Functional Implication of Cellular Prion
Protein in Antigen-Driven Interactions
between T Cells and Dendritic Cells
This information is current as
of June 18, 2017.
Clara Ballerini, Pauline Gourdain, Véronique Bachy, Nicolas
Blanchard, Etienne Levavasseur, Sylvie Grégoire, Pascaline
Fontes, Pierre Aucouturier, Claire Hivroz and Claude
Carnaud
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The Journal of Immunology is published twice each month by
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Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2006; 176:7254-7262; ;
doi: 10.4049/jimmunol.176.12.7254
http://www.jimmunol.org/content/176/12/7254
The Journal of Immunology
Functional Implication of Cellular Prion Protein in
Antigen-Driven Interactions between T Cells and Dendritic
Cells1
Clara Ballerini,2,3* Pauline Gourdain,2* Véronique Bachy,* Nicolas Blanchard,4†
Etienne Levavasseur,* Sylvie Grégoire,5* Pascaline Fontes,6* Pierre Aucouturier,*
Claire Hivroz,† and Claude Carnaud7*
T
ransmissible spongiform encephalopathies (TSE)8 are fatal neurodegenerative conditions including scrapie and
bovine spongiform encephalopathy in animal species and
Creutzfeldt-Jakob disease in humans (1). TSE are characterized by
the presence in the brain and the lymphoid tissues of a misfolded
protein termed scrapie prion protein (PrPSc), which is viewed as
*Université Pierre et Marie Curie-Paris6 and Unité Mixte de Recherche (UMR) Institut National de la Santé et de la Recherche Médicale (INSERM) Unité (U)-712,
Paris, France; and †INSERM U-653, Institut Curie, Paris, France
Received for publication August 8, 2005. Accepted for publication March 28, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by INSERM and Université Pierre et Marie Curie-Paris6,
and by specific grants from Groupement d’Intéret Scientifique-Maladies à Prions and
European Union Grant no. QLKS-CT-2002-01044. C.B. was the recipient of a poste
vert INSERM and a fellowship from Université Pierre et Marie Curie-Paris6; P.G. is
the recipient of a thesis fellowship from the French Ministry of Research and Technology; N.B. was supported by a fellowship from Fondation pour la Recherche Médicale (FRM); S.G. was supported by a fellowship from FRM.
2
C.B. and P.G. contributed equally to this work.
3
Current address: Laboratory of Neuroimmunology, Department of Neurological Sciences, University of Firenze, 50134 Florence, Italy.
4
Current address: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
5
Current address: Centre National de la Recherche Scientifique UMR 7087, Hôpital
Pitié-Salpêtrière, 75005 Paris, France.
6
INSERM U-431, Université Montpellier 2, 34095 Montpellier, France.
7
Address correspondence and reprint requests to Dr. Claude Carnaud, INSERM
U-712, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France. E-mail address: [email protected]
8
Abbreviations used in this paper: TSE, transmissible spongiform encephalopathy;
PrPSc, prion protein scrapie; PrPC, cellular prion protein; LAT, linker for activation
of T cell; DC, dendritic cell; BM, bone marrow; SAF, scrapie-associated fibril.
Copyright © 2006 by The American Association of Immunologists, Inc.
the major (if not the unique) pathogenic element responsible for
the neurodegenerative process and disease transmissibility (2).
PrPSc proceeds from the posttranslational conversion of a highly
conserved, host-encoded glycoprotein, termed cellular prion protein (PrPC) (3), constitutively expressed on many tissues, including the CNS and cell subsets of hemopoietic origin. PrPC is essentially present at the cell surface, concentrated in sphingolipid
and cholesterol-enriched microdomains, and linked to the plasma
membrane by a GPI moiety (4).
The normal biological function of PrPC is still enigmatic (5, 6).
Besides a complete resistance to TSE infectious propagation (7),
mice lacking PrPC (PrP⫺) display only minor phenotypic anomalies (8, 9). Yet, the remarkable conservation of Prnp, the PrPCencoding gene (⬎85% homology between mouse and human sequences) and its universal expression in vertebrate species (10,
11), suggests that the gene product fulfills either directly or indirectly, some vital function(s). Deciphering the biological role of
PrPC is therefore a major challenge for an evolutionary interpretation of the Prnp gene conservation and for a better understanding
of TSE pathogenesis.
The GPI insertion of PrPC suggests at least three putative functions: capture of an exogenous ligand, adhesion to cells or to extracellular matrices, and signaling. All three possibilities have been
abundantly documented (12). Several groups have reported that PrPC
binds and internalizes copper ions that in turn enhance the activity of
superoxide dismutase enzymes, resulting in better resistance against
oxidative stress (13–15). Other groups have shown that PrPC might
exert neuroprotection through alternative pathways including the
binding to laminin or to the precursor of the laminin-specific receptor
(16, 17), to chaperones and stress proteins (18 –20), or to members of
the antiapoptotic Bcl-2 family (21). Signaling has been demonstrated
using anti-PrP Ab as a substitute of a presumptive natural ligand. The
0022-1767/06/$02.00
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The cellular prion protein (PrPC) is a host-encoded, GPI-anchored cell surface protein, expressed on a wide range of tissues
including neuronal and lymphoreticular cells. PrPC may undergo posttranslational conversion, giving rise to scrapie PrP, the
pathogenic conformer considered as responsible for prion diseases. Despite intensive studies, the normal function of PrPC is still
enigmatic. Starting from microscope observations showing an accumulation of PrPC at the sites of contact between T cells and
Ag-loaded dendritic cells (DC), we have studied the contribution of PrPC in alloantigen and peptide-MHC-driven T/DC interactions. Whereas the absence of PrPC on the DC results in a reduced allogeneic T cell response, its absence on the T cell partner
has no apparent effect upon this response. Therefore, PrPC seems to fulfill different functions on the two cell partners forming the
synapse. In contrast, PrPC mobilization by Ab reduces the stimulatory properties of DC and the proliferative potential of
responding T cells. The contrasted consequences, regarding T cell function, between PrPC deletion and PrPC coating by Abs,
suggests that the prion protein acts as a signaling molecule on T cells. Furthermore, our results show that the absence of PrPC
has consequences in vivo also, upon the ability of APCs to stimulate proliferative T cell responses. Thus, independent of neurological considerations, some of the evolutionary constraints that may have contributed to the conservation of the Prnp gene in
mammalians, could be of immunological origin. The Journal of Immunology, 2006, 176: 7254 –7262.
The Journal of Immunology
FIGURE 1. Cell surface PrPC is up-regulated on activated T cells and maturing DC. Con A-activated spleen
T cells and BM-derived DC from either B6 or BALB/c
mice were generated as described in Materials and
Methods. CD4⫹ and CD8⫹ T cell subsets were examined at days 0, 1, and 2 of culture (A and B for B6 and
BALB/c, respectively). BM-derived DC collected at
days 4, 6, and 8 were stained and analyzed by flow cytometry after gating on CD11c-positive cells (C and D
for B6 and BALB/c, respectively).
ing conventional alloantigen or MHC-peptide-driven interactions
between T cells and DC. We have examined the impact of PrPC
genetic knockout or that of Ab-mediated coating, on either partner
of the immunological synapse, using as readout the proliferation of
the stimulated T cells. We have also investigated the impact of
PrPC upon in vivo responses. Our results show that PrPC has a
definite effect on both sides of the synapse, but that this effect
might be of a different nature depending on whether it is expressed
on DC or T cell membrane.
Materials and Methods
Mice
PrP⫺ mice were from the original Zürich strain (25) (with permission from
C. Weissmann, Institute of Neurology, Medical Research Council Prion
Unit, London, U.K.) and have been iteratively backcrossed in our facility
to the C57BL/6 (B6) background. The mice designated as PrP⫺ in this
study were homozygous offsprings derived from backcross 10. The wildtype mice used as controls came from the same B6 breeding stock and were
fully histocompatible with the knockout mice. Mutants and controls were
raised and maintained under strictly identical conditions. In some transfer
experiments, Ly5.1 mice (either PrP⫺ or wild-type) were used as recipients. These mice were generated by appropriate backcrossings with B6
Ly5.1 breeders.
TCR transgenic Marilyn B6 female mice with a RAG2 null mutation
(44) were obtained at the Centre de Développement des Techniques Avancées pour l’Expérimentation Animale (CDTA)-Centre National de la Recherche Scientifique (CNRS) (Orléans la Source, France). BALB/c mice
were purchased from a commercial supplier (Janvier). All of the animals
were housed in individual ventilated cages, in compliance with European
recommendations, and were maintained under strict specific pathogen-free
conditions. The sanitary status was regularly monitored at the CDTACNRS and the Virology Reference Center of Nimegue (Netherlands).
Cell separations
T cells were enriched from spleen and pooled lymph nodes by negative
magnetic cell sorting. Mechanically dispersed suspensions were freed from
red cells by hemolysis in ammonium chloride buffer (ACK), and then incubated with a mixture of anti-CD11b (Mac1) and anti-CD19 hybridoma
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oligomerization of PrPC on neuronal cell lines results in a succession
of events including phosphorylation of protein kinases, production of
reactive oxygen species, mobilization of protein kinase C, and, ultimately, activation of MAPKs ERK1/2 (22). These cascades are generally considered as pathways leading to neuronal differentiation or
survival, but some authors have also suggested a possible delivery of
apoptotic messages, when PrPC-mediated signaling exceeds a certain
threshold (23, 24).
Lymphoid tissues represent the second compartment, next after
the brain, where PrPC is most abundantly expressed. Although no
obvious immunological defect has been reported in PrP⫺ mice
(25), there is a good indication that PrPC might contribute to the
development and normal functioning of the immune system. The
protein appears to be tightly regulated on certain lymphoreticular
subsets such as T cell, monocytes, and medullary precursors (26 –
28), and anti-PrPC Abs cause partial inhibition of mitogen-driven
T cell proliferation (29 –32). More recent data based on confocal
imaging and immunoprecipitation have documented, shortly after
T cell polyclonal activation, a shift of GPI-anchored PrPC within
lipidic rafts, in physical association with a cohort of molecules
with signaling functions such as Src, fyn, lck, Zap70, linker for
activation of T cells (LAT), NADPH, and MAPKs (33–38).
Dendritic cells (DC), which are the natural partner of T cells in
initiating primary responses, are also good candidates for being the
support of PrPC functions. In addition to their implication in the
replication and propagation of PrPSc in the transmitted forms of
TSE (39 – 41), mature DC express significant amounts of PrPC
along with class II and costimulatory molecules (28, 42, 43). Yet,
as in the case of T cells, the precise role of the prion protein on DC
remains unclear.
Because so little is known about the role of PrPC on DC, and
because most data on T cells have been generated in polyclonal
systems of activation, we thought it was important to re-evaluate
the contribution of PrPC in more physiological conditions, imply-
7255
7256
supernatants, followed by an incubation with magnetic particles coupled to
goat anti-rat Ig Ab (Ademtech). Washed suspensions were submitted to a
magnetic field, and the nonretained cells, containing ⬎85% T cells, were
carefully decanted.
Spleen DC were purified by positive magnetic cell sorting. Spleens were
perfused with 3 ml of collagenase D (Roche) at 1 mg/ml in PBS, cut into
small fragments, and incubated for 45 min at 37°C. Cells were dispersed on
cell strainers (BD Biosciences), hemolysed in ACK, washed, and incubated
for 12 min at 4°C with magnetic particles coupled to anti-CD11c Ab (20 ␮l
for 1 ⫻ 108 cells) (Miltenyi Biotec). Cell suspensions were then deposited
on a magnetic column, washed, and the CD11c⫹-retained cells were
flushed out. Passage through columns was repeated twice for a better purity. The percentage of CD11c⫹ cells at the end of the procedure was
verified by flow cytometry and was ⬃90%.
In vitro differentiation of DC from bone marrow (BM)
precursors
In vitro stimulation of spleen DC
Spleen DC isolated as described above were plated in 96-well plates (BD
Falcon) at a concentration of 1 ⫻ 106/ml, in a total volume of 200 ␮l. Cells
were incubated for 24 h in GM-CSF containing medium supplemented
with either 1 ␮g/ml LPS (Sigma-Aldrich), 2 ␮g/ml bacterial CpG motifs
(Sigma-Aldrich), 15 ␮g/ml poly(I:C) (Amersham Biosciences), or nothing.
After 24 h, DC were collected, washed, and resuspended in FACS buffer.
DC were then immunostained with anti-CD11c-FITC, anti-IAb-PE, and
either anti-CD40-biotin, anti-CD86-biotin, or anti-CD80-FITC. Biotinylated Abs were revealed with streptavidin-APC (BD Pharmingen, BD
Biosciences).
In vitro T cell activation and proliferation assays
B6 or BALB/c T cells suspended at 1 ⫻ 106/ml in DMEM, supplemented
with 10% SVF, 1% 1 nM sodium pyruvate, 1% 2 mM L-glutamin, 1%
penicillin (100 U/ml), 1% streptomycin (100 ␮g/ml) (all reagents were
obtained from Invitrogen Life Technologies), and 0.05 mM 2-ME (SigmaAldrich) were polyclonally activated with 2 ␮g/ml Con A.
For MLR, responder T lymphocytes from either BALB/c or B6 origin,
enriched as above described, were suspended at 2 ⫻ 106/ml in supplemented DMEM. Purified stimulating DC from B6 wild-type or PrP⫺ donors or from BALB/c mice were suspended in DMEM at various concen-
FIGURE 2. Visualizing PrPC at the zones of
T/DC contacts. TNF-␣-matured BM-derived DC
loaded with the H-Y peptide were incubated together with Marilyn TCR transgenic T cells. PrPC is
stained in red. A–C, Represent attempts at colocalizing the prion protein with CD3, LFA-1, and CD43,
respectively (in green). ⴱ, Mark the Marilyn T lymphocytes associated to an Ag-loaded DC, seen in
phase contrast microscopy. White arrows point at
the sites of T/DC contacts, where an enrichment in
PrPC is seen. Blue arrows underline the accumulation or the exclusion of T cell markers associated
with the supramolecular complex.
trations (from 3 ⫻ 105 down to 3 ⫻ 103 cells/ml). Equal volumes of 100
␮l/well of responding and stimulating cells were distributed in flat-bottom,
96-microtiter plates (BD Falcon), which were incubated at 37°C in humidified 5% CO2 air for 5 days. The absence of proliferation of purified DC
populations alone, or with syngeneic T cells, made irradiation unnecessary.
Cultures were pulsed with 1 ␮Ci [3H]thymidine per well for the last 18 h
of culture (Amersham Biosciences). Incorporated thymidine was collected
on cellulose filters with an automated harvester (Tomtech MacIII;
PerkinElmer) and was measured by scintillation (MicroBeta 1450 Trimux;
Wallac).
Marilyn T cell proliferation in response to male Ag was assayed under
similar conditions. T cells from transgenic female donors were collected
from pooled lymph nodes and spleens and enriched by elimination of
CD11b⫹ (Mac1 positive) cells. They were suspended in supplemented
DMEM and distributed in flat-bottom microplates together with various
concentrations of spleen DC from female B6 mice and the H-Y peptide
(NAGFNSNRANSSRSS) (a gift from Dr. O. Lantz, Institut Curie, Paris,
France), in a total volume of 200 ␮l/well. Plates were incubated for 4 days,
pulsed with [3H]thymidine for the last 18 h, and processed as described
above.
In vitro blocking experiments with mAb
Anti-PrP mAb including clone scrapie-associated fibril (SAF)83, SAF61
(45), and Fab of SAF61 (a gift from J. Grassi, Commissariat à l’Energie
Atomique, Saclay, France) were assayed in parallel with their respective
IgG1 and IgG2a isotype controls (BD Pharmingen). The Abs were added
at the onset of the cultures, under a fixed volume of 20 ␮l/well, and left for
the whole duration of the experiment.
In vivo assay for Marilyn T cell proliferation
Enriched T lymphocytes from RAG2⫺/⫺ transgenic Marilyn mice were
labeled with CFSE at 4 ␮M (Sigma-Aldrich) for 5 min in PBS. The reaction was stopped by addition of chilled FCS. After 2 consecutive washes in
PBS, 3 ⫻ 106-labeled cells were injected i.v. into wild-type or PrP⫺ Ly5.1
B6 female recipients challenged within the next 2 h with H-Y peptide (50
␮g/mouse) in IFA injected at the base of the tail. Mice were sacrificed 3
days later. Regional (inguinal plus lumboaortic) and mesenteric nodes were
collected, homogenized separately, labeled with anti-Ly5.2 and anti-CD4
Ab, and analyzed by flow cytometry. Statistical analysis was made between
pairs of PrP⫺ and wild-type mice assayed under the same circumstances,
using the nonparametric Wilcoxon paired test.
Flow cytometry
Fluorescence analyses were performed on a two-laser FACSCalibur (BD
Biosciences). Cell samples, usually 1 ⫻ 106, were washed in FACS buffer
(PBS 1⫻, 0.5% BSA, 0,1% azide). Fc receptor blocking was achieved in
a saturating solution of 2.4G2 anti-CD16/CD32 Ab. Staining Ab directly
coupled to fluorochromes were added at pretitrated dilutions, for 20 min at
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BM-derived DC were generated from primary cultures of femoral marrow
from 8- to 10-wk-old female wild-type and PrP⫺ mice. Cells were cultivated in RPMI 1640 supplemented with 10% FCS and GM-CSF at 200
U/ml (PeproTech), added at days 0 and 3. At day 6, cells were collected
and maturated for 48 h in fresh GM-CSF containing medium plus TNF-␣
at 500 U/ml (PeproTech). Maturation was monitored by FACS analysis of
CD86 expression on electronically gated CD11c⫹ cells.
ROLE OF PrPC AT THE IMMUNOLOGICAL SYNAPSE
The Journal of Immunology
7257
⫹4°C. SAF61 and SAF83 anti-PrP Ab (45) were biotinylated according to
routine procedures (EZ link Sulfo-NHS-LC-biotin; Pierce) and revealed
with streptavidin PE or allophycocyanin. Cell fluorescence was acquired
and analyzed using CellQuest software (BD Biosciences). CFSE fluorescence acquisitions were treated in addition with FlowJo software (Tree
Star).
Confocal analysis of T/DC conjugates
Coverslips were covered with 1 ⫻ 105 BM-derived or spleen DC loaded
with 10 nM of the H-Y peptide. After 30 min at 37°C, Marilyn T cells were
added at a 1:1 ratio. After 45 min of incubation at 37°C, the coverslips were
washed with PBS, fixed 10 min with 4% paraformaldehyde, and permeabilized with a PBS solution containing 0.05% saponin (ICN Biomedicals)
and 2% BSA (Sigma-Aldrich). Primary and secondary Abs were diluted in
PBS, 2% BSA, and 0.05% saponin, and incubated for 2 h and 1 h, respectively. Abs used were as follows: biotin-conjugated hamster anti-mouse
CD3␧ (clone 145-2C11; BD Pharmingen) followed by Alexa 488-conjugated streptavidin (Molecular Probes); anti-LAT (rabbit polyclonal IgG;
Upstate Biotechnology) followed by Alexa 488-conjugated goat anti-rabbit
IgG (Molecular Probes); monoclonal rat anti-mouse LFA1 (American
Type Culture Collection; TIB-127) followed by Alexa 488-conjugated goat
anti-rat IgG (Molecular Probes); anti-PrP SAF83 followed by Cy3-conjugated donkey anti-mouse (Fab(⬘)2; Jackson ImmunoResearch
Laboratories).
Images of conjugates were acquired on a Leica TCS SP2 confocal scanning microscope (Leica), equipped with a 100⫻ 1.4 aperture HCX PL APO
oil immersion objective. “En face” view of the T-DC contact zone (xz) was
reconstructed from series of xy sections spaced by 0.3 ␮m (Metamorph
software; Universal Imaging).
Results
Up-regulation of cell surface PrPC on activating T cells and on
maturing DC
The rapid up-regulation of cell surface PrPC, following T cell activation, has been reported in several studies (29 –32). We confirmed that PrPC was increased on both CD4⫹ and CD8⫹ T cell
subsets from B6 or BALB/c mice using Con A as a polyclonal T
cell activator (Fig. 1, A and B). Although PrPC up-regulation on
differentiating DC has been less well studied, there is indication
that PrPC is tightly regulated in this lineage too (42). To provide
further evidence, we followed prion protein expression in cultures
of maturing BM-derived DC of B6 and BALB/c origin. As shown
in Fig. 1, C and D, cell surface PrPC increased steadily together
with CD86 costimulatory molecules, between day 4 and day 8. A
similar steady increase of the costimulation molecule CD80 and of
MHC class II, was observed between day 4 and day 8 (data not
shown).
PrPC migrates at the sites of contact between T cells and DC
To substantiate the idea that PrPC is involved in Ag-driven interactions, we looked at the distribution of the protein in T/DC synapses. We took advantage of the Marilyn model, where the recognition by the transgenic Marilyn TCR of DC loaded with male
H-Y peptide in the I-Ab context can be readily visualized by the
formation of conjugates (44). Complexes formed between Agloaded, fully matured BM-derived DC, and Marilyn T cells were
subsequently labeled with anti-PrP Abs and examined by confocal
microscopy. More than 70% of such complexes showed an accumulation of PrPC fluorescence at the sites of contact between T
cells and DC (Fig. 2). Marilyn T cells alone were stained as controls. They presented a diffuse and even distribution of PrPC on
their surface (data not shown).
To define more precisely where PrPC migrates within the supramolecular complex that structures the immunological synapse,
we costained the conjugates for PrPC (in red) and for molecules
such as CD3, LFA-1, or CD43 (in green), which accumulate at the
center, the periphery, or are excluded from the supramolecular
complex, respectively (46). As can be seen in Fig. 2, no clear
colocalization was evidenced between PrPC and those markers,
although PrPC was enriched in the zones of contact. No colocalization was observed either with LAT, another central marker of
the complex, or with CD90, a GPI-anchored T cell marker associated with activation (data not shown). Thus, PrPC appears to be
mobilized at the immunological synapse, at a still unidentified
location.
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FIGURE 3. Whether PrPC is absent on DC or
on T cells has a different impact upon allogeneic
MLR. Enriched T cells were stimulated in a fully
allogeneic MLR model. PrPC was missing on
stimulating DC (A and B) or on responding T
cells (C and D). A, A typical experiment comparing PrPC-positive DC (u) vs PrP-deprived
DC (䡺) (mean cpm of triplicate wells ⫾ SD). B,
The compilation of five independent experiments showing comparative responses induced
by PrP-positive vs PrP-negative allogeneic DC
at a concentration of 5 ⫻ 104 cells/well. C, A
typical experiment comparing PrPC-positive (u)
vs PrPC-deprived (䡺) responding T cells. D, A
compilation of four identical experiments.
7258
ROLE OF PrPC AT THE IMMUNOLOGICAL SYNAPSE
Different impact of PrPC absence on the two partners of the
MLR
The absence of PrPC does not affect DC maturation
A trivial explanation for the lower efficiency of PrPC-deprived DC
could have been that the gene invalidation indirectly affected maturation, reducing the expression of both MHC class II or costimulatory molecules. To rule out this possibility, we compared the
phenotypes of spleen DC isolated from PrP⫺ or wild-type mice
and matured in vitro for 24 h with LPS. Starting from comparable
populations of positively selected CD11c⫹ DC, we found that the
absence of PrPC had no detectable influence upon the expression
of MHC class II, CD80 and CD40 costimulation molecules (Fig.
4). Other agents of DC maturation such as TNF-␣, oligo-CpG, or
poly(I:C) led to similar conclusions (data not shown), suggesting
that, irrespective of the TLR pathway being used, the absence of
PrPC does not interfere with DC maturation. IL-12p70 production
by LPS or CpG-activated spleen DC was not altered either by the
absence of PrPC (data not shown).
PrPC coating by Ab partially blocks MLR
To gain further insight into the respective roles of PrPC on both
partners of allogeneic MLR, we looked at the effects of SAF83, an
IgG1 mAb that binds to cell surface PrPC. An isotype control was
used in parallel to rule out a possible implication of Fc receptors
expressed on DC and activated T cells. As can be seen in Fig. 5,
Ab inhibited in a dose-dependent manner, alloantigen-driven T cell
proliferation. It did so in MLR, where PrPC was expressed on both
cell partners (Fig. 5A) or on the stimulating DC only (Fig. 5B). But
rather unexpectedly, anti-PrP Ab were also effective under conditions where PrPC was expressed on T cells only, thus revealing an
implication of the prion protein on both sides of the synapse and
notably on T cells where the mere absence of PrPC had shown no
effect (Fig. 5C). Finally, to rule out a destabilizing effect of the Abs
on the immunological synapse, we tested in parallel Fab and total
Ig of SAF61, an IgG2a mAb with similar specificity as SAF83 for
mouse PrPC. The results of such an experiment, shown in Fig. 5D,
indicate clearly that Fab are as effective as total Ig in inhibiting
FIGURE 4. Absence of PrPC on spleen DC does not affect their maturation. CD11c⫹ DC were isolated from spleens of wild-type or PrP⫺ mice
and cultured overnight with GM-CSF plus LPS (bold histogram) or GMCSF alone (light histogram) as a control. Cells were phenotyped on the
following day for class II, CD80, and CD40 markers.
allogeneic MLR. Therefore, it seems that anti-PrP Abs do not mediate their effect by steric hindrance, and that PrPC does not necessarily need to be cross-linked to modify the proliferative T cell
response.
Absence of PrPC on DC or its mobilization by Ab affects the in
vitro response of Marilyn T cells
To extend the conclusions from allogeneic to peptide-MHC driven
T/DC interactions, we came back to the Marilyn model where
PrPC accumulation at the sites of conjugation had been initially
observed. Naive transgenic T cells were cocultured for 4 days with
PrP⫺ or wild-type female DC loaded with H-Y peptide. Experiments shown in Fig. 5, A and B, replicated the results of allogeneic
MLR, in that DC devoid of PrPC were systematically less efficient
in stimulating T cells than wild-type DC. This was true at all the
experimental conditions tested, whether the doses of peptide (Fig.
6A) or the numbers of loaded DC were varied (Fig. 6B).
The reciprocal experiment was unfortunately not feasible, because the PrP null mutation could not be passed onto Marilyn
RAG2⫺/⫺ mice due to the close vicinity of RAG and Prnp loci on
chromosome 2. Still, it was possible to study the effects of Ab
coating in situations where PrPC was present either on both partners or on T cells only. As shown in Fig. 6, C and D, SAF83
caused definite inhibition of T cell proliferation, irrespective of
whether PrPC was present on both partners (Fig. 6C) or on the T
cells only (Fig. 6D). Here again, the isotype control did not cause
significant inhibition, thus ruling out an effect of anti-PrP Ab due
to Fc receptor binding.
Absence of PrPC on APCs affects Ag-driven T cell proliferation
in vivo
Having found that PrPC-deprived DC stimulated T cells less efficiently in allogenic and peptide-MHC-driven in vitro interactions,
we sought to extend this result in vivo, by comparing the efficiency
of the APCs in PrP⫺ vs wild-type mice. Recipients of both types
were first transferred i.v. with purified CFSE-labeled Marilyn T
cells, and subsequently challenged with H-Y peptide in IFA. Control mice received emulsified PBS instead. Ag-driven T cell proliferation was evaluated 3 days later by measuring the decrease in
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As a first attempt to evaluate the contribution of PrPC in Ag-driven
T/DC interactions, we examined the consequences of prion protein
invalidation on allogeneic MLR. Having verified that B6 and
BALB/c strains behaved similarly in terms of PrPC expression
(see Fig. 1), we first compared the stimulating potential of wildtype vs PrPC-null DC of B6 origin cultured with responding
BALB/c T lymphocytes. As shown in Fig. 3A, T cell stimulation
induced by DC deprived of PrPC was less vigorous than that
caused by wild-type DC. This was true at all tested concentrations
of stimulating cells, ruling out a marginal effect due to suboptimal
conditions of stimulation. Fig. 3B shows the results of five independent experiments, each time confirming the lower stimulating
efficiency (from 30 to 55% decrease) of PrPC-deprived allogeneic
DC. Interestingly, the release of IL-2 by the same responding T
cells was not affected, suggesting that the reduced proliferation
was not a direct consequence of a lack of growth factor (data not
shown).
In reciprocal experiments, we compared PrPC-deficient vs wildtype B6 T cells stimulated by allogeneic BALB/c DC. Here, at
variance with what had been previously observed, the absence of
prion protein on the T cell partner had no impact on the proliferative response, neither in the experiment shown in Fig 3C, nor in
four similar assays compiled in Fig. 3D. These experiments therefore revealed a difference in the functional status of dendritic vs
lymphocytic PrPC.
The Journal of Immunology
7259
FIGURE 5. Anti-PrP Ab reveal a functional implication of PrPC on both stimulating DC and responder T cells. T cells were stimulated as in Fig. 3.
A, The inhibitory effect of anti-PrP mAb SAF83 in a
MLR where PrPC is expressed on both partners. In
B, PrPC is present on DC only and in C on T cells
only. F, Represent values of thymidine uptake in
cultures with SAF83; E, represent values in control
cultures with the IgG1 isotype control (mean cpm of
triplicates ⫾ SD). D, Fab and total Ig of SAF61 have
been tested in parallel with an IgG2a isotype control.
F, SAF61; E, isotype control; f, Fab of SAF61.
Discussion
The objective of this study was to assess the functional implication
of PrPC in T/DC interactions, by examining the behavior of cell
partners on which the prion protein was either missing or was
coated by mAb. Several teams are currently contributing to the
elucidation of PrPC function(s) in the immune system. The novelty
of our approach resides in the following points. First, we have
considered simultaneously the two cell types of the immunological
synapse, T cells and DC, whereas most studies published so far
have focused on the T cell partner only (29 –37). Second, at variance with other studies examining polyclonal T cell activation by
mitogenic lectins (29, 30), Ab cross-linking (35–37), or hypothermic shock (38), we have looked at more physiological conditions.
Even if allogeneic stimulation or MHC-peptide activation of TCR
transgenic T cells mimic only the normal development of an immune response, the two models imply the formation of T/DC synapses, specific recognition of antigenic patterns, and physiological
signal transduction pathways. Last, we demonstrate that the lack of
PrPC has in vivo repercussions, which could provide an explanation for the selective advantage of the Prnp gene.
An interesting result from the present study is that the absence
of PrPC does not have the same consequences on T cells and DC.
Lack of PrPC on T lymphocytes has no visible influence on their
capacity to proliferate in response to allogeneic APCs, whereas the
lack of PrPC on DC results in a significant reduction of proliferation by the responding T cells. This difference may account for
some of the discrepancies noted in the literature regarding the consequences of Prnp gene knockout on polyclonal T cell responses
(30 –31). It probably reflects differences in function and in signaling properties of dendritic vs lymphocytic PrPC.
Regarding the DC side, we have ruled out an effect of PrPC
absence on the expression of MHC and CD80/CD86 or CD40
costimulation molecules. The production of IL-12p70 by DC is not
modified either by the absence of PrPC. A more likely eventuality,
comforted by the observation that the prion protein is mobilized at
the supramolecular complex, could be that PrPC stabilizes the synapse, affecting in turn the duration and the efficiency of T/DC
interactions. In a recent study using the Marilyn transgenic model
(44), it was shown that the dynamics of conjugation, which differs
between immature and full-fledged DC, had an impact on T lymphocyte activation. One of our future objectives will be to document, through imaging experiments, the possibility that the absence of PrPC on DC affects the quality of T/DC conjugates. The
GPI anchoring, which confers flexibility and mobility to the prion
protein, would certainly be compatible with a role of PrPC in the
physical shaping of the synapse. Furthermore, it will be important
to find out whether PrPC acts exclusively as an element of physical
cohesion between T cells and DC or also as a signaling molecule
transducing messages inside the DC. Preliminary data regarding
synapses formed between Marilyn T cells and DC from knockout
mice suggest that lymphocytic PrPC migrates more readily when
PrPC is also present on the DC partner (data not shown).
The discrepancy between the lack of functional effect of PrPC
invalidation on T lymphocytes and the inhibition of their proliferation after Ab-mediated PrPC recruitment is a strong indication
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
CFSE fluorescence of the transferred T cells, collected in the draining lumboaortic and inguinal lymph nodes or in the mesenteric
chain. The percentages of retrieved T cells were similar in wildtype and PrP⫺ recipients. As expected, the T cells in control mice
that had not received male Ag, manifested maximal fluorescence
intensity, whereas in mice challenged with the H-Y peptide, they
displayed several peaks of decreasing fluorescence corresponding
to successive waves of cell division (Fig. 7A). A close comparison
of the patterns seen in a PrP⫺ vs a wild-type recipient mouse (Fig.
7B) showed, however, a delayed proliferation of the T cells implanted in the PrP-deficient host. There are, for instance, four times
more cells (32 vs 9%) in peak 1 of the PrPC-deprived mouse than
in the wild-type control, whereas the reverse is seen (5 vs 21%) in
peak 4 corresponding to T cells that have undergone more divisions. This experiment was repeated four times, with a total of six
PrP⫺ and five wild-type mice. In five of the six PrPC-deficient
recipients, Marilyn T cells proliferated less promptly than in the
wild-type controls. The difference between the two groups was
statistically significant (Fig. 7C). Thus, the absence of PrPC on
APCs has a definite impact upon in vivo Ag-driven proliferation of
responding T cells.
7260
ROLE OF PrPC AT THE IMMUNOLOGICAL SYNAPSE
that lymphocytic PrPC exerts signaling functions. Abs do not simply mask or strip off PrPC on T cells, like genetic invalidation. By
mobilizing PrPC, they probably induce a cascade of biochemical
events resulting in partial inhibition of T cell proliferation. Results
already exist, both in neuron and in lymphocyte cell lines, suggesting that the mobilization of PrPC leads to signaling pathways
(22, 37, 47). Still, the physiological consequences of PrPC engagement, whether it results into differentiation, expansion, acquisition
or inhibition of functions, or to apoptosis, remain to be properly
evaluated. The latter possibility is of particular interest in view of
the fact that both pro- and antiapoptotic effects have been attributed to PrPC in neuronal cells (23, 24). Thus, it is possible that
similar pathways are at work in T lymphocytes, depending upon
the intensity, duration, and timing of PrPC signaling, together with
an eventual synergy with TCR/CD3 signaling. Another line of
thoughts is provided by studies dealing with the Ab-mediated recruitment of GPI-anchored proteins on T cells. Such studies have
revealed profound similarities between all these molecules, and
notably their capacity, following Ab-mediated mobilization, to inhibit clonal T cell expansion through the IL-2R pathway, while
preserving the functions of the lymphocytes (48, 49). An important
issue will be to find out whether PrPC follows the signaling pathway common to most GPI-anchored proteins on T cells, a pathway
that results in clonal size control, while leaving intact effector
functions such as cytotoxicity or lymphokine production, or
whether PrPC initiates its own specific signaling pathway.
FIGURE 7. Marilyn T cells proliferate less
readily upon antigenic challenge in a PrP⫺ host.
CFSE-labeled Marilyn T cells were injected into
PrP⫺ or wild-type female mice and subsequently
stimulated with the H-Y peptide in IFA. Control
mice received emulsified PBS. A, CFSE fluorescence profiles of transferred, but not stimulated,
Marilyn T cells vs Ag-stimulated T cells retrieved 3
days later from the mesenteric nodes of recipients.
B, A quantitative cycle analysis of the Marilyn T
cells retrieved from Ag-challenged PrP⫺ vs wildtype mice. C, The compilation of four independent
experiments comparing pairs of PrP⫺ vs wild-type
female mice assayed in parallel. Statistical difference between the two groups was assessed by Wilcoxon paired test.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Implication of PrPC in peptideMHC-driven T/DC interactions. A and B, The in
vitro proliferation, expressed as stimulation index,
of Marilyn T cells stimulated with Ag-loaded female
spleen DC expressing either PrPC (F) or PrPC⫺
(E). A, T cells were stimulated with a constant number of spleen DC pulsed with decreasing concentrations of H-Y peptide. B, The amount of peptide was
maintained constant at 10 nM, while the number of
DC was decreased (mean stimulation indexes of
triplicates ⫾ SD). C and D compare T cell proliferation at two concentrations of H-Y peptide, in the
presence of SAF83 (䡺), isotype control (u), or no Ig
(f). C, PrPC is present on both partners, whereas in
D PrPC is present on T cells only (mean stimulation
indexes of triplicates ⫾ SD).
The Journal of Immunology
Acknowledgments
We thank I. Renault for the mouse breeding and the management of the
animal facility; Dr. C. Weissmann for the PrP⫺ breeders; Dr. O. Lantz for
advice, discussion, and gift of H-Y peptide; Dr. J. Grassi for the gift of
anti-PrP mAbs and Fab; and Dr. M. Rosset-Bruley for critical reading of
the manuscript.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
Disclosures
The authors have no financial conflict of interest.
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The last part of this study was aimed at evaluating the effects of
Prnp gene invalidation in vivo. Although the present experimental
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More focused experiments will have to be performed to find out
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