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IMMUNOBIOLOGY
From the Department of Pathology, Case Western Reserve
University, Cleveland, OH.
The activation of discrete T-cell responses depends on the
triggering of individualized threshold numbers of T-cell receptors (TCRs). The results of this study indicate that the lipocalin placental
protein 14 (PP14), a T-cell inhibitor produced by cells of the
reproductive and hematopoietic systems, mediates its anti-inflammatory activity by elevating the T-cell activation threshold, thereby rendering T cells less sensitive to stimulation. Significantly, the
data demonstrate hierarchical sensitivity of selected cytokine responses to PP14-mediated inhibition, with the hierarchy reflecting their respective activation thresholds. These findings suggest a novel
paradigm for immunoinhibition wherein negative regulators can finely
tune, rather than inactivate, T-cell responses, and thereby skew the
cytokine output of immunologic responses.
(Blood. 2001;98:3727-3732) The T-cell activation threshold model proposes that
(1) T-cell activation ensues once a threshold number of receptors have been triggered, as measured by down-modulation of T-cell receptors (TCRs)1; (2) distinct cytokine and other functional
responses within individual T cells are pegged to different levels of
TCR occupancy, and hence, there is a hierarchy of individualized
thresholds at the single cell level2,3; and (3) the
threshold number is flexible, in that costimulation can significantly
lower the number of receptors required for individual
responses.1,3
Whereas the effect of costimulation on T-cell activation thresholds has
been evaluated, the possibility that T-cell inhibitors might also
function through the tuning of such thresholds has not been explored.
Hence, although T-cell inhibitors are generally presumed to act by
blocking activation pathways in an all-or-none fashion, the possibility
emerges that at least some of these inhibitors might function in a
"rheostatic" manner to desensitize TCR signaling and elevate the
number of TCRs required for activation of specific T-cell responses.
Placental protein 14 (PP14; progesterone-associated endometrial
protein; glycodelin) is a 28-kd glycoprotein of the lipocalin structural superfamily with documented immunoinhibitory
properties.4-7 This glycoprotein is produced by cells of
the female and male reproductive tracts and is present at especially
high levels in amniotic fluid (AF) and maternal serum.8,9
Interestingly, we have demonstrated that PP14 is also present in
platelets7 and is released from activated platelets (N. Xiong and M.L.T., unpublished observations, 1996).
Significantly, we have recently demonstrated that PP14 directly
inhibits human T cells and accounts for the T-cell inhibitory activity
of AF.10 Thus, PP14 joins transforming growth factor Interestingly, in contrast to TGF- Cell culture
As a source of accessory cells (ACs), monocytes were isolated
from mononuclear cell populations by adherence to plastic. PBMCs (2 × 106 cells/mL) were incubated in serum-free medium
for 1 hour 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-hour incubation period, adherent
monocytes were removed by gentle scraping with a plastic cell scraper.
Cell viability was more than 80% as determined by trypan blue
exclusion. Cells were washed with phosphate-buffered saline (PBS) and
fixed for 5 minutes with 0.4% paraformaldehyde (Sigma) in PBS at room temperature. The reaction was quenched by adding an equal volume of 0.2 M L-lysine solution (in PBS). The fixed cells were then washed 3 times with medium and incubated in culture medium at 37°C
for 1 hour before adding them to cultures. The Jurkat cell line was
obtained from the American Type Culture Collection (ATCC; Manassas, VA).
Cytokine production
Jurkat cells (5 × 105) were cultured in a 1-mL volume of RPMI containing 10% FBS in individual wells of 24-well culture plates, and stimulated with different PHA concentrations, with or without AF, or cyclosporine A (CSA; Sigma) for 20 hours. Chinese hamster ovary (CHO) cells transfected with the glutamine synthetase amplification/expression construct pEE14/hB7-1 were fixed with 0.4% paraformaldehyde for 5 minutes at room temperature. Jurkat cells were stimulated as described above in the presence or absence of 5 × 105/well of these human B7-1-expressing CHO cell transfectants. Flow cytometry Down-modulation of TCR was measured by direct immunofluorescence using fluorescein isothiocyanate (FITC)-anti-CD3 monoclonal antibody (mAb; UCTH1). Similar results were obtained with indirect immunofluorescence (unpublished data, February 1998). The data were calculated using the mean fluorescence values of the cell. For intracellular cytokine staining, monensin (2 mM) was added in the last 5 hours of a 48-hour culture. Cells were fixed and coimmunostained with phycoerythrin (PE)-anti-IL-2 and FITC-anti-IFN- antibodies
(Pharmingen, San Diego, CA) as described,3 and
then analyzed, (1 × 104 cells/sample) on a FACScan flow
cytometer (Becton Dickinson, San Jose, CA) using Lysis II software.
Cell sorting Jurkat cells were stained with FITC-conjugated UCTH1 mAb and then sorted into 2 fractions of low and high expressors. Cells were incubated for 4 days, and the level of CD3 expression was confirmed by flow cytometric analysis. Cells were cultured as described above and were stimulated with a combination of protein A beads bearing immobilized anti-CD3 mAb (OKT3) and soluble anti-CD28 mAb (9.3; 1 µg/mL).AF samples and PP14 immunoabsorption Discarded human AF samples were obtained from the Center for Human Genetics Laboratory at University Hospitals of Cleveland 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 antibodies7 were coupled to protein A-Sepharose beads (Sigma) to generate an immunoabsorbent. Control beads consisted of protein A-Sepharose beads coupled to preimmune rabbit serum. Immunoabsorption was carried out by adding AF
to antibody-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  .12 pPP14 · Fc 1/REP7
was transfected into 293 cells (ATCC). Stable transfectants secreting
PP14 · Fc1 were grown in Ultraculture (Whittaker
Bioproducts) supplemented with Hygromycin (200 µg/mL; Calbiochem, La
Jolla, CA) at 37°C with 5% CO2. To purify the protein, conditioned media was 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 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).
To test whether PP14 elevates T-cell activation thresholds, we
evaluated the effects of AF on IL-2 secretion by Jurkat cells stimulated with increasing concentrations of PHA. In the absence of AF,
PHA induced dose-dependent IL-2 secretion (reaching a plateau at
approximately 1.25 µg/mL PHA) (Figure
1A). AF increased the concentration of
PHA required for a minimal measurable IL-2 response (Figure 1A).
Interestingly, and as expected for a threshold-centered mechanism, the
AF-mediated inhibitory effect was inversely correlated with activator
concentration, with the highest concentrations of PHA totally
attenuating the inhibitory effect of AF (Figure 1A). Higher AF
concentrations (50%) resulted in a requirement for higher PHA
concentration (5 µg/mL) to override the inhibition (data not shown).
Similar results were obtained with cells stimulated with solid-phase
OKT3 and CD28 mAbs (data not shown). Moreover, immunodepletion of PP14
from the AF abrogated its immunoinhibitory activity, verifying the PP14
dependence of the observed AF inhibitory effect (Figure 1A).
We next determined whether the pattern of inhibition in Figure 1A, characterized by override of inhibition with increased TCR triggering, is unique to PP14, or instead, is associated with any T-cell inhibitory agent used at nonsaturating concentrations. To test this possibility, we used CSA, an intensively studied inhibitor of the Ca++-dependent phosphatase, calcineurin, which blocks IL-2 induction.13,14 When used at saturating concentrations, CSA totally blocked IL-2 secretion at all PHA doses tested (data not shown). IL-2 secretion in the presence of nonsaturating CSA concentrations, which attenuate without fully blocking signal transmission, reached a plateau at a similar PHA concentration to untreated cells, but as expected, at lower absolute IL-2 levels (Figure 1B). In marked contrast to AF, the CSA inhibitory effect was not inversely correlated to PHA concentration, and most significantly, could not be overcome by increasing TCR occupancy. The difference between PP14 and CSA is highlighted in Figure 1, panel C, in which changes in IL-2 levels, as a function of PHA concentration, have been recalculated from the data in Figure 1, panels A and B. The plot demonstrates the marked difference between the 2 T-cell inhibitors. Whereas CSA results in greater reduction in IL-2 production with increasing PHA concentrations, AF/PP14-mediated inhibition is characterized by less of a change in the IL-2 response as the PHA stimulus increases. In light of these results (Figure 1A), which showed a shift in the
dose-response curve typical of an increase in activation threshold, the
effect of AF/PP14 on the number of triggered TCRs was next tested. We
simultaneously evaluated the effects of AF on TCR triggering and IL-2
secretion by Jurkat cells stimulated with increasing concentrations of
PHA. In the absence of AF, PHA induced dose-dependent TCR
down-modulation (as measured by loss of surface CD3) (Figure
2A), consistent with previous
observations using other TCR ligands.1,15 AF, though
inhibiting IL-2 secretion (Figure 1A), did not significantly change the
number of engaged TCRs (Figure 2A). Similar results were obtained when
peripheral T cells were stimulated with various concentrations of
either PHA (data not shown) or solid-phase OKT3 (Figure 2B). Together, these observations establish that the number of triggered TCRs required
for inducing a response is elevated in the presence of AF (Figure 2C).
These results clearly demonstrate that AF increases the relative TCR occupancy required for elicitation of a specific cytokine (IL-2) response. Therefore, by increasing the absolute number of triggered TCRs (via increasing stimulus concentrations), one can override the AF-mediated inhibitory effect. This AF effect, dependent on the presence of PP14, contrasts with the previously described effects of costimulators, which function in an opposite fashion to lower the number of triggered TCRs required for T-cell activation.1,3 Furthermore, the AF/PP14 effect differs from that of partial agonists, with the lower response of the latter attributable to fewer triggered TCRs,15 and from that of CSA, the inhibitory activity of which does not depend on the extent of TCR occupancy. Additional experiments were performed to support the proposed
functional link between AF/PP14 and T-cell activation thresholds. According to the threshold model, the capacity of a given T cell to
respond to a defined concentration of stimulus is positively correlated
with the absolute number of TCRs on that cell.1,16 Jurkat
cells were preparatively sorted into high and low TCR
expressors, and the cells were then stimulated with solid-phase
OKT3 and CD28 mAbs. The respective susceptibilities of these sorted
Jurkat cell subpopulations to inhibition by AF/PP14 were compared. As
expected, low TCR expressors secreted significantly lower levels of
IL-2 at each OKT3 concentration, as compared to Jurkat cells expressing high levels of TCR; at low concentrations of OKT3, the low TCR expressors did not respond at all (data not shown). Significantly, Jurkat cells expressing low levels of TCR were more sensitive to
AF-mediated inhibition of IL-2 secretion at all OKT3 concentrations (data not shown), and this was most prominent at OKT3 concentrations in
the lower range, where inhibition by AF reached 100% in the low-TCR
cells (Figure 3A). Thus, sensitivity to
inhibition is inversely proportional to TCR numbers at the cell
surface. The sensitivity to inhibition by AF was inversely correlated
with agonist concentration in the earlier experiments (Figure 1A), and
with the number of available TCRs in the latter experiments (Figure
3A). In both instances, the magnitude of activation and the sensitivity
to inhibition is eventually determined by the number of TCRs engaged.
Next, the interplay between AF/PP14-mediated inhibition and costimulation was examined. Because costimulatory signals delivered to CD28 on T cells are known to lower the activation threshold,1,3 we predicted that such threshold-lowering costimulatory signals would counteract any increase in activation threshold mediated by AF/PP14. To this end, PHA-stimulated Jurkat cells were inhibited with AF in the presence or absence of human B7-1-expressing CHO cell transfectants. Addition of B7-1 transfectants markedly augmented IL-2 secretion at each level of TCR stimulus (data not shown). Importantly, the presence of B7-1 costimulator markedly reduced AF-mediated inhibition of IL-2 secretion (Figure 3B). Similar results were obtained when anti-CD28 mAbs (9.3) were used in similar assays, instead of B7-expressing CHO cells (data not shown), and hence, the observed effects are not attributable to the CHO cells per se. Taken together, the findings that AF-mediated inhibition can be reduced by either increasing TCR numbers on the cell surface or by lowering activation thresholds through costimulation, are consistent with the notion that this inhibition operates via rheostatic modulation of activation thresholds. Subsequent experiments used peripheral blood T cells in place of Jurkat
cells as responders. As with Jurkats, the number of TCRs down-modulated
in anti-CD3-induced T cells was not significantly changed in the
presence of AF (Figure 2B). In the case of Jurkat cells, there is no
significant difference in the activation thresholds for the cytokines
IL-2 and IFN-
To verify that the observed pattern of inhibition (ie, inversely
related to stimulus concentration) is due to PP14 per se, and not a
consequence of the AF milieu, a recombinant PP14 · Fc
The previous set of experiments demonstrated the effect of AF/PP14 on
activation thresholds of PHA- and CD3-induced cells. We next determined
the effect of AF/PP14 on antigen-induced cells. For this purpose PBMCs
were stimulated with the influenza virus matrix peptide antigen (MA
58-66),17 and the number of IFN-
This study clearly demonstrates that AF, and its active immunoregulatory factor PP14, function to increase the number of triggered TCRs required for T-cell activation. As predicted by a model wherein PP14 increases T-cell activation thresholds, the present findings establish that inhibition is sensitive to: (1) the extent of TCR triggering (and thus can be overridden by increasing the dose of stimulus); (2) the number of TCRs expressed on the cell surface; (3) the combined presence of costimulators that reciprocally lower activation thresholds; and (4) the hierarchical threshold of the particular response being measured. In contrast to AF/PP14, the inhibitory activity of CSA, even at nonsaturating concentrations, was not attenuated with increasing PHA doses. Hence, activation threshold modulation is not a common feature of all T-cell inhibitors, although this functional property may ultimately prove to extend beyond PP14. CSA functions by blocking the activity of the phosphatase calcineurin, resulting in a failure to activate IL-2.14 By contrast, it would seem that the mode of action of PP14 is to attenuate rather than to eliminate or block downstream signaling events, resulting in functionally increased activation thresholds. In the present study we demonstrated a PP14-mediated shift in the
dose-response curve to a TCR stimulus. This shift does not stem from a
lower number of engaged TCR molecules, because the same number of TCR
molecules were down-regulated in the absence or presence of PP14.
Likewise, costimulation through CD28, which also functions by
modulating the TCR activation threshold (in this case, lowering
it1), was shown to do so without affecting the
number of TCR triggering events.11 That said, it should be
noted that TCR down-regulation does not necessarily give a complete
picture of TCR engagement, because only those engagement events that
result in full Our mechanistic insight into PP14's threshold modulation activity at the cellular level provides a useful framework for further exploring the intracellular pathways influenced by this molecular factor. In fact, in a parallel series of experiments, we have recently shed some light on the molecular underpinnings of the threshold effect and the consequent desensitization of receptor signaling that we have formally documented here. Specifically, our other data demonstrate that PP14 decreases the half-lives of TCR-triggered phosphotyrosines by promoting their dephosphorylation in a CD45-dependent fashion (J.R. et al, manuscript submitted for publication). In this way, PP14's effect on T cells contrasts with that of the B7/CD28 costimulation axis that stabilizes tyrosine phosphates linked to TCR activation.11 This is significant from our standpoint because, as we have shown here, PP14 and costimulation also have opposing effects on TCR activation thresholds (Figure 3).1,3 Modulation of the stability of TCR-triggered phosphoproteins provides a compelling explanation at the molecular level for TCR activation threshold modulation by PP14, whereby the balance between tyrosine kinase and phosphatase activity sets the T-cell activation threshold. In aggregate, the data point to a novel immunoregulatory mechanism for
T-cell inhibition based on the modulation of T-cell activation
thresholds. By this mechanism, immunoregulatory factors (in this case,
PP14) may dictate not only quantitative, but also qualitative, aspects
of immune responses. This is highlighted by our demonstration that PP14
differentially influences cytokines according to their activation
thresholds (such as IFN-
The authors thank Drs M. Tary-Lehmann, P. V. Lehmann, and R. Trezza for assistance with ELISAspot assays. We appreciate Dr R. Germain's (National Institutes of Health) and Dr M. I. Greene's (University of Pennsylvania) review of the manuscript. We thank Dr M. Lamm for his long-term support, and Drs A. Hochberg and N. de Groot for helpful discussions. We also thank Ms Susi Brill for her expert secretarial assistance.
Submitted November 28, 2000; accepted June 28, 2001.
Supported by grant RO1 AI 38960 from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mark L. Tykocinski, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 6 Gates Bldg, 3400 Spruce St, Philadelphia, PA, 19104-4283; e-mail: mlt4{at}mail.med.upenn.edu.
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© 2001 by The American Society of Hematology.
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  B. Toth, K. Roth, C. Kunert-Keil, C. Scholz, S. Schulze, I. Mylonas, K. Friese, and U. Jeschke Glycodelin Protein and mRNA Is Downregulated in Human First Trimester Abortion and Partially Upregulated in Mole Pregnancy J. Histochem. Cytochem., May 1, 2008; 56(5): 477 - 485. [Abstract] [Full Text] [PDF]
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 M. Seppala, H. Koistinen, R. Koistinen, P.C.N. Chiu, and W.S.B. Yeung Glycosylation related actions of glycodelin: gamete, cumulus cell, immune cell and clinical associations Hum. Reprod. Update, May 1, 2007; 13(3): 275 - 287. [Abstract] [Full Text] [PDF]
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 E. Ish-Shalom, A. Gargir, S. Andre, Z. Borovsky, Z. Ochanuna, H.-J. Gabius, M. L. Tykocinski, and J. Rachmilewitz {alpha}2,6-Sialylation promotes binding of placental protein 14 via its Ca2+-dependent lectin activity: insights into differential effects on CD45RO and CD45RA T cells Glycobiology, March 1, 2006; 16(3): 173 - 183. [Abstract] [Full Text] [PDF]
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K. Lapid and N. Sharon Meet the multifunctional and sexy glycoforms of glycodelin Glycobiology, March 1, 2006; 16(3): 39R - 45R. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) or presence of either AF (25% 
) or PP14-depleted AF
(
) (A); or in the presence of CSA (100 pg/mL [
]) (B). IL-2 secretion was determined in the conditioned medium.
The data represent the mean of triplicate samples. Comparable results
were obtained in 4 separate experiments. (C) Change in absolute IL-2
levels as a function of PHA concentration in the presence of either AF
(25% 
). (A) Percentage of TCR down-modulation in Jurkat
cells as a function of PHA concentration, as measured by CD3
immunofluorescence and flow cytometry. (B) Percentage of TCR
down-modulation in T cells as a function of OKT3 concentrations. (C)
IL-2 secretion by Jurkat cells as a function of the percentage of TCR
down-modulation, as calculated from the data in Figure 
), in conjunction with anti-CD28
mAb (
-chain and ZAP-70 phosphorylation lead to receptor
down-regulation.