Functional Implication of Cellular Prion in Antigen-Driven Interactions between T Cells and Dendritic Cells

This information is current as Clara Ballerini, Pauline Gourdain, Véronique Bachy, Nicolas of September 25, 2021. Blanchard, Etienne Levavasseur, Sylvie Grégoire, Pascaline Fontes, Pierre Aucouturier, Claire Hivroz and Claude Carnaud J Immunol 2006; 176:7254-7262; ;

doi: 10.4049/jimmunol.176.12.7254 Downloaded from http://www.jimmunol.org/content/176/12/7254

References This article cites 54 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/176/12/7254.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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*Ve´ronique Bachy,* Nicolas Blanchard,4† Etienne Levavasseur,* Sylvie Gre´goire,5* Pascaline Fontes,6* Pierre Aucouturier,* Claire Hivroz,† and Claude Carnaud7*

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 Downloaded from Ag-loaded dendritic cells (DC), we have studied the contribution of PrPC in alloantigen and peptide-MHC-driven T/DC inter- actions. Whereas the absence of PrPC on the DC results in a reduced allogeneic 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 http://www.jimmunol.org/ has consequences in vivo also, upon the ability of APCs to stimulate proliferative T cell responses. Thus, independent of neuro- logical 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.

ransmissible spongiform encephalopathies (TSE)8 are fa- the major (if not the unique) pathogenic element responsible for tal neurodegenerative conditions including scrapie and the neurodegenerative process and disease transmissibility (2). T bovine spongiform encephalopathy in animal species and PrPSc proceeds from the posttranslational conversion of a highly

Creutzfeldt-Jakob disease in (1). TSE are characterized by conserved, host-encoded glycoprotein, termed cellular prion pro- by guest on September 25, 2021 the presence in the brain and the lymphoid tissues of a misfolded tein (PrPC) (3), constitutively expressed on many tissues, includ- protein termed scrapie prion protein (PrPSc), which is viewed as ing the CNS and cell subsets of hemopoietic origin. PrPC is es- sentially present at the cell surface, concentrated in sphingolipid and cholesterol-enriched microdomains, and linked to the plasma *Universite´Pierre et Marie Curie-Paris6 and Unite´Mixte de Recherche (UMR) In- membrane by a GPI moiety (4). stitut National de la Sante´et de la Recherche Me´dicale (INSERM) Unite´(U)-712, The normal biological function of PrPC is still enigmatic (5, 6). Paris, France; and †INSERM U-653, Institut Curie, Paris, France Besides a complete resistance to TSE infectious propagation (7), Received for publication August 8, 2005. Accepted for publication March 28, 2006. mice lacking PrPC (PrPϪ) display only minor phenotypic anom- The costs of publication of this article were defrayed in part by the payment of page alies (8, 9). Yet, the remarkable conservation of Prnp, the PrPC- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. encoding gene (Ͼ85% between and se- 1 This work was supported by INSERM and Universite´Pierre et Marie Curie-Paris6, quences) and its universal expression in vertebrate species (10, and by specific grants from Groupement d’Inte´ret Scientifique-Maladies a`Prions and 11), suggests that the gene product fulfills either directly or indi- European Union Grant no. QLKS-CT-2002-01044. C.B. was the recipient of a poste vert INSERM and a fellowship from Universite´Pierre et Marie Curie-Paris6; P.G. is rectly, some vital function(s). Deciphering the biological role of the recipient of a thesis fellowship from the French Ministry of Research and Tech- PrPC is therefore a major challenge for an evolutionary interpre- nology; N.B. was supported by a fellowship from Fondation pour la Recherche Me´di- tation of the Prnp gene conservation and for a better understanding cale (FRM); S.G. was supported by a fellowship from FRM. of TSE pathogenesis. 2 C.B. and P.G. contributed equally to this work. The GPI insertion of PrPC suggests at least three putative func- 3 Current address: Laboratory of Neuroimmunology, Department of Neurological Sci- ences, University of Firenze, 50134 Florence, Italy. tions: capture of an exogenous ligand, adhesion to cells or to extra- cellular matrices, and signaling. All three possibilities have been 4 Current address: Department of Molecular and Cell Biology, University of Califor- nia, Berkeley, CA 94720. abundantly documented (12). Several groups have reported that PrPC 5 Current address: Centre National de la Recherche Scientifique UMR 7087, Hoˆpital binds and internalizes copper ions that in turn enhance the activity of Pitie´-Salpeˆtrie`re, 75005 Paris, France. superoxide dismutase enzymes, resulting in better resistance against 6 INSERM U-431, Universite´Montpellier 2, 34095 Montpellier, France. oxidative stress (13–15). Other groups have shown that PrPC might 7 Address correspondence and reprint requests to Dr. Claude Carnaud, INSERM exert neuroprotection through alternative pathways including the U-712, Hoˆpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571 Paris Ce- binding to laminin or to the precursor of the laminin-specific receptor dex 12, France. E-mail address: [email protected] (16, 17), to chaperones and stress (18–20), or to members of 8 Abbreviations used in this paper: TSE, transmissible spongiform encephalopathy; PrPSc, prion protein scrapie; PrPC, cellular prion protein; LAT, linker for activation the antiapoptotic Bcl-2 family (21). Signaling has been demonstrated of T cell; DC, dendritic cell; BM, bone marrow; SAF, scrapie-associated fibril. using anti-PrP Ab as a substitute of a presumptive natural ligand. The

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 7255 oligomerization of PrPC on neuronal cell lines results in a succession ing conventional alloantigen or MHC-peptide-driven interactions of events including phosphorylation of protein kinases, production of between T cells and DC. We have examined the impact of PrPC reactive oxygen species, mobilization of protein kinase C, and, ulti- genetic knockout or that of Ab-mediated coating, on either partner mately, activation of MAPKs ERK1/2 (22). These cascades are gen- of the immunological synapse, using as readout the proliferation of erally considered as pathways leading to neuronal differentiation or the stimulated T cells. We have also investigated the impact of survival, but some authors have also suggested a possible delivery of PrPC upon in vivo responses. Our results show that PrPC has a apoptotic messages, when PrPC-mediated signaling exceeds a certain definite effect on both sides of the synapse, but that this effect threshold (23, 24). might be of a different nature depending on whether it is expressed Lymphoid tissues represent the second compartment, next after on DC or T cell membrane. the brain, where PrPC is most abundantly expressed. Although no obvious immunological defect has been reported in PrPϪ mice Materials and Methods (25), there is a good indication that PrPC might contribute to the Mice development and normal functioning of the immune system. The PrPϪ mice were from the original Zu¨rich strain (25) (with permission from protein appears to be tightly regulated on certain lymphoreticular C. Weissmann, Institute of Neurology, Medical Research Council Prion subsets such as T cell, monocytes, and medullary precursors (26– Unit, London, U.K.) and have been iteratively backcrossed in our facility Ϫ 28), and anti-PrPC Abs cause partial inhibition of mitogen-driven to the C57BL/6 (B6) background. The mice designated as PrP in this study were homozygous offsprings derived from backcross 10. The wild- T cell proliferation (29–32). More recent data based on confocal type mice used as controls came from the same B6 breeding stock and were imaging and immunoprecipitation have documented, shortly after fully histocompatible with the knockout mice. Mutants and controls were

T cell polyclonal activation, a shift of GPI-anchored PrPC within raised and maintained under strictly identical conditions. In some transfer Downloaded from Ϫ lipidic rafts, in physical association with a cohort of molecules experiments, Ly5.1 mice (either PrP or wild-type) were used as recipi- with signaling functions such as Src, fyn, lck, Zap70, linker for ents. These mice were generated by appropriate backcrossings with B6 Ly5.1 breeders. activation of T cells (LAT), NADPH, and MAPKs (33–38). TCR transgenic Marilyn B6 female mice with a RAG2 null mutation Dendritic cells (DC), which are the natural partner of T cells in (44) were obtained at the Centre de De´veloppement des Techniques Avan- initiating primary responses, are also good candidates for being the ce´es pour l’Expe´rimentation Animale (CDTA)-Centre National de la Re- cherche Scientifique (CNRS) (Orle´ans la Source, France). BALB/c mice support of PrPC functions. In addition to their implication in the http://www.jimmunol.org/ were purchased from a commercial supplier (Janvier). All of the animals replication and propagation of PrPSc in the transmitted forms of were housed in individual ventilated cages, in compliance with European TSE (39–41), mature DC express significant amounts of PrPC recommendations, and were maintained under strict specific pathogen-free along with class II and costimulatory molecules (28, 42, 43). Yet, conditions. The sanitary status was regularly monitored at the CDTA- as in the case of T cells, the precise role of the prion protein on DC CNRS and the Virology Reference Center of Nimegue (Netherlands). remains unclear. Cell separations Because so little is known about the role of PrPC on DC, and T cells were enriched from spleen and pooled lymph nodes by negative because most data on T cells have been generated in polyclonal magnetic cell sorting. Mechanically dispersed suspensions were freed from systems of activation, we thought it was important to re-evaluate red cells by hemolysis in ammonium chloride buffer (ACK), and then in- the contribution of PrPC in more physiological conditions, imply- cubated with a mixture of anti-CD11b (Mac1) and anti-CD19 hybridoma by guest on September 25, 2021

FIGURE 1. Cell surface PrPC is up-regulated on ac- tivated 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. CD4ϩ and CD8ϩ T cell subsets were exam- ined 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 cy- tometry after gating on CD11c-positive cells (C and D for B6 and BALB/c, respectively). 7256 ROLE OF PrPC AT THE IMMUNOLOGICAL SYNAPSE

supernatants, followed by an incubation with magnetic particles coupled to trations (from 3 ϫ 105 down to 3 ϫ 103 cells/ml). Equal volumes of 100 goat anti- Ig Ab (Ademtech). Washed suspensions were submitted to a ␮l/well of responding and stimulating cells were distributed in flat-bottom, magnetic field, and the nonretained cells, containing Ͼ85% T cells, were 96-microtiter plates (BD Falcon), which were incubated at 37°C in humid-

carefully decanted. ified 5% CO2 air for 5 days. The absence of proliferation of purified DC Spleen DC were purified by positive magnetic cell sorting. Spleens were populations alone, or with syngeneic T cells, made irradiation unnecessary. perfused with 3 ml of collagenase D (Roche) at 1 mg/ml in PBS, cut into Cultures were pulsed with 1 ␮Ci [3H]thymidine per well for the last 18 h small fragments, and incubated for 45 min at 37°C. Cells were dispersed on of culture (Amersham Biosciences). Incorporated thymidine was collected cell strainers (BD Biosciences), hemolysed in ACK, washed, and incubated on cellulose filters with an automated harvester (Tomtech MacIII; for 12 min at 4°C with magnetic particles coupled to anti-CD11c Ab (20 ␮l PerkinElmer) and was measured by scintillation (MicroBeta 1450 Trimux; for 1 ϫ 108 cells) (Miltenyi Biotec). Cell suspensions were then deposited Wallac). on a magnetic column, washed, and the CD11cϩ-retained cells were Marilyn T cell proliferation in response to male Ag was assayed under flushed out. Passage through columns was repeated twice for a better pu- similar conditions. T cells from transgenic female donors were collected rity. The percentage of CD11cϩ cells at the end of the procedure was from pooled lymph nodes and spleens and enriched by elimination of verified by flow cytometry and was ϳ90%. CD11bϩ (Mac1 positive) cells. They were suspended in supplemented DMEM and distributed in flat-bottom microplates together with various In vitro differentiation of DC from bone marrow (BM) concentrations of spleen DC from female B6 mice and the H-Y peptide precursors (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, BM-derived DC were generated from primary cultures of femoral marrow 3 Ϫ pulsed with [ H]thymidine for the last 18 h, and processed as described from 8- to 10-wk-old female wild-type and PrP mice. Cells were culti- above. vated 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 In vitro blocking experiments with mAb

and maturated for 48 h in fresh GM-CSF containing medium plus TNF-␣ Downloaded from Anti-PrP mAb including clone scrapie-associated fibril (SAF)83, SAF61 at 500 U/ml (PeproTech). Maturation was monitored by FACS analysis of ϩ (45), and Fab of SAF61 (a gift from J. Grassi, Commissariat a`l’Energie CD86 expression on electronically gated CD11c cells. Atomique, Saclay, France) were assayed in parallel with their respective In vitro stimulation of spleen DC 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 Spleen DC isolated as described above were plated in 96-well plates (BD the whole duration of the experiment. Falcon) at a concentration of 1 ϫ 106/ml, in a total volume of 200 ␮l. Cells In vivo assay for Marilyn T cell proliferation were incubated for 24 h in GM-CSF containing medium supplemented http://www.jimmunol.org/ ␮ ␮ Ϫ Ϫ with either 1 g/ml LPS (Sigma-Aldrich), 2 g/ml bacterial CpG motifs Enriched T lymphocytes from RAG2 / transgenic Marilyn mice were ␮ (Sigma-Aldrich), 15 g/ml poly(I:C) (Amersham Biosciences), or nothing. labeled with CFSE at 4 ␮M (Sigma-Aldrich) for 5 min in PBS. The reac- After 24 h, DC were collected, washed, and resuspended in FACS buffer. tion was stopped by addition of chilled FCS. After 2 consecutive washes in b DC were then immunostained with anti-CD11c-FITC, anti-IA -PE, and PBS, 3 ϫ 106-labeled cells were injected i.v. into wild-type or PrPϪ Ly5.1 either anti-CD40-biotin, anti-CD86-biotin, or anti-CD80-FITC. Biotinyl- B6 female recipients challenged within the next 2 h with H-Y peptide (50 ated Abs were revealed with streptavidin-APC (BD Pharmingen, BD ␮g/mouse) in IFA injected at the base of the tail. Mice were sacrificed 3 Biosciences). days later. Regional (inguinal plus lumboaortic) and mesenteric nodes were collected, homogenized separately, labeled with anti-Ly5.2 and anti-CD4 In vitro T cell activation and proliferation assays Ab, and analyzed by flow cytometry. Statistical analysis was made between Ϫ 6 pairs of PrP and wild-type mice assayed under the same circumstances, B6 or BALB/c T cells suspended at 1 ϫ 10 /ml in DMEM, supplemented by guest on September 25, 2021 with 10% SVF, 1% 1 nM sodium pyruvate, 1% 2 mM L-glutamin, 1% using the nonparametric Wilcoxon paired test. penicillin (100 U/ml), 1% streptomycin (100 ␮g/ml) (all reagents were obtained from Invitrogen Life Technologies), and 0.05 mM 2-ME (Sigma- Aldrich) were polyclonally activated with 2 ␮g/ml Con A. Fluorescence analyses were performed on a two-laser FACSCalibur (BD For MLR, responder T lymphocytes from either BALB/c or B6 origin, Biosciences). Cell samples, usually 1 ϫ 106, were washed in FACS buffer enriched as above described, were suspended at 2 ϫ 106/ml in supple- (PBS 1ϫ, 0.5% BSA, 0,1% azide). Fc receptor blocking was achieved in mented DMEM. Purified stimulating DC from B6 wild-type or PrPϪ do- a saturating solution of 2.4G2 anti-CD16/CD32 Ab. Staining Ab directly nors or from BALB/c mice were suspended in DMEM at various concen- coupled to fluorochromes were added at pretitrated dilutions, for 20 min at

FIGURE 2. Visualizing PrPC at the zones of T/DC contacts. TNF-␣-matured BM-derived DC loaded with the H-Y peptide were incubated to- gether with Marilyn TCR transgenic T cells. PrPC is stained in red. A–C, Represent attempts at colocal- izing the prion protein with CD3, LFA-1, and CD43, -Mark the Marilyn T lym ,ء .(respectively (in green phocytes 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 accumula- tion or the exclusion of T cell markers associated with the supramolecular complex. The Journal of Immunology 7257

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 com- paring PrPC-positive DC (u) vs PrP-deprived DC (Ⅺ) (mean cpm of triplicate wells Ϯ SD). B, The compilation of five independent experi- ments showing comparative responses induced by PrP-positive vs PrP-negative allogeneic DC at a concentration of 5 ϫ 104 cells/well. C,A typical experiment comparing PrPC-positive (u) vs PrPC-deprived (Ⅺ) responding T cells. D,A Downloaded from compilation of four identical experiments. http://www.jimmunol.org/

ϩ4°C. SAF61 and SAF83 anti-PrP Ab (45) were biotinylated according to of maturing BM-derived DC of B6 and BALB/c origin. As shown routine procedures (EZ link Sulfo-NHS-LC-biotin; Pierce) and revealed in Fig. 1, C and D, cell surface PrPC increased steadily together with streptavidin PE or allophycocyanin. Cell fluorescence was acquired and analyzed using CellQuest software (BD Biosciences). CFSE fluores- with CD86 costimulatory molecules, between day 4 and day 8. A cence acquisitions were treated in addition with FlowJo software (Tree similar steady increase of the costimulation molecule CD80 and of Star). MHC class II, was observed between day 4 and day 8 (data not

. by guest on September 25, 2021 Confocal analysis of T/DC conjugates shown) Coverslips were covered with 1 ϫ 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 PrPC migrates at the sites of contact between T cells and DC 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 perme- To substantiate the idea that PrPC is involved in Ag-driven inter- abilized with a PBS solution containing 0.05% saponin (ICN Biomedicals) actions, we looked at the distribution of the protein in T/DC syn- 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, respec- apses. We took advantage of the Marilyn model, where the rec- tively. Abs used were as follows: biotin-conjugated hamster anti-mouse ognition by the transgenic Marilyn TCR of DC loaded with male CD3␧ (clone 145-2C11; BD Pharmingen) followed by Alexa 488-conju- H-Y peptide in the I-Ab context can be readily visualized by the gated streptavidin (Molecular Probes); anti-LAT (rabbit polyclonal IgG; formation of conjugates (44). Complexes formed between Ag- Upstate Biotechnology) followed by Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes); monoclonal rat anti-mouse LFA1 (American loaded, fully matured BM-derived DC, and Marilyn T cells were Type Culture Collection; TIB-127) followed by Alexa 488-conjugated goat subsequently labeled with anti-PrP Abs and examined by confocal anti-rat IgG (Molecular Probes); anti-PrP SAF83 followed by Cy3-conju- microscopy. More than 70% of such complexes showed an accu- Ј gated donkey anti-mouse (Fab( )2; Jackson ImmunoResearch Laboratories). mulation of PrPC fluorescence at the sites of contact between T Images of conjugates were acquired on a Leica TCS SP2 confocal scan- cells and DC (Fig. 2). Marilyn T cells alone were stained as con- ning microscope (Leica), equipped with a 100ϫ 1.4 aperture HCX PL APO trols. They presented a diffuse and even distribution of PrPC on oil immersion objective. “En face” view of the T-DC contact zone (xz) was their surface (data not shown). reconstructed from series of xy sections spaced by 0.3 ␮m (Metamorph software; Universal Imaging). To define more precisely where PrPC migrates within the su- pramolecular complex that structures the immunological synapse, Results we costained the conjugates for PrPC (in red) and for molecules Up-regulation of cell surface PrPC on activating T cells and on such as CD3, LFA-1, or CD43 (in green), which accumulate at the maturing DC center, the periphery, or are excluded from the supramolecular The rapid up-regulation of cell surface PrPC, following T cell ac- complex, respectively (46). As can be seen in Fig. 2, no clear tivation, has been reported in several studies (29–32). We con- colocalization was evidenced between PrPC and those markers, firmed that PrPC was increased on both CD4ϩ and CD8ϩ T cell although PrPC was enriched in the zones of contact. No colocal- subsets from B6 or BALB/c mice using Con A as a polyclonal T ization was observed either with LAT, another central marker of cell activator (Fig. 1, A and B). Although PrPC up-regulation on the complex, or with CD90, a GPI-anchored T cell marker asso- differentiating DC has been less well studied, there is indication ciated with activation (data not shown). Thus, PrPC appears to be that PrPC is tightly regulated in this lineage too (42). To provide mobilized at the immunological synapse, at a still unidentified further evidence, we followed prion protein expression in cultures location. 7258 ROLE OF PrPC AT THE IMMUNOLOGICAL SYNAPSE

Different impact of PrPC absence on the two partners of the MLR 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 wild- type 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 inde- pendent 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). Downloaded from In reciprocal experiments, we compared PrPC-deficient vs wild- FIGURE 4. Absence of PrPC on spleen DC does not affect their mat- type B6 T cells stimulated by allogeneic BALB/c DC. Here, at uration. CD11cϩ DC were isolated from spleens of wild-type or PrPϪ mice variance with what had been previously observed, the absence of and cultured overnight with GM-CSF plus LPS (bold histogram) or GM- prion protein on the T cell partner had no impact on the prolifer- CSF alone ( histogram) as a control. Cells were phenotyped on the ative response, neither in the experiment shown in Fig 3C, nor in following day for class II, CD80, and CD40 markers. four similar assays compiled in Fig. 3D. These experiments there- http://www.jimmunol.org/ fore revealed a difference in the functional status of dendritic vs allogeneic MLR. Therefore, it seems that anti-PrP Abs do not me- lymphocytic PrPC. diate their effect by steric hindrance, and that PrPC does not nec- essarily need to be cross-linked to modify the proliferative T cell The absence of PrPC does not affect DC maturation response. A trivial explanation for the lower efficiency of PrPC-deprived DC Absence of PrPC on DC or its mobilization by Ab affects the in could have been that the gene invalidation indirectly affected mat- vitro response of Marilyn T cells uration, reducing the expression of both MHC class II or costimu- To extend the conclusions from allogeneic to peptide-MHC driven latory molecules. To rule out this possibility, we compared the by guest on September 25, 2021 Ϫ T/DC interactions, we came back to the Marilyn model where phenotypes of spleen DC isolated from PrP or wild-type mice PrPC accumulation at the sites of conjugation had been initially and matured in vitro for 24 h with LPS. Starting from comparable ϩ observed. Naive transgenic T cells were cocultured for 4 days with populations of positively selected CD11c DC, we found that the PrPϪ or wild-type female DC loaded with H-Y peptide. Experi- absence of PrPC had no detectable influence upon the expression ments shown in Fig. 5, A and B, replicated the results of allogeneic of MHC class II, CD80 and CD40 costimulation molecules (Fig. MLR, in that DC devoid of PrPC were systematically less efficient ␣ 4). Other agents of DC maturation such as TNF- , oligo-CpG, or in stimulating T cells than wild-type DC. This was true at all the poly(I:C) led to similar conclusions (data not shown), suggesting experimental conditions tested, whether the doses of peptide (Fig. that, irrespective of the TLR pathway being used, the absence of 6A) or the numbers of loaded DC were varied (Fig. 6B). PrPC does not interfere with DC maturation. IL-12p70 production The reciprocal experiment was unfortunately not feasible, be- by LPS or CpG-activated spleen DC was not altered either by the cause the PrP null mutation could not be passed onto Marilyn absence of PrPC (data not shown). RAG2Ϫ/Ϫ mice due to the close vicinity of RAG and Prnp loci on 2. Still, it was possible to study the effects of Ab PrPC coating by Ab partially blocks MLR coating in situations where PrPC was present either on both part- To gain further insight into the respective roles of PrPC on both ners or on T cells only. As shown in Fig. 6, C and D, SAF83 partners of allogeneic MLR, we looked at the effects of SAF83, an caused definite inhibition of T cell proliferation, irrespective of IgG1 mAb that binds to cell surface PrPC. An isotype control was whether PrPC was present on both partners (Fig. 6C)orontheT used in parallel to rule out a possible implication of Fc receptors cells only (Fig. 6D). Here again, the isotype control did not cause expressed on DC and activated T cells. As can be seen in Fig. 5, significant inhibition, thus ruling out an effect of anti-PrP Ab due Ab inhibited in a dose-dependent manner, alloantigen-driven T cell to Fc receptor binding. 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 Absence of PrPC on APCs affects Ag-driven T cell proliferation rather unexpectedly, anti-PrP Ab were also effective under condi- in vivo tions where PrPC was expressed on T cells only, thus revealing an Having found that PrPC-deprived DC stimulated T cells less effi- implication of the prion protein on both sides of the synapse and ciently in allogenic and peptide-MHC-driven in vitro interactions, notably on T cells where the mere absence of PrPC had shown no we sought to extend this result in vivo, by comparing the efficiency effect (Fig. 5C). Finally, to rule out a destabilizing effect of the Abs of the APCs in PrPϪ vs wild-type mice. Recipients of both types on the immunological synapse, we tested in parallel Fab and total were first transferred i.v. with purified CFSE-labeled Marilyn T Ig of SAF61, an IgG2a mAb with similar specificity as SAF83 for cells, and subsequently challenged with H-Y peptide in IFA. Con- mouse PrPC. The results of such an experiment, shown in Fig. 5D, trol mice received emulsified PBS instead. Ag-driven T cell pro- indicate clearly that Fab are as effective as total Ig in inhibiting liferation was evaluated 3 days later by measuring the decrease in The Journal of Immunology 7259

FIGURE 5. Anti-PrP Ab reveal a functional im- plication of PrPC on both stimulating DC and re- sponder 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 Ϯ 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. Downloaded from http://www.jimmunol.org/ CFSE fluorescence of the transferred T cells, collected in the drain- signal transduction pathways. Last, we demonstrate that the lack of ing lumboaortic and inguinal lymph nodes or in the mesenteric PrPC has in vivo repercussions, which could provide an explana- chain. The percentages of retrieved T cells were similar in wild- tion for the selective advantage of the Prnp gene. Ϫ type and PrP recipients. As expected, the T cells in control mice An interesting result from the present study is that the absence that had not received male Ag, manifested maximal fluorescence of PrPC does not have the same consequences on T cells and DC. intensity, whereas in mice challenged with the H-Y peptide, they Lack of PrPC on T lymphocytes has no visible influence on their displayed several peaks of decreasing fluorescence corresponding capacity to proliferate in response to allogeneic APCs, whereas the to successive waves of cell division (Fig. 7A). A close comparison lack of PrPC on DC results in a significant reduction of prolifer- Ϫ of the patterns seen in a PrP vs a wild-type recipient mouse (Fig. ation by the responding T cells. This difference may account for by guest on September 25, 2021 7B) showed, however, a delayed proliferation of the T cells im- some of the discrepancies noted in the literature regarding the con- planted in the PrP-deficient host. There are, for instance, four times sequences of Prnp gene knockout on polyclonal T cell responses more cells (32 vs 9%) in peak 1 of the PrPC-deprived mouse than (30–31). It probably reflects differences in function and in signal- in the wild-type control, whereas the reverse is seen (5 vs 21%) in ing properties of dendritic vs lymphocytic PrPC. peak 4 corresponding to T cells that have undergone more divi- Regarding the DC side, we have ruled out an effect of PrPC sions. This experiment was repeated four times, with a total of six absence on the expression of MHC and CD80/CD86 or CD40 PrPϪ and five wild-type mice. In five of the six PrPC-deficient costimulation molecules. The production of IL-12p70 by DC is not recipients, Marilyn T cells proliferated less promptly than in the modified either by the absence of PrPC. A more likely eventuality, wild-type controls. The difference between the two groups was comforted by the observation that the prion protein is mobilized at statistically significant (Fig. 7C). Thus, the absence of PrPC on the supramolecular complex, could be that PrPC stabilizes the syn- APCs has a definite impact upon in vivo Ag-driven proliferation of apse, affecting in turn the duration and the efficiency of T/DC responding T cells. interactions. In a recent study using the Marilyn transgenic model Discussion (44), it was shown that the dynamics of conjugation, which differs between immature and full-fledged DC, had an impact on T lym- The objective of this study was to assess the functional implication of PrPC in T/DC interactions, by examining the behavior of cell phocyte activation. One of our future objectives will be to docu- partners on which the prion protein was either missing or was ment, through imaging experiments, the possibility that the ab- coated by mAb. Several teams are currently contributing to the sence of PrPC on DC affects the quality of T/DC conjugates. The elucidation of PrPC function(s) in the immune system. The novelty GPI anchoring, which confers flexibility and mobility to the prion of our approach resides in the following points. First, we have protein, would certainly be compatible with a role of PrPC in the considered simultaneously the two cell types of the immunological physical shaping of the synapse. Furthermore, it will be important synapse, T cells and DC, whereas most studies published so far to find out whether PrPC acts exclusively as an element of physical have focused on the T cell partner only (29–37). Second, at vari- cohesion between T cells and DC or also as a signaling molecule ance with other studies examining polyclonal T cell activation by transducing messages inside the DC. Preliminary data regarding mitogenic lectins (29, 30), Ab cross-linking (35–37), or hypother- synapses formed between Marilyn T cells and DC from knockout mic shock (38), we have looked at more physiological conditions. mice suggest that lymphocytic PrPC migrates more readily when Even if allogeneic stimulation or MHC-peptide activation of TCR PrPC is also present on the DC partner (data not shown). transgenic T cells mimic only the normal development of an im- The discrepancy between the lack of functional effect of PrPC mune response, the two models imply the formation of T/DC syn- invalidation on T lymphocytes and the inhibition of their prolif- apses, specific recognition of antigenic patterns, and physiological eration after Ab-mediated PrPC recruitment is a strong indication 7260 ROLE OF PrPC AT THE IMMUNOLOGICAL SYNAPSE

FIGURE 6. Implication of PrPC in peptide- MHC-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 PrPCϪ (E). A, T cells were stimulated with a constant num- ber of spleen DC pulsed with decreasing concentra- tions 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 Ϯ SD). C and D compare T cell prolifer- ation at two concentrations of H-Y peptide, in the presence of SAF83 (Ⅺ), 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 Downloaded from indexes of triplicates Ϯ SD). http://www.jimmunol.org/ that lymphocytic PrPC exerts signaling functions. Abs do not sim- the intensity, duration, and timing of PrPC signaling, together with ply mask or strip off PrPC on T cells, like genetic invalidation. By an eventual synergy with TCR/CD3 signaling. Another line of mobilizing PrPC, they probably induce a cascade of biochemical thoughts is provided by studies dealing with the Ab-mediated re- events resulting in partial inhibition of T cell proliferation. Results cruitment of GPI-anchored proteins on T cells. Such studies have already exist, both in and in lymphocyte cell lines, sug- revealed profound similarities between all these molecules, and gesting that the mobilization of PrPC leads to signaling pathways notably their capacity, following Ab-mediated mobilization, to in-

(22, 37, 47). Still, the physiological consequences of PrPC engage- hibit clonal T cell expansion through the IL-2R pathway, while by guest on September 25, 2021 ment, whether it results into differentiation, expansion, acquisition preserving the functions of the lymphocytes (48, 49). An important or inhibition of functions, or to , remain to be properly issue will be to find out whether PrPC follows the signaling path- evaluated. The latter possibility is of particular interest in view of way common to most GPI-anchored proteins on T cells, a pathway the fact that both pro- and antiapoptotic effects have been attrib- that results in clonal size control, while leaving intact effector uted to PrPC in neuronal cells (23, 24). Thus, it is possible that functions such as cytotoxicity or lymphokine production, or similar pathways are at work in T lymphocytes, depending upon whether PrPC initiates its own specific signaling pathway.

FIGURE 7. Marilyn T cells proliferate less readily upon antigenic challenge in a PrPϪ host. CFSE-labeled Marilyn T cells were injected into PrPϪ or wild-type female mice and subsequently stimulated with the H-Y peptide in IFA. Control mice received emulsified PBS. A, CFSE fluores- cence 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 PrPϪ vs wild- type mice. C, The compilation of four independent experiments comparing pairs of PrPϪ vs wild-type female mice assayed in parallel. Statistical differ- ence between the two groups was assessed by Wil- coxon paired test. The Journal of Immunology 7261

The last part of this study was aimed at evaluating the effects of and 9 avian PrPs reveals high conservation of flexible regions of the prion pro- Prnp gene invalidation in vivo. Although the present experimental tein. J. Mol. Biol. 289: 1163–1178. 11. Cotto, E., M. Andre, J. Forgue, H. J. Fleury, and P. J. Babin. 2005. Molecular setting gives only a partial view on this issue, by focusing exclu- characterization, phylogenetic relationships, and developmental expression pat- sively on the Ag-presenting side of immune responses, it shows terns of prion genes in zebrafish (Danio rerio). FEBS J. 272: 500–513. 12. Martins, V. R., A. F. Mercadante, A. L. Cabral, A. R. Freitas, and R. M. Castro. nevertheless that the absence of PrPC on APCs has a definite im- 2001. Insights into the physiological function of cellular prion protein. Braz. pact on Ag-driven T cell proliferation. Whether this is sufficient to J. Med. Biol. Res. 34: 585–595. qualify PrPϪ mice as immunocompromised is still too preliminary. 13. Brown, D. R., K. Qin, J. W. Herms, A. Madlung, J. Manson, R. Strome, P. E. Fraser, T. Kruck, A. von Bohlen, W. Schulz-Schaeffer, et al. 1997. The More focused experiments will have to be performed to find out cellular prion protein binds copper in vivo. Nature 390: 684–687. whether, at variance with what had been initially observed (25), 14. Milhavet, O., and S. Lehmann. 2002. Oxidative stress and the prion protein in the knockout of the Prnp gene represents a true selective disad- transmissible spongiform encephalopathies. Brain Res. Brain Res. Rev. 38: 328–339. vantage, especially when PrPC-deprived mice are, for instance, 15. Kuwahara, C., A. M. Takeuchi, T. Nishimura, K. Haraguchi, A. Kubosaki, confronted by harmful pathogens. A better understanding of PrPC Y. Matsumoto, K. Saeki, Y. Matsumoto, T. Yokoyama, S. Itohara, and implication in vivo might provide a clue regarding the evolution- T. Onodera. 1999. Prions prevent neuronal cell-line death. Nature 400: 225–226. 16. Rieger, R., F. Edenhofer, C. I. Lasmezas, and S. Weiss. 1997. The human 37-kDa ary conservation of a gene whose only known function so far is its laminin receptor precursor interacts with the prion protein in eukaryotic cells. contribution to a fatal neurodegenerative condition. Nat. Med. 3: 1383–1388. 17. Graner, E., A. F. Mercadante, S. M. Zanata, O. V. Forlenza, A. L. Cabral, The fact that the same molecule might be involved in immuno- S. S. Veiga, M. A. Juliano, R. Roesler, R. Walz, A. Minetti, et al. 2000. Cellular logical and neurological synapses is not unprecedented. MHC prion protein binds laminin and mediates neuritogenesis. Brain Res. Mol. Brain class I molecules and agrin, a glycoprotein present at synaptic and Res. 76: 85–92. 18. Edenhofer, F., R. Rieger, M. Famulok, W. Wendler, S. Weiss, and Downloaded from neuromuscular junctions as well as in the sphingolipid microdo- E. L. Winnacker. 1996. Prion protein PrPc interacts with molecular chaperones of mains of lymphoid cells, are obvious examples of molecules with the Hsp60 family. J. Virol. 70: 4724–4728. functional properties in the two systems (50, 51). Semaphorins and 19. Jin, T., Y. Gu, G. Zanusso, M. Sy, A. Kumar, M. Cohen, P. Gambetti, and N. Singh. 2000. The chaperone protein BiP binds to a mutant prion protein and their receptors, which were originally identified as molecules in- mediates its degradation by the proteasome. J. Biol. Chem. 275: 38699–38704. volved in axonal guidance during CNS development, also seem to 20. Zanata, S. M., M. H. Lopes, A. F. Mercadante, G. N. Hajj, L. B. Chiarini, modulate T/DC interactions (52, 53). For instance, expression of R. Nomizo, A. R. Freitas, A. L. Cabral, K. S. Lee, M. A. Juliano, et al. 2002. Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers plexin-A1, a receptor of semaphorins present at the surface of ma- neuroprotection. EMBO J. 21: 3307–3316. http://www.jimmunol.org/ ture DC, is tightly regulated with transcription factor CIITA, 21. Bounhar, Y., Y. Zhang, C. G. Goodyer, and A. LeBlanc. 2001. Prion protein protects human against Bax-mediated apoptosis. J. Biol. Chem. 276: which controls MHC class II expression and optimizes T lympho- 39145–39149. cyte activation. Abs against -1, another member of the 22. Schneider, B., V. Mutel, M. Pietri, M. Ermonval, S. Mouillet-Richard, and semaphorin family present at the T and DC surfaces, inhibit T cell O. Kellermann. 2003. NADPH oxidase and extracellular regulated kinases 1/2 are targets of prion protein signaling in neuronal and nonneuronal cells. Proc. Natl. proliferation in a way reminiscent of our present results (54). The Acad. Sci. USA 100: 13326–13331. implication of PrPC in two independent physiological systems 23. Paitel, E., C. Alves da Costa, D. Vilette, J. Grassi, and F. Checler. 2002. Over- does not necessarily mean that the protein fulfills equivalent func- expression of PrPc triggers caspase 3 activation: potentiation by proteasome in- hibitors and blockade by anti-PrP . J. Neurochem. 83: 1208–1214. tions, but it provides the ground for future investigations aimed at 24. Solforosi, L., J. R. Criado, D. B. McGavern, S. Wirz, M. Sanchez-Alavez, a better understanding of the physiological and eventually the S. Sugama, L. A. DeGiorgio, B. T. Volpe, E. Wiseman, G. Abalos, et al. 2004. by guest on September 25, 2021 pathogenic role of the prion protein. Cross-linking cellular prion protein triggers neuronal apoptosis in vivo. Science 303: 1514–1516. 25. Bueler, H., M. Fischer, Y. Lang, H. Bluethmann, H. P. Lipp, S. J. DeArmond, Acknowledgments S. B. Prusiner, M. Aguet, and C. Weissmann. 1992. Normal development and We thank I. Renault for the mouse breeding and the management of the behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356: Ϫ 577–582. animal facility; Dr. C. Weissmann for the PrP breeders; Dr. O. Lantz for 26. Durig, J., A. Giese, W. Schulz-Schaeffer, C. Rosenthal, U. Schmucker, advice, discussion, and gift of H-Y peptide; Dr. J. Grassi for the gift of J. Bieschke, U. Duhrsen, and H. A. Kretzschmar. 2000. Differential constitutive anti-PrP mAbs and Fab; and Dr. M. Rosset-Bruley for critical reading of and activation-dependent expression of prion protein in human peripheral blood the manuscript. leucocytes. Br. J. Haematol. 108: 488–495. 27. Kubosaki, A., S. Yusa, Y. Nasu, T. Nishimura, Y. Nakamura, K. Saeki, Y. Matsumoto, S. Itohara, and T. Onodera. 2001. Distribution of cellular isoform Disclosures of prion protein in T lymphocytes and bone marrow, analyzed by wild-type and The authors have no financial conflict of interest. prion protein gene-deficient mice. Biochem. Biophys. Res. Commun. 282: 103–107. 28. Liu, T., R. Li, B. S. Wong, D. Liu, T. Pan, R. B. Petersen, P. Gambetti, and References M. S. Sy. 2001. Normal cellular prion protein is preferentially expressed on 1. Collinge, J. 2001. Prion diseases of humans and animals: their causes and mo- subpopulations of murine hemopoietic cells. J. Immunol. 166: 3733–3742. lecular basis. Annu. Rev. Neurosci. 24: 519–550. 29. Cashman, N. R., R. Loertscher, J. Nalbantoglu, I. Shaw, R. J. Kascsak, 2. Prusiner, S. B. 1998. Prions. Proc. Natl. Acad. Sci. USA 95: 13363–13364. D. C. Bolton, and P. E. Bendheim. 1990. Cellular isoform of the scrapie agent 3. Basler, K., B. Oesch, M. Scott, D. Westaway, M. Walchli, D. F. Groth, protein participates in lymphocyte activation. Cell 61: 185–192. M. P. McKinley, S. B. Prusiner, and C. Weissmann. 1986. Scrapie and cellular 30. Mabbott, N. A., K. L. Brown, J. Manson, and M. E. Bruce. 1997. T-lymphocyte PrP isoforms are encoded by the same chromosomal gene. Cell 46: 417–428. activation and the cellular form of the prion protein. Immunology 92: 161–165. 4. Sarnataro, D., V. Campana, S. Paladino, M. Stornaiuolo, L. Nitsch, and 31. Kubosaki, A., Y. Nishimura-Nasu, T. Nishimura, S. Yusa, A. Sakudo, K. Saeki, C. Zurzolo. 2004. PrPC association with lipid rafts in the early secretory pathway Y. Matsumoto, S. Itohara, and T. Onodera. 2003. Expression of normal cellular stabilizes its cellular conformation. Mol. Biol. Cell 15: 4031–4042. prion protein (PrPc) on T lymphocytes and the effect of copper ion: analysis by 5. Derrington, E. A., and J. L. Darlix. 2002. The enigmatic multifunctionality of the wild-type and prion protein gene-deficient mice. Biochem. Biophys. Res. Com- prion protein. Drug News Perspect. 15: 206–219. mun. 307: 810–813. 6. Aguzzi, A., and M. Polymenidou. 2004. Mammalian prion biology: one century 32. Bainbridge, J., and K. B. Walker. 2005. The normal cellular form of prion protein of evolving concepts. Cell 116: 313–327. modulates T cell responses. Immunol. Lett. 96: 147–150. 7. Bueler, H., A. Aguzzi, A. Sailer, R. A. Greiner, P. Autenried, M. Aguet, and 33. Stuermer, C. A., M. F. Langhorst, M. F. Wiechers, D. F. Legler, C. Weissmann. 1993. Mice devoid of PrP are resistant to scrapie. Cell 73: S. H. Von Hanwehr, A. H. Guse, and H. Plattner. 2004. PrPc capping in T cells 1339–1347. promotes its association with the lipid raft proteins reggie-1 and reggie-2 and 8. Collinge, J., M. A. Whittington, K. C. Sidle, C. J. Smith, M. S. Palmer, leads to signal transduction. FASEB J. 18: 1731–1733. A. R. Clarke, and J. G. Jefferys. 1994. Prion protein is necessary for normal 34. Sunyach, C., A. Jen, J. Deng, K. T. Fitzgerald, Y. Frobert, J. Grassi, synaptic function. Nature 370: 295–297. M. W. McCaffrey, and R. Morris. 2003. The mechanism of internalization of 9. Tobler, I., S. E. Gaus, T. Deboer, P. Achermann, M. Fischer, T. Rulicke, glycosylphosphatidylinositol-anchored prion protein. EMBO J. 22: 3591–3601. M. Moser, B. Oesch, P. A. McBride, and J. C. Manson. 1996. Altered circadian 35. Brugger, B., C. Graham, I. Leibrecht, E. Mombelli, A. Jen, F. Wieland, and activity rhythms and sleep in mice devoid of prion protein. Nature 380: 639–642. R. Morris. 2004. The membrane domains occupied by glycosylphosphatidyli- 10. Wopfner, F., G. Weidenhofer, R. Schneider, A. von Brunn, S. Gilch, nositol-anchored prion protein and Thy-1 differ in lipid composition. J. Biol. T. F. Schwarz, T. Werner, and H. M. Schatzl. 1999. Analysis of 27 mammalian Chem. 279: 7530–7536. 7262 ROLE OF PrPC AT THE IMMUNOLOGICAL SYNAPSE

36. Mattei, V., T. Garofalo, R. Misasi, A. Circella, V. Manganelli, G. Lucania, clonal antibodies for their capacity to inhibit PrPSc replication in infected cells. A. Pavan, and M. Sorice. 2004. Prion protein is a component of the multimo- J. Biol. Chem. 280: 11247–11258. lecular signaling complex involved in T cell activation. FEBS Lett. 560: 14–18. 46. Blanchard, N., M. Decraene, K. Yang, F. Miro-Mur, S. Amigorena, and 37. Hugel, B., M. C. Martinez, C. Kunzelmann, T. Blattler, A. Aguzzi, and C. Hivroz. 2004. Strong and durable TCR clustering at the T/dendritic cell im- J. M. Freyssinet. 2004. Modulation of signal transduction through the cellular mune synapse is not required for NFAT activation and IFN-␥ production in prion protein is linked to its incorporation in lipid rafts. Cell Mol. Life Sci. 61: human CD4ϩ T cells. J. Immunol. 173: 3062–3072. 2998–3007. 47. Mouillet-Richard, S., M. Ermonval, C. Chebassier, J. L. Laplanche, S. Lehmann, 38. Wurm, S., C. Paar, A. Sonnleitner, M. Sonnleitner, O. Hoglinger, C. Romanin, J. M. Launay, and O. Kellermann. 2000. Signal transduction through prion pro- and C. Wechselberger. 2004. Co-localization of CD3 and prion protein in Jurkat tein. Science 289: 1925–1928. lymphocytes after hypothermal stimulation. FEBS Lett. 566: 121–125. 39. Aucouturier, P., F. Geissmann, D. Damotte, G. P. Saborio, H. C. Meeker, 48. Wu, Y., Y. Guo, C. A. Janeway, Jr., and Y. Liu. 1995. Signaling by a new R. Kascsak, R. Kascsak, R. I. Carp, and T. Wisniewski. 2001. Infected splenic anti-Thy 1 monoclonal inhibits T cell proliferation and interferes with dendritic cells are sufficient for prion transmission to the CNS in mouse scrapie. T-cell-mediated induction of costimulatory molecule B7-2. Cell. Immunol. 165: J. Clin. Invest. 108: 703–708. 266–277. 40. Huang, F. P., C. F. Farquhar, N. A. Mabbott, M. E. Bruce, and G. G. MacPherson. 49. Marmor, M. D., M. F. Bachmann, P. S. Ohashi, T. R. Malek, and M. Julius. 1999. 2002. Migrating intestinal dendritic cells transport PrPSc from the gut. J. Gen. Immobilization of glycosylphosphatidylinositol-anchored proteins inhibits T cell Virol. 83: 267–271. growth but not function. Int. Immunol. 11: 1381–1393. 41. Rosicarelli, B., B. Serafini, M. Sbriccoli, M. Lu, F. Cardone, M. Pocchiari, and 50. Huh, G. S., L. M. Boulanger, H. Du, P. A. Riquelme, T. M. Brotz, and C. J. Shatz. F. Aloisi. 2005. Migration of dendritic cells into the brain in a mouse model of 2000. Functional requirement for class I MHC in CNS development and plastic- prion disease. J. Neuroimmunol. 165: 114–120. ity. Science 290: 2155–2159. 42. Burthem, J., B. Urban, A. Pain, and D. J. Roberts. 2001. The normal cellular 51. Khan, A. A., C. Bose, L. S. Yam, M. J. Soloski, and F. Rupp. 2001. Physiological prion protein is strongly expressed by myeloid dendritic cells. Blood 98: regulation of the immunological synapse by agrin. Science 292: 1681–1686. 3733–3738. 52. Kikutani, H., and A. Kumanogoh. 2003. Semaphorins in interactions between T 43. Ford, M. J., L. J. Burton, R. J. Morris, and S. M. Hall. 2002. Selective expression cells and antigen-presenting cells. Nat. Rev. Immunol. 3: 159–167. of prion protein in peripheral tissues of the adult mouse. Neuroscience 113: 177–192. 53. Wong, A. W., W. J. Brickey, D. J. Taxman, H. W. van Deventer, W. Reed, Downloaded from 44. Benvenuti, F., C. Lagaudriere-Gesbert, I. Grandjean, C. Jancic, C. Hivroz, J. X. Gao, P. Zheng, Y. Liu, P. Li, J. S. Blum, et al. 2003. CIITA-regulated A. Trautmann, O. Lantz, and S. Amigorena. 2004. Dendritic cell maturation plexin-A1 affects T-cell-dendritic cell interactions. Nat. Immunol. 4: 891–898. controls adhesion, synapse formation, and the duration of the interactions with 54. Tordjman, R., Y. Lepelletier, V. Lemarchandel, M. Cambot, P. Gaulard, naive T lymphocytes. J. Immunol. 172: 292–301. O. Hermine, and P. H. Romeo. 2002. A neuronal receptor, neuropilin-1, is es- 45. Feraudet, C., N. Morel, S. Simon, H. Volland, Y. Frobert, C. Creminon, sential for the initiation of the primary immune response. Nat. Immunol. 3: D. Vilette, S. Lehmann, and J. Grassi. 2005. Screening of 145 anti-PrP mono- 477–482. http://www.jimmunol.org/ by guest on September 25, 2021