.3) treatment inhibited calcium flux in anti-CD3 mAb-stimulated H45L13, it did not do so in anti-CD3 mAb-stimulated H45XL2 (EGFR:CD45) cells,2 validating the experimental procedure. Significantly, the addition of AF (used as an abundant source of PP14) to H45L13 cells (expressing the wild-type CD45) substantially inhibited TCR-induced calcium mobilization. In contrast, there was only a minor decrease in the calcium response of H45XL2 cells (expressing the EGFR·CD45 chimeric protein) despite the fact that the concentration of AF used (50%) was significantly higher than that required for the inhibition of T cell function (10-25%) (Fig. 4A). Of note, we have demonstrated previously that the immunoinhibitory activities of AF are attributable to PP14 (37). In accord with these findings, AF that was immunodepleted of its PP14 demonstrated a significantly reduced capacity to inhibit calcium fluxes (Fig. 4A). The minor inhibitory activity that is still observed in this experiment can be attributed to the residual amounts of PP14 present in the immuno-depleted AF (37), which was used here at a relatively high concentration (i.e. 50%).


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  		Fig. 4.   Effects of PP14 on T cell calcium fluxes, surface activation marker expression, and bead conjugation require the presence of intact CD45. A, H45L13 (CD45-expressing) and H45XL2 (EGFR·CD45-expressing) cells cultured without or with either AF (50%, v/v) or AF that had been subjected to pre-clearing with anti-PP14 Ab were induced with anti-CD3 mAb (OKT3 ascites, 1:1000). Intracellular free calcium levels were measured using the calcium-sensitive dye Indo-1. B, H45L13 and H45XL2 cells were stimulated with solid-phase anti-CD3 mAb and soluble anti-CD28 mAb in the absence or presence of AF (50%, v/v). After 48 h, cells were harvested, and the expression of CD40L and CD69 were detected separately by immunofluorescence and flow cytometry. The data were calculated as the percentage of positive cells in the cell populations. Comparable results were obtained in four experiments. C, H45L13 and H45XL2 cells were stimulated as for panel B in the absence or presence of PP14·Fcgamma 1, and the percentage of CD69 positive cells was determined after 48 h. Results are presented as the percentage of inhibition of CD69-expressing cells. Comparable results were obtained in three experiments. D, photomicrographs are shown of the H45L13 (a and b) and H45XL2 (c and d) cells in panel C (L13 and XL2, respectively) conjugated to the stimulating anti-CD3 mAb-coated beads 24 h after stimulation in the absence (a and c) or presence (b and d) of PP14·Fcgamma 1.

As another functional readout, we examined the expression of two T cell surface activation markers, CD69 and CD40L. Immobilized anti-CD3 mAbs, in combination with soluble anti-CD28 mAbs, were used to trigger H45L13 and H45XL2 cells, and the expression of CD69 and CD40L was determined by direct immunofluorescence and flow cytometry. AF significantly reduced the number of H45L13 cells expressing CD69 and CD40L but had little effect on the percentage of H45XL2 (EGFR·CD45) cells expressing these activation markers (Fig. 4B). To verify that the differential AF effects on the cell lines are indeed attributable to its PP14, PP14·Fcgamma 1 was used in an analogous experiment. We have previously demonstrated that PP14·Fcgamma 1 inhibits interleukin-2 secretion from phytohemagglutinin (PHA)-induced Jurkat and T cells, interleukin-2 secretion from T cells stimulated with superantigen-pulsed monocytes (38), and proliferation of OKT3-induced T cells (39). The pattern and extent of inhibition by PP14·Fcgamma 1 is similar to that seen with AF containing comparable amounts of PP14. As shown in Fig. 4C, the addition of PP14·Fcgamma 1 to cells stimulated with anti-CD3 mAb-coated beads in the presence of soluble anti-CD28 mAb resulted in a significantly lower percentage of CD69-expressing cells for the H45L13, but not the H45XL2 cell derivatives.

Another interesting observation is that the conjugates which form between anti-CD3 mAb-coated beads (serving as artificial antigen-presenting cells or APCs) and H45L13 versus H45XL2 cells are differentially affected by PP14. Both AF2 and PP14·Fcgamma 1 (Fig. 4D) significantly reduced the number of H45L13 cells attached to the anti-CD3 mAb-coated beads, whereas PP14 had little inhibitory effect on the number of H45XL2 (CD45lo) cells attached to such beads. This bead conjugation phenomenon is activation-dependent, because it requires the presence of anti-CD3 mAb on the beads as well as the presence of soluble anti-CD28 mAb in the system.2 Taken together, these calcium flux, activation marker, and bead conjugation data establish a functional link between the inhibitory activity of PP14 and the presence of intact CD45 phosphatase.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PP14 is one of a relatively limited set of immunoregulatory proteins known to target T cells directly. Our previous findings have suggested that PP14 acts on an early step of T cell activation to dephosphorylate TCR-induced phosphoproteins (38) and desensitize TCR signaling (39). The present study provides insights into surface molecular interactions that may underlie these PP14-mediated inhibitory effects, pointing specifically to the tyrosine phosphatase receptor CD45 as a critical mediator. The specific findings supporting this CD45 link include the following. 1) Exogenous PP14 co-caps with CD45 on the T cell surface; and 2) PP14 inhibition requires the presence of intact CD45 at the cell surface, as demonstrated via three complementary readouts for T cell activation (calcium fluxes, activation marker expression, and bead-to-cell conjugate formation).

Domain-swapping experiments have established that the cytoplasmic phosphatase domain of CD45, when dissociated from its native extracellular and transmembrane domains, retains the capacity to couple the TCR to its signaling cascade (25). Nonetheless, the very existence of multiple CD45 extracellular domain isoforms suggests that they probably contribute in fundamentally important ways to CD45 functions, presumably via their engaging one or more membrane-associated and soluble ligands. CD22 is one such ligand; direct interaction between this B cell transmembrane protein and CD45 has been demonstrated in cell-cell binding assays (26, 45). However, whereas a soluble derivative of CD22 can, by engaging CD45, inhibit early steps in TCR-triggered activation (27, 45), this inhibition requires saturable binding and extensive cross-linking of CD22, conditions that are not likely to occur in vivo with this naturally membrane-associated protein (25). In turn, this has prompted others to search for soluble CD45 ligands. Galectin-1, an endogenous lectin secreted by thymic epithelial cells, activated macrophages, and antigen-activated T cells (46-50), has emerged as one such soluble CD45 ligand (28-30) with the demonstrated capacity to induce CD45-dependent apoptosis in Jurkat T cells (28-30). The present study points to PP14 as yet another soluble CD45 ligand, in this case arising from outside the immune system. Of note, whereas our co-capping findings, along with our functional analyses, are all consistent with direct PP14 to CD45 binding, these data still do not definitively rule out the possibility that there is an indirect association bridged by CD45-associated proteins.

An interesting aspect of this study relates to the binding potential of PP14. As a lipocalin, PP14 is expected to bind lipophiles in the hydrophobic pocket of its predicted calyx-like beta -barrel (51). Although the lipophiles bound by native amniotic PP14 are unknown, certain retinoids bind to recombinant PP14 when added exogenously.3 In addition, PP14 binds to the large serum carrier protein alpha 2-macroglobulin, which potentiates the T cell inhibitory activity of PP14 (52). The present study suggests yet another binding interaction involving PP14 that is based upon carbohydrate recognition, with asialofetuin most effectively blocking PP14 binding to T cells. Based upon this latter observation, the intriguing possibility emerges that PP14 may function as a lectin and, in so doing, bind to multiple glycoproteins at cells surfaces (including the abundant T cell surface glycoprotein, CD45). Although the details of how PP14 might function as a lectin and engage carbohydrates remains to be explored, it is nonetheless noteworthy that two lysine-rich glycosaminoglycan-binding motifs, normally associated with heparin-binding, can be identified in the primary sequence of PP14 (amino acid positions 79-84 and 81-90). However, regardless of the molecular anatomy of PP14 and carbohydrate interaction, the potential parallels to the other two known CD45-binding proteins, which are both lectins, are provocative. Thus, each of the three putative CD45 ligands (CD22, galectin-1, and now PP14) is dependent on carbohydrates for binding to T cells and binds to multiple glycosylated surface proteins, CD45 among them (28-30).

The conception of PP14 functioning through carbohydrate recognition may have implications of dual roles for PP14 in the immune and reproductive systems. In proposing an overlap between lymphoid and gamete recognition, the similarities between the glycans implicated in mediating gamete recognition and those on the CD45 of T cells have been noted (reviewed in Ref. 53). Thus, PP14-mediated T cell inhibition through CD45, as suggested by our data, as well as its ability to inhibit human sperm-egg binding (54, 55), may both be mediated via lectin-like interactions.

The role of CD45 in T cell activation is not straightforward. On the one hand, CD45 is required for coupling the TCR to its intracellular signaling machinery (56). On the other hand, when CD3 and CD45 are co-ligated with bridging Ab, the TCR response is blunted (11, 12, 15). It has been suggested that TCR signaling might be modulated by altering the access of the CD45 phosphatase to TCR-associated phosphoproteins that are essential for T cell activation (3). An analogous inhibitory role for CD45 comes from studies of cytokine receptor signaling. CD45 negatively regulates this latter signaling by suppressing the Janus kinases and signal transducer and activators of transcription (STAT) proteins (16, 17), and, in cell-free systems, CD45 binds to and directly dephosphorylates Janus kinases (17). Furthermore, blocking phosphatase function with pervanadate mimics TCR triggering (7, 8). This body of data establishing a negative signaling role for CD45 fits in well with a role for this phosphatase in mediating the inhibitory signaling of PP14, as suggested here.

There may be a supramolecular basis for the opposing roles of CD45 in T cell activation. According to the model developed in a recent study (18), antigen engagement induces a dynamic process of CD45 redistribution at the APC:T cell interface. CD45 is first excluded from the central region of the APC:T cell contact site, and. subsequently, a portion of the CD45 pool is recruited back to the center of the contact site. This observation has suggested that T cell activation may be driven by sequestering CD45 away from its substrates as opposed to increasing kinase activity per se (20). It is now tempting to build upon this CD45 sequestration mechanism in developing our own model for the PP14 mode of action. We have recently demonstrated that PP14 migrates to the APC:T cell interface following conjugate formation and functions from within these sites (38), observations that are consistent with a contact site-centered mode of action. It is conceivable that, within the contact site, PP14 limits clustering and sequestration of receptors critical for T cell activation, predominantly CD45, but possibly other glycoprotein receptors as well. For example, by retaining the CD45 phosphatase within the central core of the immune synapse, PP14 could directly perturb the kinase to phosphatase balance and decrease the stability of TCR-induced phosphoproteins. This would conveniently explain the unique capacity of PP14 to elevate TCR activation thresholds and, in so doing, functionally desensitize the signaling of this critical receptor (39). This also fits in well with the antagonism between CD28-mediated costimulation and PP14-mediated inhibition (39). CD28 signaling, which functionally lowers T cell activation thresholds (57), leads to increased stability of phosphorylation, possibly as a result of lower activity or accessibility of phosphatases, a process that is mediated by reorganization of membrane microdomains (41).

These two alternative suggestions for CD28 function may also be applied to the opposing effect of PP14. In this regard, our data now establish that PP14 treatment has no effect on CD45 phosphatase activity in both resting and polyhydroxyalkanoic acid-stimulated cells.4 Thus, our proposed model for PP14 action consists of PP14 modulating the post-TCR triggering events by altering the dynamic sequestration of CD45 away from TCR-triggered phosphoproteins (rather than by altering CD45's phosphatase activity per se). This model would account for three principal findings, namely, the PP14-mediated shortening of the half-lives of the TCR-triggered phosphoproteins (38), the migration of PP14 into contact sites (38), and the dependence of PP14 immunoinhibition upon the presence of intact CD45 (as demonstrated in the present study). This is a testable hypothesis that will require systematic confocal microscopic analyses focusing on the effects of PP14 upon the segregation of CD45 and other components of T cell activation at the contact sites.

From a broader perspective, contact site perturbation induced by soluble glycosylated proteins that interact with cell surface-associated glycoproteins in a lectin-like mode may represent a more general mechanism for interfering with normal contact site clustering phenomena and thereby down-regulating T cell activation. Interestingly, it has been proposed that galectin-3, which binds to glycans on the TCR, forms a galectin-glycoprotein lattice that interferes with receptor clustering and elevates TCR activation thresholds (58). We suggest that PP14 may function in a similar fashion, in this case targeting CD45 instead.

Potentially, a PP14-glycoprotein lattice might form that distorts kinase and phosphatase relationships within the contact sites by limiting receptor clustering, as has been proposed for galectin-glycoprotein lattices (58, 59). Additionally, it is possible that when high doses of PP14 are present, either systemically (as in early pregnancy) or locally (as in sites of platelet degranulation), PP14, like galectin-3, may be constitutively bound to T cells, tonically desensitizing them and favoring Th2 responses. Lastly, it is tempting to speculate that the potentiation of the PP14 inhibitory function by multivalent alpha 2-macroglobulin (52) is explained by alpha 2-macroglobulin induction of a higher order of PP14-glycoprotein lattices (59). These intriguing mechanistic possibilities invite in depth exploration and point to new routes for T cell immunoregulation.

    ACKNOWLEDGEMENT

We thank Dr. A. Weiss (University of California, San Francisco, CA) for providing H45L13 and H45XL2 transfectant cells.

    FOOTNOTES

* This study was supported by National Institutes of Health Grant R01 AI-38960 and the Greensboro Community Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Goldyne Savad Inst. of Gene Therapy, Hadassah University Hospital, P.O.B. 12000, Jerusalem 91120, Israel. Tel.: 972-2-677-7848; Fax: 972-2-643-0982; E-mail: rjacob@hadassah.org.il.

Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M211716200

2 J. Rachmilewitz and Z. Borovsky, unpublished data.

3 N. Xiong and M. C. Tykocinski, unpublished data.

4 J. Rachmilewitz, Z. Borovsky, and M. L. Tykocinski, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: TCR, T cell receptor; Ab, antibody; mAb, monoclonal Ab; EGF, epidermal growth factor; EGFR, EGF receptor; PP14, placental protein 14; AF, amniotic fluid; APC, antigen-presenting cell.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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